Negative regulation of the inflammasome: keeping inflammation under control.

Gustavo Pedraza-Alva Leonor Perez-Martınez Laura Valdez-Hernandez Karla F. Meza-Sosa Masami Ando-Kuri

Negative regulation of the inflammasome: keeping inflammation under control

Authors’ address Gustavo Pedraza-Alva1, Leonor Perez-Martınez1, Laura ValdezHernandez1, Karla F. Meza-Sosa1, Masami Ando-Kuri1 1 Laboratorio de Neuroinmunobiologıa, Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologıa, Universidad Nacional Aut onoma de Mexico, Cuernavaca, Mexico.

Summary: In addition to its roles in controlling infection and tissue repair, inflammation plays a critical role in diverse and distinct chronic diseases, such as cancer, metabolic syndrome, and neurodegenerative disorders, underscoring the harmful effect of an uncontrolled inflammatory response. Regardless of the nature of the stimulus, initiation of the inflammatory response is mediated by assembly of a multimolecular protein complex called the inflammasome, which is responsible for the production of inflammatory cytokines, such as interleukin-1b (IL1b) and IL-18. The different stimuli and mechanisms that control inflammasome activation are fairly well understood, but the mechanisms underlying the control of undesired inflammasome activation and its inactivation remain largely unknown. Here, we review recent advances in our understanding of the molecular mechanisms that negatively regulate inflammasome activation to prevent unwanted activation in the resting state, as well as those involved in terminating the inflammatory response after a specific insult to maintain homeostasis.

Correspondence to: Gustavo Pedraza-Alva Departamento de Medicina Molecular y Bioprocesos Instituto de Biotecnologıa Universidad Nacional Aut onoma de Mexico Av. Universidad 2001 Cuernavaca, Morelos 62210, Mexico Tel.:+1 52 777 3290869 e-mail: [email protected]

Keywords: inflammation, inflammasome, NLRP, Caspase-1, IL-1b, IL-18 Acknowledgements L. V.-H. and K. M.-S. are supported by CONACyT/Mexico Graduate Scholarships. Research in the author’s laboratory is supported by grants from DGAPA/UNAM (IN209212 and IN209513), from CONACYT/Mexico (155290, 154542), and from ICGEB/Italy (CRP/MEX11-01). The authors have no conflicts of interest to declare.

This article is part of a series of reviews covering Inflammasomes appearing in Volume 265 of Immunological Reviews.

Immunological Reviews 2015 Vol. 265: 231–257

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Immunological Reviews 0105-2896

Introduction Inflammation is a response mounted by an organism to combat a plethora of insults, ranging from external noxious agents to internal danger signals released following trauma or cellular malfunction. The first cytokines produced in response to such noxious stimuli are interleukin-1b (IL-1b) and IL-18. Although many stimuli promote IL-1b and IL-18 gene expression and protein synthesis, a second stimulus is required to promote their processing and secretion. The processing of pro-IL-1b and pro-IL-18 into mature cytokines is mediated by select caspases, specifically caspase-1 and caspase-5 (1). In turn, the activation of these pro-inflammatory caspases depends on the formation of a macromolecular complex called the inflammasome (2), whose activity is controlled by members of a large NLR [nucleotide-binding and oligomerization domain (NOD)-like receptor] family of intracellular receptors or by PYHIN [pyrin domain (PYD)

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

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Pedraza-Alva et al Negative regulation of inflammasomes

and HIN domain-containing] family members. Upon activation, the NLR receptor undergoes conformational changes that promote its oligomerization and interaction with caspase-1, either directly or indirectly through interaction with adapter molecules, such as ASC or CARDINAL. The formation of this macromolecular complex favors caspase-1 selfprocessing into an active form capable of leading to inflammatory cytokine production (reviewed in 3) or inflammatory cell death, called pyroptosis (4) (Fig. 1). Although inflammation is in most cases initiated by cells of the immune system in response to invading pathogens, any cells of any type are capable of triggering an inflammatory response when confronted with an insult. It has been shown that intestinal and gingival epithelial cells produce IL-1b and IL-18 in response to pathogenic bacteria (5, 6) that neurons can produce IL-1b in response to injury (7), and that b cells in the pancreas produce IL-1b in response to high glucose levels in the blood (8). Thus, in addition to the roles of IL1b and IL-18 in the context of an immune response either against exogenous antigens or in response to self-antigens (autoimmunity), it is now clear that activation of the inflammasome is linked to many inflammatory diseases including vitiligo, gouty arthritis, type I and type II diabetes, PAMPs DAMPs

TLR

ADP UTP UDP

P2Y

K+

ATP

and a group of lesser known disorders referred to as cryopyrinopathies (9). Thus, mechanisms underlying the regulation of the inflammasome have been investigated extensively. Although many studies have focused on the initiation or promotion of inflammasome assembly and activation, the mechanisms involved in its negative regulation remain largely unknown. Here, we review the current understanding of the processes inhibiting inflammasome activation in resting conditions that prevent unwanted activation and terminate the inflammatory response to preserve homeostasis. We also review the mechanisms that pathogens have acquired through evolution to dampen the inflammatory response and ensure infection. Finally, the efforts to identify compounds with anti-inflammasome activity and their potential for controlling the inflammatory process associated with different diseases are reviewed. Endogenous inhibitors of inflammasome activation COPs and POPs Given the importance of preventing inflammation in the absence of noxious stimuli and of its termination once such stimuli have been removed, cells have acquired different

K+

P2X7 Pannexin

1. Priming

Particles (MSU,Silica,Alum) Pore formed by bacterial toxin

[K+]

ROS NLR

Lysosomal disruption

mtDNA

Pro-IL-18

NF-kB

2. Activation ER

UPR

Pro-IL NLR

ILASC Caspase-1 Pro-IL 18

IL-18

Fig. 1. Overview of inflammasome activation. Priming (left). Binding of LPS to a Toll-like receptor (TLR) activates NFjB, which then initiates the transcription of genes encoding for components of the inflammasome and inflammatory cytokines. Activation (right). Assembly of the NLRP inflammasome can be induced by different danger signals, including pore-forming toxins, exogenous ATP, K+ exit from the cells, lysosomal damage caused by silica, uric acid, or asbestos crystals, the unfolded protein response, reactive oxygen species and oxidized mitochondrial DNA produced as consequence of mitochondrial dysfunction. PAMPs, pathogen-associated molecular patterns; DAMPs, danger-associated molecular patterns; ER, endoplasmic reticulum; UPR, unfolded protein response; NLR, NOD-like receptor; ASC, apoptosis-associated speck-like protein containing a CARD; ROS, reactive oxygen species; NFjB, nuclear factor j-light-chain-enhancer of activated B cells; MSU, monosodium urate; P2X7, Purinergic 2X7 receptor; P2Y, Purinergic P2Y receptor.

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mechanisms to regulate inflammasome activation and inactivation. Interestingly many proteins containing a single CARD domain [CARD-only proteins (COPs)] or a single pyrin domain [PYD-only proteins (POPs)], which function as dominant-negative molecules and therefore regulate the inflammatory response, are encoded by the human but not the mouse genome, suggesting a more complex regulation of inflammation in the human. The first COPs identified were ICEBERG and pseudo-ICE (COP1) (10). Both proteins interact with the caspase-1 CARD domain and restrict caspase-1 interaction with ASC, thereby preventing inflammasome activation. In a similar fashion, INCA (inhibitory CARD) and caspase-12 interact with caspase-1 and prevent its recruitment to the inflammasome (11–13). Interestingly, a short version of Nod2 (Nod2-s), which contains only the first CARD domain, also functions as a COP (14) (Fig. 2). Likewise, endogenous POPs such as POP1 and POP2 bind to the PYD domains of ASC and NLRs (NLRP2 and NLRP7), thereby sequestering them and halting inflammasome assembly (15). The expression of several COPS and POPS is upregulated during inflammation, suggesting that these molecules are part of the negative feedback loops that control the strength and duration of the inflammatory response. However, inflammasome assembly and function are inhibited not only by COPs or POPs but also by anti-apoptotic proteins, which can negatively regulate the activation of the NLRP1 inflammasome. It was recently shown that Bcl-2 and Bcl-XL bind directly to NLRP1 through its LRR domains to prevent ATP binding and subsequent oligomerization, thereby keeping NLRP1 in the inactive state (16). A proteomic approach to identifying new ASC-interacting proteins has shown that PML (promyelocytic leukemia protein), a nuclear scaffold protein that controls important cellular process such as transcriptional and cell cycle regulation, antiviral response, DNA damage response and repair, apoptosis, and metabolism (17), is a binding partner of ASC. Further studies have demonstrated that THP-1derived macrophages lacking PML possess enhanced NLRP3 and AIM2 inflammasome activity as well as IL-1b production compared to wildtype macrophages but show normal pyroptosome activation in response to Salmonella infection, suggesting that PML negatively regulates ASC participation in inflammasome-induced IL-1b production but not in pyroptosome activation (18). Consistent with these results, unstimulated PML-deficient macrophages showed altered ASC localization, with more ASC molecules present in the © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

cytosol than in the nucleus. This implies that by restricting ASC access to the cytoplasm, PML prevents inflammasome activation in resting cells, although the molecular mechanism underlying the release of ASC from the nucleus upon stimulation remains to be determined. The interaction of PML with ASC through a sequence encompassing its Ring, BBox1, and BBox2 domains and the fact that it is the target of multiple post-translational modifications (17) suggests that upon cellular stimulation, PML might undergo specific post-translational modifications to permit release of ASC and form a functional inflammasome. In a similar proteomic approach involving the immunoprecipitation of NLRP3 rather than ASC, it was found that the leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) associates with NLRP3 through its N-terminal domain, recruiting the caspase-1 pseudosubstrate Flightless-I to the inflammasome complex via its LRRFIP2 Coil motif, thus favoring caspase-1 inhibition (19) (Fig. 2). Whether the same mechanism is used to inhibit other inflammasomes is an open question. Pyrin and HIN200 domain-containing proteins Negative regulation of the AIM2 inflammasome limits IL-1b production and cell death in response to viral or bacterial dsDNA, allowing the production of type I interferon. Recently, it has been suggested that members of the pyrin and HIN200 domain-containing family [(PYHIN) also known as p200 or HIN200] of proteins might mediate inhibition of the AIM2 inflammasome. Given that these molecules lack the PYD domain and therefore cannot interact with ASC, it has been proposed that upon DNA binding, p200 prevents AIM2 inflammasome assembly and caspase-1 activation (20). Supporting experimental evidence has been published recently by Yin et al. (21), who showed that binding of p200 to dsDNA does not prevent AIM2 binding to dsDNA, but does prevent AIM2 clustering by separating AIM2 molecules, thus preventing ASC oligomerization and inflammasome assembly. In addition, AIM2 can be negatively regulated by repeated telomeric DNA sequences (22). Previous studies showed that repeated telomeric sequences, such as TTAGGG, negatively modulate the inflammatory response in mouse models of arthritis, toxic shock, atherosclerosis, and silica-induced pulmonary inflammation (23–25). Kaminski et al. (22) showed that suppressive oligonucleotides bind AIM2 and prevent the binding of stimulatory dsDNA sequences, thus inhibiting the AIM2 inflammasome. Accordingly, suppressive oligonucleotides reduced IL-1b and IL-18 production in dendritic cells infected with cytomegalovirus (22).

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Immunoglobulins Recent studies have pointed to an anti-inflammatory function for immunoglobulins when administered intravenously (26). Stroke is a major cause of neuronal cell death and disability, and conditions such as nutrient deficiency, oxidative stress, and inflammation following stroke have been associated with neuron degeneration and death. Accordingly, it has been shown that levels of inflammasome proteins (NLRP1, NLRP3, ASC, and caspase-1) were increased in the brains of human stroke survivors and ischemic animals, which correlated with elevated mature IL-1b and IL-18 levels and neuronal cell death. Strikingly, intravenous administration of IgG reduced IL-1b and IL-18 levels in ischemic brains, accompanied by clear reductions in the levels of inflammasome proteins and neuronal death (27). Thus, the anti-inflammatory function of intravenous IgG administration was confirmed. Given that this treatment inhibits NFjB and MAPK activation (27), it is possible that intravenous IgG prevents expression of the genes encoding inflammasome proteins in response to ischemia. However, the possibility that intravenous IgG triggers the post-translational modification of inflammasome components leading to their proteosomal or autophagic degradation has not been disproven. The pro-neural effects of intravenous IgG administration can be explained by the fact that IgG is able to induce the expression of genes with anti-apoptotic activities, such as Bcl-2 and Bcl-XL. Furthermore, as already mentioned, the direct protein–protein interactions of both Bcl-2 and Bcl-XL negatively regulate NLRP1 activation (16). Thus, by inhibiting NLRP1, IgG could also prevent inflammasome activation and inflammation. Nonetheless, the molecular mechanisms through which IgG blocks NFjB activity and promotes NLRP1, NLRP3, and caspase-1 degradation while promoting Bcl-2 and Bcl-XL expression are completely unknown. Thrombomodulin Cumulative experimental evidence has shown that in addition to its function as an anticoagulant, thrombomodulin, through its extracellular domain, regulates the inflammatory response elicited by different insults. Specifically, the thrombomodulin extracellular domain (TED) negatively modulates inflammation by: (i) binding to LPS and preventing its interaction with CD14 and TLR4, thus reducing the inflammatory response of macrophages (28); (ii) negatively regulating the expression of adhesion molecules, which prevents the binding of polymorphonuclear leukocytes to endothelial

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cells, thereby reducing neutrophil-mediated tissue damage (29); (iii) interacting with HMBG1 (high-mobility box protein 1), which prevents leukocyte activation, ultraviolet irradiation-induced cutaneous inflammation, and LPS-induced lethality (30); and (iv) obstructing both the classical and the lectin complement activation pathways (31). In a recent study (32), delivery of the TED by adenovirus vector was shown to reduce the inflammatory response in glycemic db/ db mice (a model of obesity-induced nephropathy) by targeting inflammasome activation. Interestingly, TED reduced NLRP3 expression levels and caspase-1 activity in the kidneys of TED-treated mice compared with those in control mice, and the reduction was correlated with reduced IL-1b and IL-18 cytokine serum levels. Reduced ROS production and NFjB activation was also observed in the kidneys of db/ db mice receiving TED (32), suggesting that the TED domain prevents inflammation by blocking NFjB activation and therefore NLRP3 expression and that it may also prevent thioredoxin-mediated NLRP3 inflammasome activation by reducing ROS levels. Although further experiments are required to elucidate the molecular mechanism by which TED prevents NLRP3 inflammasome activation, viral delivery of TED might be a promising therapeutic tool for controlling deleterious inflammatory responses (Fig. 2). Molecular interactions with NLRPs The protein–protein interactions between NLRPs have been suggested as a mechanism underlying NLRP function and inflammasome activation (33). The NLRP3 inflammasome mediates IL-1b production in response to the presence of fatty acids (34). Accordingly, diets rich in fat result in chronic inflammation, which leads to NLRP3 inflammasome activation in adipose tissue that triggers IL-1b production that in turn leads to glucose intolerance and insulin resistance in C57BL/6 obese mice (34). Interestingly, the alteration in glucose metabolism resulting from a high-fat diet is considerably delayed in Balb/c mice (35). Unlike C57BL/6 mice that express the Nlrp1b2 gene, Balb/c mice express the Nlrp1b1 gene, suggesting that NLRP1B1 inflammasome may negatively modulate lipid-induced NLRP3 inflammasome activation. Accordingly, preliminary data from our laboratory indicate that expression of the Nlrp1b1 allele in a C57BL/6 background results in reduced adipose tissue inflammation as well as improved glucose metabolism (L. Valdez-Hernandez, J. Salazar, M. Iriart, L. Perez-Martınez and G. Pedraza-Alva, unpublished data). The fact that NLRP3 inflammasome activation in response to ATP in LPS-primed © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


PAMPs DAMPs

IFN

-3 FAs

CRH

TLR

IFNGR

NMDA

GPR120 GPR40 ARRB2

25-HC

Thrombomodulin

NLRP6 NLRP3

Pro-IL

IL-

Caspase-1 Pro-IL 18

Pro-IL-18

IL-18

F1

ASC NLR

p202

SREBP

AIM2 TTAGGG

ODNs miR-223 miR-BART15

ARRB2 PML

Bcl-xl TRIM30

COP

Bcl-2 LRRFIP2

mNLRP3 GPSM3 HSPA8

F1

POP

SGT1 HSP90

Fig. 2. Extracellular and intracellular signals that modulate inflammasome activation. Cells have different mechanisms to downregulate inflammasome activation: hormones and miRNAs, inhibit the transcription of inflammasome components; Omega-3 fatty acids and survival signals, promote the interaction of NRLPs, ASC, and caspase-1 with several proteins to inhibit the assembly of inflammasome. TLR, Toll-like receptor 4; PAMPs, pathogen-associated molecular patterns; DAMPs, danger-associated molecular patterns; NLR, NOD-like receptor; ASC, apoptosis-associated speck-like protein containing a CARD; AIM2, absent in melanoma 2; NFjB; nuclear factor j-light-chain-enhancer of activated B cells; CRH, corticotropin releasing hormone; x-3FAs, Omega-3 fatty acids; PML, promyelocytic leukemia protein; ARRB2, b-arrestin-2; TRIM30, tripartite-motif protein-30; LRRFIP2, leucine-rich repeat Fli-I-interacting protein 2; F1, Flightless-I; P202; member of the pyrin and HIN200 domain-containing protein family (PYHIN, also known as p200 or HIN200 proteins); ODNs, oligodeoxynucleotides; miRNA-223, microRNA-223; miRNA-BART15, microRNA-BART15; GPSM3, protein signaling modulator-3; HSP90, heat shock protein 90; STG1, suppressor of G2 allele of SKP1; HSPA8, heat shock protein 8; SREBP, sterol regulatory element-binding protein; 25-HC, 25-hydroxycholesterol; mNLRP3, NLRP3 messenger RNA; INFGR, interferon-c receptor.

Balb/c bone marrow-derived macrophages (BMDMs) is comparable to that observed in C57BL/6 BMDMs suggests that there are no functional differences in NLRP3 inflammasome activation between macrophages derived from these mouse strains. Current experiments in our laboratory aim to define the molecular mechanism by which NLRP1B1, but not NLRP1B2, prevents NLRP3 inflammasome activation in response to fatty acids. Inflammasome negative regulation by phosphorylation IKKa has historically been viewed as a proinflammatory molecule because its activation by a variety of signals results in the NFjB-mediated expression of inflammatory cytokines. Recently, however, it has been shown that basal IKKa kinase activity in resting macrophages prevents spontaneous inflammasome activation (36). Martin et al. (36) elegantly demonstrated that IKKa associates with ASC and phosphorylates its residue S293, thus restricting ASC to the nucleus. Upon macrophage stimulation, IKKι mediates IKKa/ASC complex translocation to the perinuclear space, where IKKa keeps ASC in its inactive state. It is not until the cell receives a second © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

signal that phosphatase PP2A, and possible other phosphatases, releases ASC from the negative influence of IKKa, thereby allowing inflammasome activation. Interestingly, it has been shown that IKKa negatively regulates NLRP3, NLRC4, and AIM2 inflammasomes through this mechanism, thus providing a clear explanation for the early observations that mice with impaired IKKa kinase activity developed inflammatory diseases, enhanced susceptibility to LPS-induced shock (37), and even cancer (38). The fact that kinases, such as Syk and JNK, promote inflammasome activation through ASC phosphorylation (39) suggests that these kinases phosphorylate ASC once IKKa interaction has been lost. However, it is possible that ASC phosphorylation by Syk or JNK promotes IKKa/ASC complex dissociation and inflammasome assembly. Further experiments are required to test this possibility. Inflammasome inhibition by innate and adaptive immune mediators As with other cellular responses triggered by a specific signal, inflammation must eventually be terminated to avoid undesired damage to bystander cells and tissues. Inability to termi-

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T Cell CD4+

RANKL 41BBL CD40L CD30L LIGHT OX40L

CD45RO+ IVIg

FasL

DAMPs PAMPs

IFNAR/ IFNGR

TLR

NLR P2X7 Caspase-1 Pro-IL-18

NF-kB

K+

IFN IFN

IL-10R

Fas

TNFRSF

ATP

LTx

P2X7 Pore Particles (MSU,Alum, Asbesto)

Mtb

2. Activation

T3 STA

NLRP1 NLRP3

T1 STA

IKK

Pro-IL IL-

ASC

1. Priming

SNO

Caspase-1 Pro-IL 18

IL-18

Fig. 3. Inflammasome regulation by innate and adaptive immune mediators. Memory or effector CD4+ T cells are able to inhibit NLRP inflammasomes by T cell:antigen-presenting cell contact via the expression of membrane-bound TNF family ligands like RANKL and CD-40L or independently of cell:cell contact, which involves the release of soluble Fas-ligand (Fas-L). PAMPs, pathogen-associated molecular patterns; DAMPs, danger-associated molecular patterns; NLRP1/3, NOD-like receptor (NLR) family pyrin domain containing 1–3; NFjB, nuclear factor j-light-chain-enhancer of activated B cells; TLR, Toll-like receptor; ASC, apoptosis-associated speck-like protein containing a CARD; IL-10, interleukin-10; TNFRSF, tumor necrosis factor receptor superfamily; MSU, monosodium urate; Mtb, Mycobacterium tuberculosis; LTx, Anthrax lethal toxin; IFNb-c, interferon b, Interferon-c; CD40L, CD40 ligand; IFNAR, interferon-a/b receptor; IFNGR, interferon-c receptor.

nate inflammation results in chronic diseases such as type 2 diabetes, arthritis, and death by septic shock. Nitric oxide (NO) is produced by macrophages to kill and eliminate infectious agents in response to c interferon (INFc) produced by activated T cells. However, Hernandez-Cuellar et al. (40) recently showed that in addition to its pathogenic activity, NO suppresses the NLRP3 inflammasome (and to a lesser extent, the AIM2 and NLRC4 inflammasomes) through a mechanism that involves direct NLRP3 and caspase-1 S-nitrosylation, thereby reducing IL-1b production and inflammation. Interestingly, INFc produced by T-helper 1 (Th1) cells during M. tuberculosis infection suppresses production of IL-1b by alveolar macrophages (41). It is tempting to speculate that in addition to cognate T-cell activation, the INFc secreted by lung effector Th1 cells to destroy mycobacterium also prevents an uncontrolled inflammatory response by reducing IL1b production in the lung environment. However, given that neither NLRP3 nor caspase-1 are responsible for IL-1b production in the lungs of mice infected with M. tuberculosis, it is possible that NO released by macrophages in response to

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INFc could also promote S-nitrosylation and inhibition of the neutrophil proteases responsible for IL-1b production in response to M. tuberculosis. Interestingly, NO is also produced by endothelial cells in response to cholinergic innervations (42). Thus, the CNS anti-inflammatory response might also involve acetylcholine-mediated NO production and S-nitrosylation of inflammasome components to control inflammatory cytokine production in the periphery. Another example of T cell-mediated negative regulation of inflammasome function requires T-cell activation and antigen stimulation (Fig. 3). Memory or effector T cells are able to inhibit the NLRP1 and NLRP3 inflammasomes. The negative effect on inflammasome activation involves contact between the T cell and antigenpresenting cell as well as the expression of membrane-bound TNF family ligands such as RANKL and CD-40L on activated T cells (43). However, the signal transduction pathway leading to inflammasome inactivation remains to be elucidated. Contrary to the data discussed above, another study claimed that the negative effect of memory T cells on the NLRP3 inflammasome is independent of cell–cell contact © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


PAMPs DAMPs

TLR2 TLR4

Autophagosome

LC3B PAI-2 NLRP3 Caspase-1

Lysosome

PAI-2 beclin vps34

Pro-IL-18

ER mtDNA

Autolysosome

mROS P62

AIM2 Pro-IL

NLRP

ASC

poly ubiq uitin

atio

IL-

ASC Caspase-1

IL-18

n

Autophagy

Pro-IL 18

Fig. 4. Autophagy negatively regulates the inflammasome. Several lines of evidence show that autophagy prevents inflammasome activation. The activation of TLRs pathway promotes PAI-2 expression, which stabilized the process of mitoautophagy diminishing the insults that activate the inflammasome as mROS or mtDNA. In addition, assembled inflammasomes could undergo ubiquitination and be recruited by p62 to autophagosomes. PAMPs, pathogen-associated molecular patterns; DAMPs, danger-associated molecular patterns; NLRP, NOD-like receptor (NLR) family pyrin domain containing; NFjB, nuclear factor j-light-chain-enhancer of activated B cells; LC3B, protein light chain 3; vps34, vacuolar protein sorting 34; PAI-2, plasminogen activator inhibitor type 2; ER, endoplasmic reticulum; p62, protein of 62KDa; AIM2, absent in myeloma 2; ASC, apoptosis-associated speck-like protein containing a CARD; mROS, mitochondrial reactive oxygen species.

and involves the release of soluble Fas-ligand (Fas-L). In this study, Fas-L promoted the reduction in P2X7R expression in macrophages, thus explaining why Fas-L inhibited NLRP3 inflammasome activation only in response to ATP but not to alum or monosodium urate crystals (44). Interestingly, INFb-primed memory T cells mediated inhibition of the NLRP3 inflammasome. Although type I interferons have been long used to control inflammation in autoimmune diseases and other inflammatory disorders, the molecular mechanism of their action remained a mystery. Guarda et al. (45) showed that type I interferons inhibit NLRP1 and NLRP3 inflammasomes through a STAT-1-dependent mechanism (Fig. 3). A further reduction in IL-1b production is then achieved by type I interferon-dependent IL-10 production. IL-10, through an autocrine mechanism, activates STAT-3 to inhibit pro-IL-1b expression (45). In agreement, INFb treatment reduced IL-1b production in monocytes taken from multiple sclerosis patients. Nonetheless, the molecular mechanism through which STAT-1 negatively regulates inflammasome activation remains to be determined. It is possible that by skewing cellular immunity toward a T-helper 2 response, pathogens might also inhibit IL-1b production in an IL-10-dependent manner, thus assur© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

ing infection. Hence, independent of the source, type I interferons trigger different mechanisms to limit inflammatory cytokine production, providing an explanation for the weak immune response against secondary infection generated after viral exposure. Negative regulation of inflammasome activation by heat shock chaperones and ubiquitination The function of heat shock proteins (HSP) has been associated with accurate protein folding under basal conditions or in response to a variety of cellular insults, including heat shock. However, recent reports have made clear that heat shock proteins can be secreted and can function as mediators of the immune response by promoting the expression of cytokines and cell adhesion molecules (46). Depending on cellular context, some heat shock proteins may negatively modulate inflammation. We recently showed that HSP70 negatively regulates the expression of TLR-induced pro-inflammatory cytokines (47). The first evidence that the heat shock response also regulates inflammasome activation came from studies of plant response to pathogens. In plants, pathogen-sensing molecules known as R proteins share structural homology with the LRR domain of

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PAMPs

TLR V. parahaemolyticus S. enterica M. tuberculosis VopQ,VopS Yap C. trachomatis F. tularensis P. aeruginosa CPAF mviN,ripA ExoU,ExoS Yersinia spp. S.flexneri L. pneumophila YopM,YopK,YopT OspC3 sdhA NLR Pro-IL-18

NLR AIM2

Pro-IL 18

ASC

IL-18 Caspase-1

KSHV Orf63 Shope

virus gp013 Myxoma ML013

Measles virus MV-V Vaccinia virus F1L HSV-1 ICP0

HPV E6-AP Cowpox crmA

Fig. 5. Negative regulation of inflammasome by microbial and viral molecules. Bacterial pathogens have protein export systems to transport proteins to achieve a successful colonization. It has been proved that components of these secretions systems as T3SS or T4SS modulate negatively the inflammasome assembly. Moreover, viruses also have evolved to diminish the inflammatory response by interfering with the production of IL-1b and IL-18. TLR 4, Toll-like receptor 4; NFjB, nuclear factor j-light-chain-enhancer of activated B cells; KSHV, Kaposi’s sarcoma-associated herpes virus; Orf63, open reading frame 63; NLRP, NOD-like receptor (NLR) family pyrin domain containing; HSV-1, herpes simplex virus; crmA, cytokine response modifier; MV-V, Measles virus V protein; ASC, apoptosis-associated speck-like protein containing a CARD; PAMPs, pathogen-associated molecular patterns; ExoU, Exoenzyme U; ExoS, Exoenzyme S; VopQ, Vibrio outer protein Q; VopS, Vibrio outer protein; Sdh A, succinate dehydrogenase; AIM2, absent in melanoma 2; mivN, mouse virulence N; E6-AP (E6-associated protein); CPAF, chlamydial protease-like activity factor.

mammalian NLRPs. To mount a successful response to pathogens, the R protein forms a complex with HSP90 and the ubiquitin ligase-associated protein SGT1 (48). In mammals, the interaction of NLRP3 with the HSP90–SGT1 complex maintains NLRP3 in a primed state, and upon proper stimulation, NLRP3 is released from the HSP90–SGT1 complex to initiate inflammasome assembly and activation (49). Thus, in the resting state, the HSP90–SGT1 complex prevents improper inflammasome activation by ubiquitinating NLRP3. In accord with the idea that inactive NLRP3 is ubiquitinated, Py et al. (50) showed that inhibiting deubiquitinating enzymes with the small inhibitor G5 prevented NLRP3 inflammasome activation. The deubiquitinating enzyme BRCC3 has been shown to be critical for NLRP3 deubiquitination and activation (50). Apparently, ubiquitination is not a general mechanism for the maintenance of NLRP inactive state because neither AIM2 nor NLRC4 inflammasome activity is affected by ubiquitination. Thus, primed NLRP3 needs to be deubiquitinated to be fully functional. More recently, another negative regulator of the NLRP3 inflammasome was identified using a two-hybrid

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system. The hematopoietic-restricted protein ‘G protein signaling modulator-30 (GPSM3) interacts with the LRR domain of NLRP3. Interestingly, this interaction is enhanced by HSP8 (51). GPSM3 deficiency results in enhanced IL-1b production and NLRP3 inflammasome activation in response to ATP or alum in LPS-treated macrophages, an effect that was specific for the NLRP3 inflammasome (GPSM3-deficient macrophages showed no defects in AIM2 or NLRC4 inflammasome activation in response to DNA or flagellin, respectively). Consistent with these results, alum-induced peritonitis was more severe in GPSM3-deficient mice than in wildtype mice. Thus, in resting macrophages, the GPSM3–HSP8 complex maintains NLRP3 in an inactive complex. However, LPS priming decreases GPSM3 expression, thus likely allowing the interaction of NLRP3 with the HSP90–SGT1 complex to adapt the primed conformation. Finally, upon second signal, NLRP3 is deubiquitinated by BRCC3. Whether other inflammasomes are also regulated by ubiquitination and deubiquitination remains to be determined. However, it has been shown that the heat response negatively regulates inflammasome activation. Levin et al. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


ATP

K+

LPS

EGCG Creosol Ginsenosides Polyenylpyrrole

TLR EGCG Anthraquinones Ginsenosides Resveratrol

P2X7

ROS Cathepsin B

AIM2 EGCG Arsenic trioxide

NLRP1

Ethanol

EPP-AF Glyburide Resveratrol

NLRP3 NLRP3 Caspase-1

ASC

Pro-IL 18 IL-18 Caspase-1

Pro-IL-18 EGCG Creosol Ginsenosides Polyenylpyrrole

Creosol

Polyphenols

Allopurinol Arsenic trioxide EGCG Creosol Artemisinin

Allopurinol Anthraquinones EGCG EPP-AF Ethanol Creosol Glyburide Ginsenosides Polyenylpyrrole Tymoquinone

Fig. 6. Inhibition of inflammasome by organic compounds. Certain metabolites of plants have anti-inflammatory properties, and some compounds negatively regulate the inflammasome by diminishing the assembly of the NLRP3 inflammasome complexes or blunting NFjBmediated inflammatory responses. On the other hand, polyphenols can activate NFR2 and induce antioxidant signaling thus indirectly preventing NLRP3 inflammasome activation. TLR4, Toll-like receptor 4; LPS, lipopolysaccharide; ROS, reactive oxygen species; NLRP 1–3, NOD-like receptor (NLR) family pyrin domain containing 1–3; NFjB, nuclear factor j-light-chain-enhancer of activated B cells; ASC, apoptosis-associated speck-like protein containing a CARD; NRF2, nuclear factor E2-related factor 2.

(52) showed that heat shock renders macrophages resistant to Anthrax lethal toxin-mediated cell death. This effect was not specific to the NLRP1 inflammasome because nigericinmediated NLRP3 inflammasome activation was also inhibited in heat-shocked macrophages (52). Heat shock-mediated inhibition did not involve HSP90; thus, further experiments are required to determine the composition of the protein complex that prevents caspase-1 activation. Together, these lines of evidence show that heat shock proteins control inflammasome activity during the course of the inflammatory response. Regulation of inflammasome activity by autophagy Autophagy is a key homeostatic process involved in the degradation of dysfunctional or unnecessary cellular components (e.g. organelles and proteins). Alterations in autophagy may result in pathological conditions, including cancer. Besides starvation, autophagy can be induced by infection, in an effort to limit pathogen growth (53). Accordingly, inflammatory cytokines such as INFc and TNF-a promote autophagy (54), whereas anti-inflammatory cytokines such as IL-4 and IL-13 have the opposite effect (55). Data obtained from macrophages deficient in the Atg-16 gene, a gene known to regulate autophagy that is © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

associated with Crohn’s disease, suggested that autophagy negatively regulates inflammation by controlling IL-1b and IL-18 production (56). In agreement, Harris et al. (57) have recently shown that pro-IL-1b, produced in response to the stimulation of Toll-like receptors, is sequestered in autophagosomes and that upon autophagy induction, pro-IL-1b is degraded (Fig. 4). The same group also confirmed that autophagy inhibition enhanced IL-1b and IL-18 production through a mechanism involving NLRP3 inflammasome activation by ROS, which may result from the accumulation of dysfunctional mitochondria (57). In addition, mitochondrial membrane permeability transition and the translocation of mitochondrial DNA to the cytosol that results from challenging macrophages with LPS and ATP is enhanced in BCL3-deficient and Beclin haploinsufficient macrophages, which showed enhanced inflammasome activation and IL-1 b production both in vitro and in vivo (58). The participation of the AIM2 inflammasome as a cytosolic mitochondrial DNA sensor has been ruled out. Interestingly, NLRP3 is required for mitochondrial DNA translocation to the cytosol in a mitochondrial ROS-dependent manner (58). The lack of inflammasome activation in response to ROS generated after the engagement of TLRs remained a mystery for long time. The first experimental

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evidence indicating that TLR signaling activates a mechanism to control TLR-induced ROS production came from experiments showing an inverse correlation between the expression level of tripartite-motif protein 30 (TRIM-30) and IL-1b production in macrophages stimulated with LPS and ATP. Lack of TRIM-30 enhanced LPS-mediated ROS production, whereas TRIM-30 overexpression reduced it, resulting in a respective increase and reduction in NLRP3 inflammasome activation (59). Due to its lysosomal localization, it has been suggested that TRIM-30 might induce autophagy to control inflammasome activity, but this remains to be proven. Nonetheless, Chuang et al. (60) have recently lifted the veil surrounding this mystery and have shown that induction of TLR-2 and TLR-4 promotes the expression of the serine protease inhibitor PAI-2 (plasminogen activator inhibitor type 2). PAI-2 binds to the HSP90/Beclin-1 complex, thereby preventing its proteosomal degradation and therefore promoting the autophagic degradation of mitochondria-associated and cytosolic NLRP3, thus preventing caspase-1 activation and IL-1b production (60). Autophagy is apparently triggered at the same time as inflammasome activation to control the duration and strength of the inflammatory response by targeting inflammasome components for degradation in the phagosome (61). Shi et al. (61) showed that NLR activation not only results in inflammasome assembly and caspase-1 activity but also promotes the activation of the small GTPAse RalB in a manner independent of ASC or caspase-1 activity. RalB is involved in autophagy initiation by promoting the assembly of the Exo84–Beclin-1 and Exo84– Vps34 complexes (62). Interestingly, these researchers also found that inflammasome activation promoted ASC ubiquitination, thus tagging it for interaction with p62, an autophagic adapter, and delivery to the autophagosome (61). Although these studies have clearly highlighted the relationship between inflammasome activation and the initiation of autophagy, and the consequent downregulation of inflammasome activity, additional questions arise. Because caspase-1 activity is not required for triggering autophagy, how does the cell sense when to initiate or to stop autophagic destruction of inflammasome components without risking the innate immune response? Given that HSP90 and SGT1 are associated with NLRPs under basal conditions and keep them in an inactive but competent state (49), and HSP90 associates with Beclin-1 to induce autophagosome nucleation, one wonders whether the HSP90 molecules that dissociate from the NLRP upon activation might then interact with Beclin-1 to initiate autophagy.

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Inflammasome regulation by microRNAs microRNAs (miRNAs) are small endogenous RNAs of approximately 19–21 nucleotides (nt) that mediate the posttranscriptional silencing of their target mRNAs through base pairing between 2 and 8 bases of the miRNA (seed sequence) and the mRNA 30 UTR (63–66). miRNA-mediated silencing of a target mRNA is a general cellular processes that exquisitely controls the spatio-temporal regulation of gene expression for essentially all biological processes, including development (67, 68), metabolism (63, 66), cellular differentiation (69, 70), cellular proliferation, and programmed cell death (63, 66). Therefore, it is not surprising that miRNAs also play a role in the initiation and control of the inflammatory response and that the deregulation of miRNA expression has been associated with different inflammatory disorders (71). Recent experimental evidence indicates that in addition to targeting the NFjB pathway, miRNAs also directly control inflammation by modulating the expression of inflammasome components and indirectly by modulating the levels of positive or negative inflammasome regulators. Several bioinformatics studies have predicted that NLRPs could be targeted by different miRNAs: miR-125 was predicted to target NLRP1 and NLRP3, miR-181 potentially targets NLRP8, miR-143 might regulate NLRP1, miR-200 might regulate NLRP3 and NLRC4, miR-520 could target NLRP1, NLRP3, and NLRP8, and miR-548 might target NLRP1, NLRP3, and NLRC4 (72). Although miR-125, miR143, and miR-181 have been implicated in the inflammatory response, so far only miR-223 has been proven to be a bona fide inflammasome regulator (73). miR-223 is enriched in the myeloid linage. Consistent with the fact that macrophages express higher levels of miR-223 than dendritic cells, macrophages expressed lower levels of NLRP3 protein than dendritic cells and barely responded to ATP or nigericin exposure. Interestingly, LPS induces NLRP3 protein expression without affecting miR-223 levels, indicating that an LPS-mediated increase in NLRP3 protein level results mainly from de novo gene expression. Nonetheless, negative regulation of miR-223 function by LPS cannot be ruled out (74). In contrast to LPS, the positive effect of GMSCF on NLRP3 protein levels results from negative regulation of miR-223 expression levels. However, the molecular mechanism negatively controlling miR-223 expression remains to be elucidated. It has also been suggested that miR-223 could regulate NLRP3 inflammasome activation by targeting upstream activators such as cathepsin L and cathepsin Z (75), two confirmed target genes of miR-223 (76). © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


Unlike miR-223, miR-133a-1 potentiates inflammasome activation (77). miR-133a-1 targets uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS production and inflammation (78). miR-133a-1 overexpression enhances H2O2-induced caspase-1 activation and IL-1b production in differentiated human THP-1 macrophages, which is correlated with low UCP2 protein levels. Consistent with these results, negative regulation of UCP2 by RNA interference also resulted in enhanced inflammasome activation. Interestingly, a recent study showed increased miR-133a-1 levels in circulation in sepsis patients and in mice with polymicrobial sepsis (79). Thus, miR-133a-1, through inhibiting the expression of proteins negatively controlling NLRP3 activation, promotes and sustains an inflammatory response. Whether miR-133a targets proteins other than UCP2 remains to be elucidated. Ongoing and future studies will uncover new functional relationships between miRNAs and the inflammasome components modulating inflammasome activity, leading to insights about the inflammatory response. We will also gain insight into the mechanism by which the expression of deregulated miRNAs contributes to pathologies resulting from dysfunctional inflammatory processes. With this in mind, we performed a bioinformatics analysis to identify potential miRNAs regulating some of the molecules involved in the inhibition of inflammasome activation described in this review. Only miRNAs with a high probability of interaction with the target mRNA as determined by the ΔΔG value are presented (Table 1). Among these, miR122 and miR-133 are downregulated in the adipose tissue of obese mice fed a high-fat diet (80) and in ob/ob mice, whereas miR-125 and miR-520 are downregulated in the adipose tissue of obese individuals (81). Our bioinformatic analysis indicates that miR-122 has as potential target the mRNA encoding NOD2 and ASC, and that miR-125 might negatively regulate NLRP1. In addition, miR-520 might negatively regulate the NLRP3 inflammasome directly by targeting NLRP3 and indirectly by targeting HSP90. Interestingly, miR133a is predicted to target PP2A and thus negatively regulate NLRP3. This is consistent with the observation that chronic inflammation results in reduced miR-133 levels, which correlates with increased NLRP3 levels and inflammasome activation in the adipose tissue of obese mice (82). These observations suggest that the downregulation of these miRNAs is part of the mechanism leading to chronic inflammation in obese individuals. Regarding Type 2 diabetes (T2D), several miRNAs whose predicted targets are related to inflammasome acti© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

vation have also be found to be downregulated both in mouse models and in humans (83, 84). Thus, in T2D, persistent NLRP3 inflammasome activation could be maintained by the downregulation of miR-20 (predicted to target NLRP3), miR-133 (predicted to target NLRP3 and PP2A), and miR-411 (predicted to target HSP90). In addition, the upregulation of miR-216 in T2D might reinforce PP2A activity and inflammasome activation because this miRNA has as a potential target the protein phosphatase 2 regulatory subunit B (PPP2R5C). Downregulation of miR320 (predicted to target NOD2) and miR-486 (predicted to target NLRP1) could contribute to the altered responses to different insults observed in T2D patients (85). Interestingly, among the miRNAs downregulated in b pancreatic cells in T2D patients (84) is miR-23. Our bioinformatic analysis predicts that miR-23 could negatively regulate GPCR120, which has been shown to mediate Omega-3 fatty acid (x-3 FAs) inhibition of the NLRP3 inflammasome activation in obese mice (86). Thus, it has been suggested that x-3 FAs reduce the NLRP3 inflammasome activation and b pancreatic cell death resulting from high glucose levels (87). Hence, reducing miR-23 levels might potentiate the negative effect of x-3 FA on NLRP3 inflammasome activation, resulting in b pancreatic cell survival. Nonetheless, it remains to be determined whether inflammatory cytokines negatively modulate the expression of the miRNAs that target inflammasome molecules or positive regulators as a mechanism to maintain chronic inflammation. Furthermore, whether the miRNAs described in Table 1 have a role in different inflammatory conditions has yet to be tested. Hormones and second messengers It has long been proposed that the inflammatory response is monitored and tuned by the nervous system to maintain homeostasis (88). Consistent with these observations, immune cells not only express neurotransmitters and neurotrophin receptors on their cell surfaces but might also express and secrete neurotransmitters and neurotrophins (89). It is thought that high levels of inflammatory cytokines are sensed by the afferent neurons of the vagus nerve and that the brain responds to the inflammatory status through the activation of the cholinergic anti-inflammatory pathway via the efferent vagus and the splenic nerves (90). Although it has been shown that memory T cells secrete acetylcholine to inhibit the inflammatory cytokine production (e.g. IL-1b) of macrophages upon norepinephrine release from the vagus nerve (91), so far there is no direct

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Table 1. miRNAs that have conserved binding sites in the 30 UTR of genes related to different inflammatory signaling pathways Conservation of sites (organisms)

ΔΔG (kcal/mol)

Human, chimpanzee, rhesus, and guinea pig Human, chimpanzee, and rhesus Human, chimpanzee, rhesus, bushbaby, and armadillo Human, rhesus, bushbaby, treeshrew, mouse, guinea pig, and rabbit Human, chimpanzee, rhesus, bushbaby, cat, and cow Human, chimpanzee, dog, and cat Human, chimpanzee, and rhesus Human, chimpanzee, rhesus, mouse, rat, hedgehog, horse, elephant, and tenrec Human, chimpanzee, bushbaby, dog, and horse Human and chimpanzee Human, chimpanzee, dog, cat, horse, and elephant Human, chimpanzee, rhesus, bushbaby, treeshrew, mouse, and guinea pig Human, chimpanzee, rhesus, dog, cat, horse, and elephant

7.16 9.08 12.78 12.38

1

Human, chimpanzee, rhesus, dog, cat, horse, and elephant

11.77

7 1 1 2 3 2 2 2

Human and chimpanzee Human and chimpanzee Human, chimpanzee, horse, and cat Human and chimpanzee Human and chimpanzee Human and chimpanzee Human, chimpanzee, and rhesus Human, chimpanzee, rhesus, mouse, rat, treeshrew, dog, cow, and horse Human, chimpanzee, rhesus, mouse, rat, treeshrew, dog, cow, and horse Human, chimpanzee, and rhesus Human, chimpanzee and horse Human, chimpanzee, and rhesus Human, chimpanzee, and rhesus Human, chimpanzee, and rhesus

17.73 10.57 8.05 11 16.71 15.26 10.02 11.74

Human and chimpanzee Human and chimpanzee Human and chimpanzee Human and chimpanzee Human and chimpanzee Human, chimpanzee, and rhesus Human, chimpanzee, and rhesus Human and chimpanzee Human and chimpanzee Human, chimpanzee, and rhesus Human, chimpanzee, and rhesus Human, chimpanzee, rhesus, rabbit, shrew, hedgehog, dog, cow, elephant, tenrec, opossum, and platypus Human, chimpanzee, rhesus, mouse, rat, guinea pig, rabbit, shrew, hedgehog, dog, horse, cow, armadillo, elephant, tenrec, and opossum Human, chimpanzee, rhesus, guinea pig, rabbit, shrew, hedgehog, and tenrec Human, chimpanzee, rhesus, guinea pig, rabbit, cat, horse, cow, and armadillo Human, chimpanzee, and rhesus Human, chimpanzee, and rhesus Human, chimpanzee, rhesus, rabbit, horse, cow, armadillo, elephant, and tenrec Human, chimpanzee, rhesus, mouse, rat, guinea pig, shrew, hedgehog, dog, cat, horse, cow, armadillo, tenrec, opossum, chicken, and frog

10.28 10.37 18.72 11.31 15.29 14.56 15.84 10.28 10.81 13.54 8.42 10.97

Protein (gene)

miRNA(s)

NOD1

miR-27abc miR-31 miR-485-5p miR-122

5 5 5 4

miR-215 miR-320 miR-7 miR-24-3p

2 5 6 4

miR-125ab-5p miR-486-5p miR-17 miR-22

3 2 1 2

miR-302abcde /372/373/520be /520acd-3p miR-93/17/17-5p /20ab/20b-5p /93/106ab/427 /518a-3p/519d miR-4488 miR-4697-5p miR-548v miR-181abcd miR-614 miR-663 miR-185 miR-124-5p

1

miR-761

8

miR-122 miR-383 miR-214 miR-412 miR-18b

1 1 1 1 2

NOD2

STING (TMEM173) NLRP1 NLRP3

NLRP4 NLRP8 AIM2 RIG (LY6E)

ASC (PYCARD) CASPASE-1 (CASP1) CASPASE-11 (CASP4) CASPASE-8 (CASP8)

PKR (EIF2AK2)

SYK JNK1 (MAPK8)

IKKa (CHUK)

PKCd (PRKCD)

242

Number of sites

miR-508-5p miR-661 miR-939 miR-1231 miR-1273 miR-485-5p miR-510 miR-616 miR-766 miR-635 miR-1262 miR-1244

1 1 2 1 1 29 14 7 12 8 8 9

miR-219a-2-3p

11

miR-1290

13

miR-1322

2

miR-152 miR-376c miR-942

2 4 4

miR-26a

2

10.44 14.05 8.48 11.79 8.86 13.87 10.64 8.54 11.63

10.54 10.15 16.95 10.88 8.48 7.14

12.86 10.76 13.21 9.47 8.43 9.08 14.02



Table 1. (continued)

Protein (gene)

DAPK1

PP2A (PPP2CA)

GPSM3

SGT1 (ECD) HSP70 (HSPA4)

HSP90 (HSP90AA1)

b-ARRESTIN 2 (ARRB2)

GPCR129 (FFAR4/O3FAR1) GPCR140 (MRGPRF)

miRNA(s)

Number of sites

miR-377

2

miR-637 miR-876-3p

1 2

miR-939 miR-143

1 4

miR-26a

4

miR-133a-3p

6

miR-183-5p

8

miR-1224-5p miR-1266 miR-1324 miR-377 miR-492 miR-493 miR-512-5p miR-661 miR-4311

2 3 1 2 1 3 3 1 6

miR-1233 miR-1287 miR-323-3p

3 5 2

miR-549

2

miR-633

6

miR-1271 miR-134 miR-185 miR-361-5p

2 3 4 2

miR-411 miR-515-3p /519/518d-5p /520c-5p/526 miR-1294 miR-155

2 1

miR-199ab-5p miR-331-5p miR-138 miR-23ab miR-504 miR-874 miR-138 miR-485-5p

1 1 6 8 5 7 5 10

1 1

Conservation of sites (organisms) Human, chimpanzee, rhesus, hedgehog, dog, cat, horse, cow, tenrec, and opossum Human, chimpanzee, rhesus, guinea pig, and cow Human, chimpanzee, rhesus, guinea pig, dog, cat, cow, and tenrec Human, chimpanzee, rhesus, and cow Human, chimpanzee, rhesus, guinea pig, hedgehog, dog, cat, tenrec, and opossum Human, chimpanzee, rhesus, mouse, rat, guinea pig, dog, horse, cow, tenrec, opossum, lizard, and chicken Human, rhesus, mouse, rat, guinea pig, shrew, rabbit, hedgehog, horse, cow, armadillo, elephant, tenrec, opossum, lizard, platypus, chicken, and frog Human, rhesus, mouse, rat, guinea pig, rabbit, shrew, hedgehog, dog, horse, cow, armadillo, elephant, tenrec, opossum, lizard, platypus, chicken, and frog Human, chimpanzee, tenrec, horse, and hedgehog Human, chimpanzee, and rhesus Human, chimpanzee, mouse, rat, horse, and cow Human, rhesus, and tenrec Human, chimpanzee, and rhesus Human, chimpanzee, and rhesus Human, chimpanzee, rhesus, and mouse Human, chimpanzee, and rhesus Human, chimpanzee, rhesus, bushbaby, rabbit, dog, cat, horse, cow, armadillo, and elephant Human, chimpanzee, and rhesus Human and chimpanzee Human, chimpanzee, rhesus, guinea pig, shrew, rabbit, shrew, hedgehog, dog, cat, horse, cow, armadillo, and tenrec Human, chimpanzee, rhesus, mouse, rat, guinea pig, shrew, rabbit, shrew, hedgehog, dog, cat, horse, cow, armadillo, elephant, opossum, platypus, and chicken Human, chimpanzee, rhesus, mouse, rat, shrew, hedgehog, dog, cat, horse, cow, armadillo, and elephant Human, chimpanzee, and rhesus Human, chimpanzee, rhesus, dog, horse, and cow Human, rhesus, treeshrew and rabbit Human, rhesus, mouse, rat, guinea pig, treeshrew, rabbit, dog, horse, elephant, cow, and platypus Human, chimpanzee, rhesus, and rabbit Human, chimpanzee, rhesus, and rabbit Human, chimpanzee, rhesus, mouse, horse, and cow Human, chimpanzee, rhesus, mouse, rat, guinea pig, shrew, horse, cow, opossum, and elephant Human, mouse, rat, and shrew Human, chimpanzee, rhesus, and elephant Human, chimpanzee, and rhesus Human, chimpanzee, and rhesus Human and chimpanzee Human, chimpanzee, and rhesus Human and chimpanzee Human, chimpanzee, bushbaby, mouse, and rat

ΔΔG (kcal/mol) 7.97 20.09 8.19 20.92 10.48 10.15 9.70 7.91 10.96 10.91 9.87 8.52 8.71 8.94 8.31 17.4 14.83 8.01 10.8 16.2 13.3 8.75 12.9 23.62 17.51 9.41 11.34 15 13.9 9.09 8.68 7.79 11.42 8.87 17.71 18.59 9.64 11.17

The ΔΔG value results from making the difference between the free Gibbs energies of the duplex formation (ΔGDuplex) and of the mRNA opening to liberate the site for the miRNA’s binding (ΔGOpen). A ΔΔG less than 10 kcal/mol has been reported to be a very accurated parameter to predict miRNA:mRNAs interactions. However, a ΔΔG of 7 kcal/mol is still accurated for several cases. If more than one site exists for a given miRNA on a given UTR, then the ΔΔG score appropriately sums up the DDG energies of all those sites.


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evidence that acetylcholine negatively regulates inflammasome activity and therefore IL-1b production. Nonetheless, the first direct evidence that neurotransmitters produced in response to stress negatively regulate inflammasome activation comes from experiments where mice subjected to water-avoidance stress showed a dramatic decrease in IL-1b production that correlated with reduced NLRP6 protein levels in intestinal tissue (92). The reduction in NLRP6 expression and IL-1b production in the intestine after stress was mediated by corticotropin-releasing hormone (CRH). Although CRH expression increased in the gut of stressed animals, it is not yet clear whether the regulation of NLPR6 protein levels is a direct effect of CRH or is mediated by corticosteroids because NLRP6 protein levels can be negatively modulated by CRH or corticosteroids in epithelial cells in vitro (92). Recently, it has been shown that cAMP binds to the NLRP3 nucleotide-binding domain, inhibiting NLRP3 inflammasome activity and IL-1b production (93). Given that the CRH receptor, CRHR1, activates adenylate cyclase and thus elevates cAMP levels (94), CRH might directly affect NLRP6 inflammasome activity. Thus, any hormone or neurotransmitter whose receptor promotes cAMP production may potentially inhibit inflammasome activation. Interestingly, the stress-induced negative regulation of the NLRP6 inflammasome mediated by CRH in the gut epithelia leads to dysbiosis, which is in agreement with previously published data showing that mice deficient in NLRP6 are susceptible to colitis (95). Thus, finely tuned functional NLRP6 inflammasome activity in gut epithelial cells is critical for shaping the gut microbial profile and maintaining homeostasis. The neurotransmitter aspartate also has a potent antiinflammatory effect. Farooq et al. (96) have recently shown that aspartate can prevent LPS-induced expression of IL-1b, NLRP3, and caspase-1, and ATP-mediated secretion of IL-1 b. In agreement, in vivo aspartate administration clearly reduced both hepatitis and pancreatitis in mice, resulting in increased rates of survival (96). The negative effect of aspartate on the expression of IL-1b and NLRP3 is mediated through the N-methyl-d-aspartate receptor via b-arrestin-2. In vivo b-arrestin-2 knockdown prevents the anti-inflammatory effect of aspartate and reduced in vitro IKKb/a phosphorylation (96), indicating that b-arrestin-2 might also inhibit IKKa-mediated NLRP3 perinuclear localization, thus preventing inflammasome assembly and IL-1b maturation and secretion. This is in agreement with the observation that aspartate levels increase in circulation during LPS-induced

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inflammation (97). These results suggest a physiological role for aspartate during the inflammatory response. Steroid hormones have also been suggested to control IL1b production (98). However, no direct experimental evidence exists regarding the negative modulation of inflammasome activity by these hormones. Clearly, future studies are needed to fully understand the negative regulation of inflammation imposed by the central nervous system to maintain homeostasis and avoid deleterious inflammatory responses. The molecular mechanisms negatively regulating inflammasome activity as well as IL-1b and IL-18 production in response to neurotransmitters and hormones require special attention. Viral mechanisms to negatively regulate inflammasome activity Viral infection is controlled by the early onset of cytokine production. Among cytokines, IL-1b plays a cardinal role; therefore, it is not surprising that viruses have developed different strategies to prevent inflammasome activation and IL-1b production, thus delaying or modifying the inflammatory response. The first molecule encoded by a virus identified to have caspase-1 inhibitory activity was CrmA (cytokine response modifier) in Cowpox (99). CrmA acts as a pseudosubstrate for caspase-1 as well as caspase-8 (100). Because caspase-8 is also able to promote IL-1b maturation, CrmA ensures inflammasome inactivation. In contrast, CrmA has no direct effect on caspase-3, caspase-6, and caspase-7 activity (101), and thus, CrmA has a greater anti-inflammatory activity than an anti-apoptotic one. The expression of serpine-like protease inhibitors with anti-caspase-1 activity has also been found in vaccinia (102), myxoma (103), and ectromelia viruses (104) (Fig. 5). Later, it was shown that the product of the ML013 gene of the myxoma virus is a protein containing a PYD domain that binds to ASC through the PYD–PYD domain interactions, thereby preventing inflammasome activation and IL-1b and IL-18 maturation thus favoring infection (105). Interestingly, other poxyviruses also encode PYD-containing proteins (106). The shope fibroma virus encodes the Pyrin only protein (vPOP), which is the viral form of cellular POP (cPOP). cPOP and vPOP colocalize and physically interact with ASC, preventing caspase-1 activation and IL-1b production in infected 293-T cells (107). A different strategy is used by the vaccinia virus to prevent an immune response to ensure infection. Vaccinia encodes proteins that have homology with anti-apoptotic © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


genes such as Bcl-2 (among them, A46R, A52R, B14R, C1L, C6L, C16/B22R, F1L, K7R, N1L, and N2L) (108). Of these proteins, A52R, B14R, N1L, and K7R have been shown to interfere with the NFjB signaling pathway, thus attenuating cytokine production and immune response (109). Previous results show that Bcl-2 and Bcl-XL proteins regulate proinflammatory caspase-1 activation by interaction with NLRP1 (16). It was recently shown that vaccinia F1L, in addition to its anti-apoptotic activity, interacts with NLRP1, thereby preventing caspase-1 activation (110). F1L interacts through a short amino acid sequence (32–37) on its amino terminal domain with the LRR domain of the NLRP1 molecule, thus preventing inflammasome activation. In agreement, virions lacking F1L fail to inhibit caspase-1 activation and IL-1b production both in vitro and in vivo, resulting in the inability to evade immune response. Interestingly, F1L does not interact with other NLRPs (e.g. NLRP3, NOD2), neither with AIM2 or ASC, indicative of the specific action of F1L on NLRP1. In contrast, the Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8) gene product Orf63 is able to prevent NLRP1 and NLRP3 inflammasome activation. The Orf63 gene encodes for an NLRP1-homologous protein that lacks the CARD and PYD domains and thus by itself is unable to promote caspase-1 activation. Orf63 interaction with NLRP1 involves the NBD and LRR domains of both proteins. Accordingly, Orf63 interaction with NLRP1 prevents: (i) NLRP1 oligomerization, (ii) interaction with ASC and caspase-1, and thus (iii) caspase-1 activation. Consistent with these observations, inhibition of Orf63 expression during viral infection of macrophages enhances caspase-1 activation and IL-1b production. NLRP1 inactivation by Orf63 is also required for viral reactivation. Regarding NLRP3 activity, Orf63 is able to prevent ATP and alum-induced caspase-1 activation, IL-1b production, and pyroptosis (111). The fact that different herpes viruses encode for NLRP1 orthologs suggests that blocking inflammasome formation is important for herpes virus infection and propagation. Furthermore, herpes simplex virus-1 (HSV-1) prevents caspase1 activation and production of IL-1b through different mechanisms: (i) the expression of the type 3 ubiquitin ligase ICP0 promotes the ubiquitination and degradation of the nuclear DNA sensor IFI16H a few hours after infection, thus reducing IFI16H inflammasome activity (112, 113); (ii) it prevents NLRP3 inflammasome activation by segregating NLRP3 and caspase-1 in separate clusters by an as yet undefined mechanism (113, 114); and (iii) it prevents secretion of mature IL-1b by promoting the dissociation of Rab27a from secretory vesicles containing IL-1b and its © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

localization to the trans Golgi network (113). Thus, HSV-1 has developed different mechanisms to counteract the cellular response to infection initiated by the IFI16H and NLRP3 inflammasomes. Nonetheless, further work is required to fully define the mechanism activated by HSV-1 to attenuate the inflammatory response and ensure viral propagation. However, targeting NLRPs to hinder inflammasome formation and IL-1b production is a mechanism not exclusively used by herpes viruses; the measles virus encodes the V protein that blocks the cellular interferon response by interacting with the melanoma differentiation-associated gene 5 (MDA5). Komune et al. (115) showed that in addition to its anti-interferon activities, the V protein interacts with NLRP3 and prevents ATP-induced caspase-1 activation and IL-1b production in human THP-1 differentiated macrophages. Accordingly, infection with a virus lacking the V protein results in enhanced IL-1b production (115). Although the human papillomavirus (HPV) does not directly target the NLRP3 inflammasome, it promotes IL-1b degradation in HPV-positive cells through the ubiquitin ligase E6-AP (E6 associated protein) (116). Because this study was performed in an HPV-positive tumor cell line and in stably expressing E6 and/or E7 cells, it is still an open question whether HPV possesses other strategies to block IL1b production during the infection process in vivo (e.g. inhibition of the activation of the IFI16H inflammasome). Further experiments are required to test this possibility. The Epstein–Barr virus mediates NLRP3 inflammasome inactivation through a novel mechanism involving a viral microRNA called miRBART15, which is exported via exosomes from infected B cells to non-infected cells to negatively modulate NLRP3 levels and thus preventing inflammasome activation in non-infected cells (117). Interestingly, miRBART15 targets the same sequence in the Nlrp3 30 UTR that miR-233 targets (117). Thus, preventing inflammasome complex formation and caspase-1 activity to hinder IL-1b and IL-18 production appears to be a conserved strategy to evade immune response that has shaped viral–host coevolution. Negative regulation of the inflammasome by bacterial effector molecules Chlamydia trachomatis Chlamydia trachomatis, through a type III secretion system, controls different aspects of the epithelial cell infectious process, including the establishment of a specialized vacuole where Chlamydia replicates and survives. Recent experimental evidence suggests that the serine protease CPAF might

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control caspase-1 activation to prevent precocious Chlamydia release. The negative effect of CPAF on caspase-1 activation most likely results from CPAF’s requirement to maintain vacuole integrity, thus preventing bacterial components from reaching the cytosol and inducing inflammasome assembly. Nonetheless, further experiments are required to rule out a direct effect of CPAF on caspase-1 activity, especially because the protein levels of inflammasome components were not shown in a condition where CPAF activity was inhibited (118) (Fig. 5). Francisella tularensis Francisella tularensis, a Gram-negative bacteria, infects macrophages. Early during macrophage infection, F. tularensis exits the phagosome and replicates in the cytosol. Accordingly, inflammasome activation is critical to initiate an effective immune response against F. tularensis. Mice deficient in IAM2, caspase-1, and ASC cannot control bacterial burden, resulting in enhanced mortality in response to F. tularensis infection. To overcome the innate immune response, F. tularensis has developed different strategies to shut down proinflammatory cytokine production. F. tularensis prevents TLR2-dependent MAPK and NFjB activation (119) while inhibiting cytokine production resulting from detection avoidance mediated by NLRs and inflammasome inactivation. Recently, it was shown that the mviN gen product prevents AIM2 inflammasome activation (F. tularensis lacking mviN showed enhanced AIM2 inflammasome activity and IL-1b and IL-18 production) without affecting NLRP3 or NLRC4 inflammasome activation. Accordingly, animals infected with an mviN F. tularensis mutant strain showed an attenuated phenotype with a lower bacterial burden and enhanced host survival. As expected, this phenotype was dependent on AIM2 inflammasome activation. Because mviN participates in peptidoglycan synthesis and cell wall function, the effect of mviN on inflammasome activation is probably due to preventing premature exposure of DNA to NLRs in the cytosol rather than a direct negative effect on AIM2 inflammasome assembly or caspase-1 activity (120). Nonetheless, further experiments are required to answer this question. Not surprisingly, mviN is not the only F. tularensis gene that encodes a protein that suppresses pro-inflammatory cytokine production. Macrophages infected with an F. tularensis mutant lacking the ripA gene expressed higher levels of IL-1b and IL-18 than macrophages infected with wildtype F. tularensis (121). Similar to mviN deficiency, increased IL-1b and IL-18 production resulting from ripA

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deletion was caspase-1- and ASC expression-dependent but NLRP3- and NLRC4-independent. Accordingly, mice infected with F. tularensis lacking ripA were resistant to death and showed a lower bacterial burden than mice infected with wildtype F. tularensis (121). However, whether ripA also targets the AIM2 inflammasome as mviN does remains to be determined. In addition to negatively regulating caspase-1mediated IL-1b and IL-18 production, ripA also prevented IL-1b and TNF-a expression. This effect was NFjB-independent but ERK- and JNK-dependent (121). Whether mviN also blocks these pathways to obliterate pro-inflammatory cytokine production remains to be determined. Because ripA, like mviN, is localized in the cytoplasmic membrane, the effect of ripA on inflammasome activation might not be direct. Although experimental evidence provided by Ulland et al. (120) and Huang et al. (121) suggests that mviN and ripA are part of the same molecular mechanism controlling the production of inflammatory cytokines, further experimental evidence is required to discard the possibility that these molecules play a role in distinct but redundant mechanisms. Nonetheless, F. tularensis prevents IL-1b and IL-18 production as well as pyroptosis through mviV and ripA, thus ensuring propagation and virulence in vivo. Legionella pneumophila Like F. tularensis, Legionella pneumophila is a Gram-negative bacterium that infects, replicates, and survives in macrophages. However, unlike F. tularensis, L. pneumophila resides in vacuolelike organelles called Legionella-containing vacuoles (LCVs), thus minimizing NLR recognition. Deletion of the sdhA (the translocated Dot/Icm-type IVB secretion system effector) gene enhanced L. pneumophila-induced caspase-1 activity and pyroptotic macrophage death (122). In both human and mouse macrophages, caspase-1 activity induced by sdhAdeficient L. pneumophila was independent of flagellin. Accordingly, NLRP3 and NLRC4 inflammasomes were dispensable for caspase-1 activation induced by sdhA-deficient L. pneumophila infection. ASC- and AIM2-deficient macrophages were unable to activate caspase-1 and to produce IL-1b in response to sdhA-deficient L. pneumophila exposure, indicating that SdhA prevents AIM2 inflammasome activation. ShdA does not prevent AIM2–ASC interaction nor is it able to prevent caspase-1 activity in vitro (123). This is consistent with the role of sdhA in membrane trafficking and LCV maturation and stability (123). Indeed, sdhA deficiency enhances L. pneumophila DNA exposure to the cytosolic AIM2 inflammasome. AIM2 activation induced by L. pneumophila © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


DNA is independent of type I interferon and TNF-a (122). Thus, shdA prevents activation of the inflammasome and the innate immune response by actively maintaining LCV integrity, thus avoiding L. pneumophila PAMP leakage to the cytosol. Interestingly, macrophages from C57BL/6 mice show reduced caspase-1 activation in response to sdhA-deficient L. pneumophila exposure compared to macrophages from 129S mice (122), resembling differential caspase-1 activation and pyroptosis in response to Bacillus anthracis infection. During pyroptosis, bacteria are released and vulnerable to being killed by neutrophils; C57BL/6 macrophages, which carry the Nlrp1b2 allele, are refractory to Bacillus anthracis-induced pryroptosis and thus C57BL/6 mice do not control bacterial loads, leading to their death. In contrast, macrophages from mice (129S) carrying the Nlrp1b1 allele activate caspase-1 in response to Bacillus anthracis infection and die by pyroptosis releasing bacteria that are destroyed by neutrophils, thus infected animals resolve the infection and survive. Thus, the Nlrp1b1 allele controls caspase-1 activation and survival (124). Whether the NLRP1 inflammasome plays a role in the control of L. pneumophila infection remains to be determined. The L. pneumophila effector protein SidF prevents apoptosis through its physical interaction with the pro-apoptotic factor BNIP3 (125), which promotes apoptosis through interaction with Bcl-2 (16). Interestingly, Bcl-2 negatively regulates NLRP1 (16); hence, the SidF–BNIP3 interaction might result in an increase in available Bcl-2 molecules for interaction with NLRP1 and inhibition of inflammasome activation. Thus, L. pneumophila might prevent both apoptotic and pyroptotic macrophage cell death through a SidF-dependent mechanism. Mycobacterium tuberculosis Although it has been shown that IL-1b and IL-18 production is the key to controlling in vivo M. tuberculosis infection (126), the activities of NLRP3, ASC, and caspase-1 are dispensable for in vivo IL-1b and IL-18 production (127, 128). Nonetheless, in vitro IL-1b and IL-18 production by M. tuberculosis is NLRP3-, ASC- and caspase-1-dependent. In contrast, the AIM2 inflammasome is involved in M. tuberculosis-induced IL-1b and IL-18 production both in vitro and in vivo (129). Interestingly, activation of the NLRP3 or AIM2 inflammasomes requires the M. tuberculosis ESX-1 secretion system. Accordingly, ESAT-6 and AG85 effectors and bacterial DNA inflammasome activation depend on the presence of a functional EXS-1 system (130). The fact that M. tuberculosis induced lower IL-1b production than non-virulent mycobac© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

terium led to the hypothesis that the virulent mycobacteria have developed the capacity to negatively regulate inflammasome activation to ensure infection and bacterial replication. Accordingly, Shah et al. (131) recently showed that virulent M. tuberculosis strains prevent AIM2 inflammasome activation. Interestingly, this negative effect on inflammasome activation requires a functional secretion system. Thus, effector molecules released together with DNA to the cytosol of infected macrophages prevents AIM2-dependent caspase-1 activation, and IL-1b and IL-18 production without affecting AIM2 protein levels (131). This mechanism involves in part the suppression of interferon-b signaling; however, inhibition of interferon-b signaling by M. tuberculosis is independent of the EXS-1 secretion system. Thus, the molecular mechanisms by which M. tuberculosis inhibits AIM2 inflammasome activation remain to be fully elucidated, as do the identities of the effector molecules involved. Pseudomonas aeruginosa IL-1b production by macrophages in response to infection with the opportunistic Gram-negative bacteria Pseudomonas aeruginosa depends on the P. aeruginosa type III secretion system and the NLRC4 inflammasome (132). P. aerugionosa flagellin is sensed by the NLRC4 inflammasome, and ASC is also required for flagellin-induced caspase-1 activation and IL-1b maturation. Flagellin is not the only effector molecule that reaches the cytosol of infected macrophages; ExoS, ExoT, ExoU, and ExoY are well-characterized P. aeruginosa effector molecules as well. Unlike flagellin, neither ExoS, ExoT, ExoU, nor ExoY promote inflammasome activation (132). On the contrary, it has been shown that ExoS and ExoU prevent P. aeruginosa-dependent inflammasome activation (133). ExoS is a dual enzyme, possessing an ATPase-activating N-terminal domain and an adenine dinucleotide phosphate (ADP)-ribosyltransferase (ADPRT) C-terminal domain (134), but only its ADP ribosyltransferase activity is required to inhibit inflammasome activation and IL-1b production. However, it is not clear whether ExoS prevents inflammasome activation by direct ADP-ribosylation of any of the inflammasome components or indirectly by ADP ribosylating an as yet unknown inflammasome activator. On the other hand, ExoU phospholipase A2 activity is required for negative inflammasome regulation (133). This might be an indirect effect because ExoU activity may generate an unknown product that directly prevents inflammasome assembly or caspase-1 activity. Furthermore, although ExoU has no effect on pro-IL-1b or

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caspase-1 protein levels, negative regulation of ASC or NLRC4 gene expression through signaling triggered by products of cytosolic phospholipase A2 activity, such as arachidonic acid, prostaglandins or lysophospholipid, cannot be ruled out. The fact that Syk kinase activity promotes NLRP3 inflammasome activation (135) and that Syk activation has also been implicated in P. aeruginosa infection (136) opens another interesting possibility for exploration. ExoU activity could inhibit NLRC4 inflammasome activation indirectly through prostaglandin production. In this scenario, prostaglandins produced by P. aeruginosa-infected macrophages could inhibit Syk kinase activity through PKA-mediated phosphorylation upon binding to their cognate receptor (137), thus preventing Syk-mediated NLRC4 phosphorylation and inflammasome activation. Nonetheless, the fact that the expression of ExoS or ExoU by P. aeruginosa from hospital isolates correlates with severe infection emphasizes the important role of IL-1b production and inflammasome activation in controlling P. aeruginosa infection and that virulent strains have acquired different effector molecules to prevent it. Salmonella NLRC4 activation and IL-1b and IL-18 production in response to Salmonella enterica are critical for controlling infection (138). In addition to the production of inflammatory cytokines, NLRC4 activation also controls Salmonella infection by promoting pyroptosis of infected macrophages (4, 139). As mentioned above, during pyroptosis, bacteria are released and vulnerable to being killed by neutrophils. Despite this, Salmonella infects and survives in B cells. Thus, it has been proposed that B cells are a natural Salmonella reservoir (140). Recently, it was shown that Salmonella prevents NLRC4 inflammasome activation and IL-1b production in B cells, thus promoting B-cell survival. This process requires a functional type III secretion system. Interestingly, Salmonella infection also prevents doxorubicin-mediated B-cell death (141). NLRC4 inhibition by Salmonella involves the negative regulation of NLRC4 gene expression through promotion of the phosphorylation of the YAP1 transcription factor. Phosphorylated YAP associates with Hck resulting in its retention in the cytosol of infected B cells (141). However, the Salmonella effector molecule(s) involved in YAP phosphorylation and inactivation are not known. It will be interesting to learn whether YAP inactivation is a general mechanism for the negative regulation of NLRP4 expression as well as the expression of other inflammasome components.

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Shigella flexneri The NLRC4 inflammasome also triggers caspase-1 activation and IL-1b and IL-18 production in response to Shigella flexneri infection (142). So far, it has been assumed that caspase-1 activation by NLRC4 after sensing components of the S. flexneri type III secretion system is key to controlling S. flexneri infection, by promoting macrophage pyroptosis. However, NLRC4 and caspase-1 might not be the only inflammasome components activated in response to S. flexneri. Recently it has been shown that S. flexneri prevents caspase4 activation and pyroptosis in epithelial cells (143). The authors of this study reported that the S. flexneri OspC3 effector molecule binds to cleaved caspase-4 p19 fragment and prevents its interaction with the caspase-4 p10 fragment, thus abolishing the formation of active heterodimers. Interestingly, the specific sequence of OspC3 that interacts with caspase-4 is conserved in different bacterial and viral proteins with ankyrin repeats (143). Whether these protein factors also prevent caspase-4 activation remains to be determined. Vibrio The production of IL-1b is thought to be involved in inflammatory responses and disease development during Vibrio vulnificus and Vibrio cholera infection (144). The NLRP3 inflammasome was originally implicated in caspase-1 activation and IL-1b production in response to Vibrio cytotoxins (145). However, recently it was found that in addition to NLRP3 inflammasome, the NLRC4 inflammasome is activated by the V. parahaemolyticus type III secretion system 1. Nonetheless, by promoting autophagy and negatively modulating the small GTPase CDC42, the VopQ and VopS effector molecules negatively regulate NLRC4 inflammasome activation. VopQ-induced autophagy may promote the degradation of inflammasome components, or the degradation of PAMPs that may be sensed by the NLRC4 inflammasome, thus reducing IL-1b production (146). Although VopS negatively regulates speck assembly and caspase-1 activation by altering cytoskeletal dynamics and most likely inflammasome protein localization, the exact mechanism remains to be elucidated. Nonetheless, one can imagine that the initial NLRC4 inflammasome activation, triggered by type III secretion system assembly on the cell surface, is prevented by the translocation of VopQ and VopS to the cytosol of the infected cell and that these molecules will prevent or reduce Vibrio hemolysin-dependent NLRP3 inflammasome activity, hence favoring bacterial infection. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


Yersinia pestis Triggering an innate immune response by Yersinia pestis, the causal agent of bubonic plague, as well as by Y. pseudotuberculois and Y. enterocolitica, depends on NLRP3 and NLRC4 inflammasome activation and IL-1b production in response to membrane damage by the assembly of the type III secretion system (147). However, through multiple effector molecules, Yersinia subjugates the cellular machinery to attenuate the innate immune response. The Yersinia YopP effector molecule prevents NFjB- and MAP kinase-dependent cytokine gene expression (148–151). In addition, the Yersinia YopE, YopK, and YopM effectors, through distinct mechanisms, prevent IL-1b maturation and secretion, thus favoring Yersinia pathogenesis. YopE, by a mechanism involving the inactivation of the Rac-1 small GTPase by means of its GAP activity, prevents caspase-1 activation (152). Apparently, inhibition of Rac-1 activity by YopE resulted in reduced LIM1 kinase 1 (LIMK-1) activity as well as caspase-1 and ASC oligomerization. Although this study highlights the roles of Rac-1 and LIMK-1 in inflammasome activation, the molecular mechanisms by which these molecules promote caspase-1 activity are still unknown. In contrast, it has been suggested that YopK prevents inflammasome activation by regulating the translocation of Yersinia PAMPs to the cytosol of infected cells by direct interaction with the traslocon (147) and/or by preventing traslocon recognition, thus avoiding PAMPs exposure and recognition by NLRPs. YopM, however, inhibits caspase-1 catalytic activity via a direct interaction-mediated mechanism. YopM contains an amino acid sequence that resembles a caspase-1 pseudosubstrate that interacts with the catalytic sites of both inactive and active caspase-1, thus preventing its proteolytic activity against endogenous substrates. Caspase-1 inhibition was independent of the inflammasome that initially triggered caspase-1 activation. YopM prevented NLRP1-, NLRP3-, and NLRC4-dependent caspase-1 activation induced by anthrax lethal toxin, nigericin, and Salmonella, respectively (153). These data clearly show that Yersinia obliterates caspase-1 activation by delivering different effector molecules to the infected cell via the type III secretion system, thus avoiding pyroptosis and IL-1b production and ensuring infection. Negative regulation of inflammasome activity by organic and inorganic compounds Ancient traditional medicine is based on the use of different medicinal plants, many of which have anti-inflammatory properties and are therefore used to treat different maladies © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

including gout, diabetes, skin burn, arthritis, and bites from poisonous animals. The active compounds in the plants are delivered directly, by contact as ointments, in solution by drinking potions or infusions, or by inhalation. Now, with advanced technology, many of the active compounds with anti-inflammatory properties have been identified and the molecular mechanisms by which those compounds regulate inflammation have begun to be elucidated. Here, we review the mechanisms by which these compounds regulate inflammasome activation (Fig. 6). Green tea Epigallocatechin-3-gallate (EGCG) is the major bioactive polyphenol in green tea (154). EGCG has a potent antiinflammatory effect that may be explained by its wellknown antioxidant activity and free radical-scavenging capacity as well as its ability to blunt NFjB-mediated inflammatory responses (155). Accordingly, EGCG has been shown to inhibit various inflammatory enzymes and cytokines, including iNOS, COX2, MMPs, IL-6, IL-8, IL-12, and TNF-a (156–158). Although the expression of these cytokines is induced by secreted active IL-1b (159), little evidence exists that EGCG could modulate inflammation by regulating inflammasome activation. Recently, it was shown in TPA-differentiated human THP-1 macrophages that EGCG treatment prevented ATP-induced NLRP3 inflammasome activation and IL-1b production. Interestingly, it was shown that EGCG reduced NLRP3 and caspase-1 protein levels. In another study, EGCG diminished IL-1b production in response to calcium pyrophosphate dihydrate crystals in differentiated THP-1 macrophages and in fibroblast-like synoviocytes (154). In agreement with in vitro data, in vivo administration of EGCG in a lupus mouse model prevented kidney inflammation and pathology. EGCG reduced macrophage and lymphocyte infiltration as well as IL-1b and IL-18 serum levels. This correlated with the fact that EGCG-treated animals presented reduced kidney NLRP3 protein levels compared with vehicle-treated animals. Accordingly, active caspase-1 and mature IL-1b protein levels were also reduced in EGCG-treated animals (160). The EGCG effect on inflammasome component expression levels observed in the kidneys of treated animals was mediated by EGCG antioxidant activity, which reduced ROS levels and indirectly reduced NFjB and NLRP3 activation. The negative effect of EGCG on NLRP3 inflammasome activation is apparently not specific. In a melanoma mouse

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model, EGCG administration at physiological concentrations (0.1, 1, and 10 lM) prevented IL-1b secretion but not IL1b expression from melanoma tumor cells. Interestingly, EGCG reduced NLRP1 protein levels, whereas NLRP3, ASC, and caspase-1 were not affected. Consistent with the effect of EGCG on NLRP1 protein levels, reducing NLRP1 levels by RNAi also resulted in reduced IL-1b production and tumor development (161). Together, these data show that the antiinflammatory effect of EGCG may be mediated in part by preventing inflammasome activation and therefore IL-1b and IL-18 production. Although EGCG could mediate the negative effect on inflammasome activation by preventing NFjBmediated expression of the genes encoding inflammasome components, the fact that a 30-minutes pretreatment with EGCG before ATP exposure reduces protein levels suggests a post-transcriptional regulation mechanism leading to the degradation of inflammasome components. Aloe vera Aloe vera gel has long been used for its anti-inflammatory properties. Recent studies have shown that the negative effect of aloe on inflammation might be mediated by different anthraquinons (162). These compounds prevent cytokine production, inhibit neutrophil infiltration, and promote macrophage phagocytic capacity. At the molecular level, it was shown that the negative effect of anthraquinons on IL1b and IL-18-production could be explained by the fact that this compound prevented LPS-induced NFjB and MAP kinase activation, and therefore expression of the NLRP3, caspase-1, IL-1b, and P2X7 ATP receptor (163). However, a direct effect of anthraquinons on inflammasome assembly or caspase-1 activity remains to be disproven. Bamboo vinegar Another plant product shown to have anti-inflammatory properties is bamboo vinegar, which prevents (i) monocyte MCP-1 production induced by a high-fat diet (164), (ii) LPS-induced TNF-a and IL-6 expression (165), and (iii) cysplastin-induced ROS production. Recently, it was shown that creosol, a polyphenolic compound found in bamboo vinegar, is the bioactive molecule possessing anti-inflammatory properties (166). In vitro creosol prevented iNOS and IL-6 expression but not TNF-a production in LPS-induced macrophages. Creosol also prevented ATP-mediated IL-1b production in LPS-primed macrophages. Unlike other compounds, creosol achieves negative regulation of IL-1b production without affecting LPS-induced NFjB or MAPK

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activation. However, creosol prevented LPS-induced NLRP3 but not caspase-1 expression. In contrast, creosol prevented LPS-induced PKC activation and ATP-induced ROS production. Given that it has been shown that ROS leads to NLRP3 inflammasome activation (166), the negative effect of creosol on IL-1b production in response to ATP in LPS-primed macrophages may result from both the prevention of NLRP3 activation and through a mechanism that involves PKC inhibition and therefore ROS production. The fact that creosol does not prevent LPS-mediated NFjB activation indicates that different transcription factors can also regulate NLRP3 expression in response to LPS and that the activation of those transcription factors is independent of the MAP kinase pathway. Polyenylpyrroles It has also been found that polyenylpyrrole derivatives prevent ATP-mediated NLRP3 inflammasome activation in LPSprimed murine macrophages. In addition to ROS and PKC inhibition, the negative effect of polyenylpyrrole on NLRP3 inflammasome activation also involved NFjB and MAP kinase inhibition (167). Given that polyenylpyrrole derivatives have no effect on NLRP3 or caspase-1 expression levels, their effect on NLRP3-dependent IL-1b production is probably mediated by blocking ROS production. Nonetheless, the mechanism by which polyenylpyrrole prevents NLRP3 expression and its activation by ROS is not yet defined. Polyphenols with urate-lowering activities, such as allopurinol, quercetin, and rutin, prevent fructose-induced renal inflammation; specifically, these compounds lower both renal and systemic IL-1b and IL-18 levels. This effect is partially dependent on the ability of these compounds to reduce renal NLRP3 and caspase-1 protein levels. However, it is not clear whether allopurinol, quercetin, and rutin exert this effect by regulating gene transcription or by promoting NLRP3 and caspase-1 protein degradation. Nonetheless, NLRP3 downregulation correlated with the restoration of insulin sensitivity and glucose metabolism in a model of fructose-induced hyperuricemia and dyslipidemia (168). Artemisia annual The sesquiterpene lactone artemisinin is isolated from the plant Artemisia annual, called sweet wormwood, which is an herb employed in Chinese traditional medicine. In addition to its well-known use as anti-malarial drug, it has potent anti-inflammatory effects due to its ability to inhibit the © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


NFjB pathway (169–171). Consistent with this, artemisinin has been proven to be protective in models of postinfarct myocardial remodeling (172), lupus nephritis (173), experimental autoimmune encephalomyelitis (174), and Alzheimer’s disease (175). In this case, it was shown that artemisinin administration to an Alzheimer’s disease mouse model reduced b-amyloid plaque formation. This resulted in part from the reduction in BACE1, NLRP3, ASC, and caspase-1 protein levels in the brain, which correlated with reduced NFjB activation in artemisinin treated mice (175). However, the mechanism by which artemisinin prevents NFjB activation remains unknown. Ginseng Ginsenosides exhibit anti-inflammatory properties by inhibiting NFjB activation in association with reduced secretion and/or mRNA expression of pro-inflammatory mediators (176). Red ginseng has been studied for many years as an immune-modulating substance. Previous studies have reported red ginseng as a remedy for the treatment of infectious diseases as well as many metabolic disorders (177). Recently, it was shown that ginsenosides obtained from red ginseng prevent ATP-, alum-, and nigericin-induced NLRP3 inflammasome activation in LPS-primed bone marrowderived mouse macrophages and human THP-1-differentiated macrophages (178). Interestingly, red ginseng-derived ginsenosides were also able to inhibit the AIM2 inflammasome activation induced by dsDNA and listeria exposure. In addition to their negative effect on IL-1b production in response to Listeria infection, ginsenosides also prevented listeria-induced pyroptosis. Interestingly, ginsenosides had no effect in NLRC4 inflammasome activation due to Salmonella infection. The negative effect of these ginsenosides on NLRP3 and AIM2 inflammasomes could be explained by their negative effect on ROS production and Ca2+ mobilization—two intracellular signals that trigger inflammasome activation (178). Propolis Propolis, a product of bees, is a complex mixture of plant exudates, wax, and enzymes. The anti-inflammatory properties of propolis have long been documented and have been attributed to its negative effect on NFjB activation (179). Propolis contains different anti-inflammatory compounds, such as caffeic acid derivatives, flavonoids, and phenolic compounds (180). Depending of the region from which it is obtained, propolis may contain specific anti-inflammatory © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

compounds, such as caffeic acid phenethyl ester, found in European propolis (181), or 3,5-diprenyl-4-hydroxycinnamic acid (artepillin C), found in green propolis from Brazil (182). It was shown recently that green propolis prevents the NLRP3 and NLRC4 inflammasome activation induced by the pore-forming toxin nigericin or ATP and by Legionella pneumophila, respectively, in LPS-primed mouse macrophages (183). Although the authors state that this effect might be mediated by the compound artepillin C, further studies are required to identify the compound(s) inhibiting inflammasome activation and to define the molecular mechanism of action. Red wine The anti-inflammatory effects of red wine have been attributed to its high content of polyphenols. However, in a recent study, Nurmi et al. (184) showed that ethanol reduced IL-1b and IL-18 production in response to several NLRP3 inflammasome activators including cholesterol crystals, ATP, nigericin, and b-amyloid peptides in human LPSprimed macrophages. The effect of ethanol was not due to inhibition of IL-1b expression but rather to inhibition of secretion. This effect was not the result of acetaldehyde, a product of cytochrome P450 2E1 oxidase and alcohol dehydrogenase activities; nor did it result from preventing K+ exit or ROS production. Instead, ethanol prevented lysosomal damage and cathepsin B leakage to the cytosol, thus avoiding activation of the NLRP3 inflammasome. The negative effect of ethanol on IL-1b production in response to different inflammasome stimuli was mediated through the specific inhibition of caspase-1 but not caspase-8, which has also been implicated in IL-1b maturation and secretion. Interestingly, ethanol also affected dsDNA activation of the AIM2 inflammasome (184). Given that AIM2 inflammasome does not require lysosomal damage for its activation and that ethanol had no effect on ROS production, the mechanism by which ethanol prevents AIM2-mediated caspase-1 activation remains to be determined. Given the membrane’s permeability to ethanol, it is plausible that ethanol exerts its inhibitory effect by targeting an as yet unidentified intralysosomal molecule. Arsenic Hippocrates, the father of medicine, used arsenic for medicinal purposes. Chinese traditional medicine and Western medicine have also employed arsenic therapeutically to treat different maladies with an inflammatory component, such

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as ulcers, eczema, arthritis, psoriasis, and, most recently, leukemia. In agreement with its postulated anti-inflammatory effects, two research groups working independently found that arsenic trioxide and other arsenic derivatives inhibit inflammasome activation as well as IL-1b and IL-18 production. The work of Lo et al. (185) showed that NLRP3 inflammasome activation by monosodium urate was prevented by arsenic trioxide. Maier et al. (186) extended this observation and showed that arsenic trioxide, in addition to inhibiting the NLRP3 inflammasome, also blocked the activation of the NLRP1 and NAIP5/NLRC4 inflammasomes by B. anthracis lethal toxin and flagellin, respectively. Although Lo et al. (185) claimed that the effect of arsenic trioxide was mediated through the degradation of the promyelocytic leukemia protein (PML), which they also proposed as a new NLRP3 inflammasome activator, Maier et al. (186) found no functional relationship between PML and arsenic trioxide. They also ruled out a role for arsenic trioxide in inflammasome activation resulting from proteosomal degradation, phosphorylation of a positive regulator or by direct inhibition of active caspase-1. Instead, the work of Maier et al. (186) indicates that arsenic trioxide, through ROS, generates a unique intracellular environment that attenuates inflammasome activation. This does not involve the expression of arsenic trioxide-induced heat shock proteins, which might function as negative regulators of the inflammasome (185, 186). Thus, the roles of PML and the nature of the intracellular environment induced by arsenic trioxide resulting in inflammasome inhibition remain to be determined. Nonetheless, these data indicate the importance of arsenic compounds as potential alternative pharmacological treatments for inflammatory diseases. Glyburide and omega-3 fatty acids Although synthetic antibiotics like sulfonylureas are not plant derivatives, these compounds prevent ATP-induced IL1b secretion in peritoneal macrophages. This effect was attributed to the effect of glyburides on the ABC1 transporter (187). However, it was recently shown that glyburide inhibits NLRP3 inflammasome activation in response to ATP, crystals, and pore-forming toxins. Although the mechanism of action is still unknown, it does not involve inhibition of the P2X7R ATP receptor, pannexin, ATP-sensitive K channels, the ABC transporter ABCA1, or NLRP3 ATPase activity. Furthermore, the fact that glyburide does not inhibit the NLRC4 or NLRP1b inflammasomes indicates a specific effect on the NLRP3 inflammasome (187).

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Interestingly, glyburide is the most widely used drug for the treatment of type 2 diabetes in the United States (188). The drug works by inhibiting ATP-sensitive K+ channels in pancreatic b cells, thereby promoting insulin secretion and reducing blood sugar levels in diabetic patients (189). The fact that NLRP3 inflammasome activation in response to high glucose levels promotes b cell death and hence decreased insulin levels suggests that the effect of glyburide on insulin level might also be attributed to NLRP3 inflammasome inhibition and b pancreatic cell survival. However, the fact that glyburide does not restore insulin sensitivity in humans is in sharp contrast with a study showing that the reduction in inflammation observed in the adipose tissue of obese NLRP3-deficient mice restores glucose metabolism (190). Thus, it is possible that glyburide does not prevent lipid-dependent NLRP3 inflammasome activation, and that IL-1b and IL-18 levels therefore remained elevated and insulin resistance persists. Different lines of experimental evidence clearly suggest that omega-3 fatty acids (x-3 FAs), mainly eicosapentaenoic acid and docosahexaenoic acid, have anti-inflammatory properties and are beneficial for the treatment of different diseases associated with chronic inflammation, such as arthritis, atherosclerosis, and diabetes (191–193). Indeed, x-3 FAs reduced TNF-a and IL-1b levels in the serum of healthy volunteers (194) and reduced inflammation in the adipose tissue of animals that were fed with a high-fat diet as well as enhancing insulin sensitivity (193). In agreement with the fact that G protein-coupled receptor 120 (GPCR120) can be activated by long-chain fatty acids (195) and that adipocytes and macrophages highly express GPCR120, the negative effect of x-3 FAs on inflammation is reduced in GPCR120-deficient mice. Thus, x-3 FAs inhibit lipid-induced inflammation through activation of the GPCR120. However, recently Yan et al. (86), using an in vitro model, demonstrated that x-3 FAs negatively modulate macrophage NLRP3 inflammasome activation in response to different stimuli. The NLRP1 inflammasome, but not the NLRC4 or AIM2 inflammasomes (86), was also inhibited. The negative effect of x-3 FAs on NLRP3 inflammasome activity is mediated by the GPCRs 120 and 140 and their downstream scaffold protein b-arrestin-2, which can directly interact with NLRP3. In vivo, x-3 FAs prevented lipidinduced NLRP3 inflammasome activation, thus reducing the negative effect of chronic inflammation on insulin resistance (86). In agreement with previously published results showing that x-3 FAs promote autophagy (196), recently, in © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


addition to NLRP3 inactivation by b-arrestin-2 interaction, x-3 FAs were shown to prevent inflammasome activation by promoting autophagy. ATG7-deficient macrophages were refractory to the negative effect of x-3 FAs on NLRP3 inflammasome activation (197). In conclusion, natural (whether organic or not) or synthetic compounds that prevent or inhibit inflammasome activation represent a new alternative to harness the inflammatory process underlying different chronic diseases. Concluding remarks To maintain homeostasis, an organism should possess the ability to respond to both exogenous and endogenous noxious signals, including pathogen-derived molecules, airborne particulates such as asbestos or silica, intracellular components such as ATP, DNA, peptides, and proteins such as HMBG1, and metabolites such as uric acid, cholesterol, lipids, and sugars. The discovery that sensing and responding to this milieu of stimuli is mediated by NLRPs and the assembly of the inflammasome has allowed us to understand

that inflammation is the common denominator in apparently unrelated chronic diseases, neurodegenerative diseases such as Alzheimer’s disease, and metabolic disorders such as nonalcoholic fatty liver disease, colitis, obesity, atherosclerosis, and diabetes mellitus. Further studies are required to elucidate the detailed molecular mechanisms of inflammasome assembly and function. However, the information discussed here clearly notes that understanding the mechanisms that prevent inflammasome activation in the resting state and those designed to turn off inflammation to maintain homeostasis is key to defining new therapeutic targets to control chronic inflammation. In particular, identifying the molecular arsenal that pathogens have acquired over the course of evolution to prevent or inhibit inflammasome activation is critical for controlling infectious diseases. Finally, defining the molecular structure and function of bioactive compound in plants with anti-inflammatory properties used in ancient cultures would offer the opportunity for discovery of new therapeutic approaches to treat different inflammasome-driven chronic diseases.

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Keeping inflammation at bay.

Regulation of inflammasome activation.

Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis.

Regulation of Iron Metabolism by Hepcidin under Conditions of Inflammation.

Keeping infection control in hand.

Hepatitis regulation by the inflammasome signaling pathway.

Negative endocrine control system for inflammation in rats.

The Role of Interferons in Inflammation and Inflammasome Activation.

Schwann cell proliferation in vitro is under negative autocrine control.

Correction to 'Control and regulation of pathways via negative feedback'.

Reactive oxygen species at the crossroads of inflammasome and inflammation.

Mitochondria: diversity in the regulation of the NLRP3 inflammasome.

amylin gene transcriptional control region: evidence for negative regulation.

Regulation of Nlrp3 inflammasome by dietary metabolites.

Inflammation. Potent small molecule extinguishes the NLRP3 inflammasome.

Autophagy in HCV infection: keeping fat and inflammation at bay.

Lane keeping under cognitive load: performance changes and mechanisms.

The Firepower of Work Craving: When Self-Control Is Burning under the Rubble of Self-Regulation.

Celastrol ameliorates inflammation through inhibition of NLRP3 inflammasome activation.

Kinases: a remote control in inflammasome activity.

Reciprocal Regulation between Enterovirus 71 and the NLRP3 Inflammasome.

Control your anger! The neural basis of aggression regulation in response to negative social feedback.

Inflammasome, IL-1 and inflammation in ozone-induced lung injury.

Dietary cholesterol directly induces acute inflammasome-dependent intestinal inflammation.