Molecular activation of the NLRP3 Inflammasome in fibrosis: common threads linking divergent fibrogenic diseases.

Antioxidants & Redox Signaling Molecular Activation of the NLRP3 Inflammasome in Fibrosis: Common Threads Linking Divergent Fibrogenic Diseases (doi: 10.1089/ars.2014.6148) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 1 of 38

1

FORUM ISSUE: ANTIOXIDANTS & REDOX SIGNALING on INFLAMMASOME

Molecular Activation of the NLRP3 Inflammasome in Fibrosis: Common Threads Linking Divergent Fibrogenic Diseases

Carol M. Artlett1, PhD and James D. Thacker2, PhD

1

Drexel University College of Medicine Department of Microbiology and Immunology, Philadelphia, PA; 2TherimuneX Pharmaceuticals, Inc., Plymouth Meeting PA. Address correspondence to: Carol M. Artlett, Ph.D Associate Professor Department of Microbiology and Immunology Drexel University College of Medicine 2900 Queen Lane, Philadelphia PA 19129 (215) 991-8585 (215) 848-2271 [email protected] Key Words: fibrosis, inflammasome, calcium signaling, ER stress Author disclosure Statement: CMA has no conflict of interest with the content of the article; JDT is president and CSO of TherimuneX Pharmaceuticals, Inc. Words: 5477 Figures: 6 Running heading: Redox regulation NLRP3 during fibrosis

1


Page 2 of 38

2 Abstract Significance: Over the past 10 years there has been a plethora of investigations centering on the NLRP3 inflammasome and its role in fibrosis and other disease pathologies. To date, the signaling pathways from the inflammasome to myofibroblast differentiation and chronic collagen synthesis have not been fully elucidated and there are many questions left to be answered. Recent Advances: Recent studies have demonstrated the significant and critical role of reactive oxygen species and calcium signaling in the assembly of the inflammasome and this may result in autocrine signaling maintaining the myofibroblast phenotype leading to fibrotic disease. Critical Issues: Traditionally, myofibroblasts under tight regulation, aid in wound healing and then, once the wound has closed, undergo apoptosis and the collagen in the wound remodels. During fibrosis, however, the myofibroblast maintains an activated state via a chronically activated inflammasome, leading to the continual synthesis of collagens and other extracellular matrix proteins that result in damage to the tissue or organ. The mechanism that is driving this abnormality has not been fully elucidated. Future Directions: However, studies have been conducted to suggest that modulating the calcium or the reactive oxygen species axis may be of therapeutic value in regulating inflammasome activation. A number of novel drugs are currently being developed that may prove beneficial to patients suffering from fibrotic diseases.

2


Page 3 of 38

3 Fibrosis is a commonly encountered end stage pathology Thomas Wynn has estimated that about 45% of all deaths in the Western world can be attributed to some type of fibrotic disease (106). Fibrosis is the abnormal over expression of collagens resulting in the failure of the function of the visceral organs and tissues. Once damage to a tissue has been established, additional cells are recruited to the site to aid in “repair” of the tissue and these cells can also contribute to the increased deposition of collagens. Fibrosis can be systemic, affecting many organs, or localized to a single organ. However, the excessive deposition of collagen causes irreversible damage to the tissue. Often the initiating insult driving fibrosis is unknown, but once fibrosis has been established there are no FDA approved drugs that can control the accumulation of collagens in tissues. Many of the pathological features occurring in fibrosis are also observed in wound healing. Wound healing is a critical and tightly regulated mechanism inducing the transient upregulation of collagen synthesis. Once the wound is closed, collagen synthesis declines and the tissues are remodeled. In contrast, fibrosis is wound healing that has gone awry with uncontrolled collagen synthesis. It has been proposed that fibrosis occurs due to the dysregulation of the wound healing process at either the proliferative or remodeling stages; or if the irritant persists in the tissues, it continually drives the process (107). Fibrosis can occur with or without inflammation, and with it carries a high mortality rate depending on the organ affected. Understanding the differences between wound healing and fibrosis would be crucial in understanding the mechanism(s) that drive fibrosis. Recently, innate immune signaling driven by the inflammasomes has been implicated in fibrosis, and the role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) is currently being elucidated in this process.

3


Page 4 of 38

4 Fibroblasts are a heterogeneous population of cells and when activated they differentiate into myofibroblasts, participating in matrix deposition and remodeling. Other resident or migratory cells once activated can also differentiate into myofibroblasts. Residential cells with the ability to become myofibroblasts include hepatic stellate cells (22), biliary duct fibroblasts (83), mesangial cells (115), mesenchymal cells (61, 73, 117), epithelial cells (100), and endothelial cells (51). Circulating cells that can differentiate upon activation into myofibroblasts include pericytes and fibrocytes (28, 46, 53, 79). The fate of the myofibroblast is the key to whether there is normal tissue repair or fibrosis. The mechanism(s) that results in myofibroblasts apoptosis and resolution of wound healing remain unknown; similarly the mechanisms that promote apoptosis resistant myofibroblasts and progressive fibrosis aren’t well defined, either. However, TGF-β1 appears to be central to inducing myofibroblast differentiation and their survival (99). TGF-β1 activates focal adhesion kinase and the AKT pathway and both signaling events promote pro-survival, cell adhesion, and release of growth factors (34, 96). Once activated myofibroblasts secrete a wide range of lipid mediators, chemokines, cytokines, and ROS; and can be considered an inflammatory cell (81). The signaling that drives fibrosis is complex and to date not fully elucidated however it is known that the inflammasome is part of this complex signaling pathway. ROS also appears to be a central player driving the fibrotic process as it can be induced by a number of growth factors and cell stressors that are involved in fibrosis possibly leading to feed forward signaling that maintains inflammasome activation and the fibrotic response by myofibroblasts. Furthermore, the inflammasome is responsive to ROS levels in the cell further driving the cellular signaling responses and secretion of cytokines. A number of different cytokines and growth factors are

4


Page 5 of 38

5 involved in fibrosis and these factors also promote ROS synthesis. For example, TGF-β, IL-1 and angiotensin II all promote ROS and fibrosis (21, 58, 68). Furthermore, other pathogen associated molecular pattern sensors such as the toll-like receptors have been reported to be involved in signaling that can lead to fibrosis. All of the toll-like receptors induce ROS (56) and signaling ROS this is known to activate the inflammasome (58). This will be discussed in more detail in following sections. The hypothesis is that cellular sensing of PAMPs or DAMPs can increase ROS levels activating the NLRP3 inflammasome leading to processing of IL-1 and IL-18. Depending on the cell making IL-1 and/or IL-18, autocrine signaling could promote synthesis and secretion of growth factors and cytokines leading to further production of ROS that could also enhance inflammasome activation. This would lead to an “auto” inflammatory response that is difficult to control. Understanding this orchestration between ROS and inflammasome activation needs to be further elucidated in fibrosis.

The NLRP3 Inflammasome Our laboratories have been investigating the role of the inflammasome in fibrotic disease pathology and in particular its role in systemic sclerosis. Inflammasomes containing a NOD-like receptor (NLR) recognize a diverse range of conserved molecular motifs unique to viral, parasitic, and bacterial pathogens. However, inflammasomes are not just sensors of pathogenic invasion of cells; the NLRP3 inflammasome, for example, senses the overall integrity and health of the cell and can become activated upon ER stress. ER stress is often associated with host infection and tissue damage (63).

5


Page 6 of 38

6 The NLR region of an NLR inflammasome is a sensor motif. NLRs are comprised of an N-terminal effector domain, a central NOD or NACHT domain, and a ligand sensing C-terminal leucine rich repeat domain (35, 36). Currently, twenty-three NLRs containing NOD and leucine rich repeat domains have been identified and have been sub-classified by their N-terminal region. NLRC1-5 contain a caspase recruitment domain (CARD) protein, NLRP1-14 contain pyrin, and NAIPs contain a Baculovirus inhibitor of apoptosis repeat domain (41). With the discovery of the inflammasome, it was found that leucine rich repeats contained within NLRs are a new class of pattern recognition receptor consisting of varying numbers of leucine repeats that allow the protein to fold into three dimensional curved structures (5, 33). These curved structures bind specific ligands found on pathogens and metabolic danger signals (4). The mechanism of detection of the signal is yet to be fully elucidated but once the NLR receptor is activated; it induces the formation of the complex called the inflammasome. The NLRP3 inflammasome associates with the adaptor protein apoptosis speck like protein containing a CARD (ASC) through the pyrin domain. ASC associates with pro-caspase-1 and the resulting association of these proteins induces the auto-catalytic cleavage of pro-caspase-1 and its subsequent activation. Activated caspase-1 cleaves pro-IL-1 and pro-IL-18 to their mature form that is secreted. One of the main functions of the NLRP3 inflammasome is to initiate the innate immune response via the secretion of IL-1β and IL-18 (Figure 1). Expression of inflammasome proteins can be found in a wide variety of immune and nonimmune cells, including monocytes/macrophages (89), T cells (50), myofibroblasts/fibroblasts (2), keratinocytes (12, 18), and hepatic stellate cells (103). Because non-immune cells bearing inflammasome proteins are found in all organs and systems of the body, we propose that they are

6


Page 7 of 38

7 often involved in the initiation of inflammation to further enhance the recruitment of immune cells to the site where there is infection or damage (94). For three inflammasomes studied, NLRP1, NLRP3 and IPAF, it has been hypothesized that there are different states of expression of the inflammasome components depending on the cells and/or tissues; however this observation has only been reported by one group (110). Tier one has been assigned to tissues that have constitutive expression of all inflammasome components in a stand-by state, that is ready to assemble into a functional inflammasome upon detection of a stimulus. Tier two tissues and cells require the induced expression of one inflammasome component having all the other components in a stand-by state; whereas, tier three tissues and cells require the upregulation of more than one component for inflammasome activation. NLRP3 inflammasome is one of the most well studied inflammasomes and is involved in many disease pathologies. The critical role for the NLRP3 inflammasome is activation of caspase-1. During the recruitment of inflammasome proteins into the complex, the assembly of the inflammasome localizes caspase-1 proteins in close proximity allowing for the auto-catalytic cleavage and activation of pro-caspase-1 (105). Once activated, caspase-1 itself is then able to process a wide variety of protein precursors, inducing unconventional or leaderless protein secretion that is independent of the ER/Golgi pathway (45). IL-1β secretion is thought to involve P2X7 (20). IL-1β and IL-18 are two of the most studied proteins known to be cleaved by active caspase-1. IL-1β is initially translated as a 30.7 kDa inactive protein that is cleaved to the active 17.5 kDa form. Likewise, IL-18 is initially processed as a larger 22.3 kDa inactive protein and is cleaved to a 17.3 kDa active form. Once cleaved by caspase-1, both IL-1β and IL-18 are then

7


Page 8 of 38

8 secreted from the cell where they can, depending on the local environment and cell type, be involved in autocrine and paracrine signaling. IL-1β and IL-18 are structurally similar and both contain predominantly β-pleated sheets (15) which is much less common than α-helical structures. IL-1β is a pleiotropic cytokine and has been found to be involved in localized inflammation targeting inflammatory responses to parasitic, bacterial, or viral infections. It plays a role in systemic inflammation, as well as many chronic diseases. IL-1β is produced by many different inflammatory and non-inflammatory cell types also in response to cellular damage. IL1β signals via p38 MAPK phosphorylation and NFκB activation (14). IL-1β release is tightly regulated requiring two signals for the activation and secretion of the protein. The first signal, usually obtained via toll-like receptor signaling or IL-1 receptor signaling upregulates IL-1β gene transcription and the second signal is obtained via inflammasome activation, where caspase-1 induces the maturation of IL-1β for secretion (14). The acute release of IL-1β into the peripheral blood upregulates the expression of other proinflammatory cytokines such as IL-6; it also raises cortisol levels, and acts on the hypothalamus to induce fever (23). However, in some chronic diseases, the amount of IL-1β that is secreted may act locally to enhance inflammatory gene response. This could promote disease resolution or disease progression as in the case of fibrotic diseases and some cancers. Furthermore, IL-1β has been found to promote collagen synthesis leading to fibrotic tissues (48, 77, 97) (Figure 2). The function of IL-18 (interferon- inducing factor) also participates in downstream inflammasome signaling, but this cytokine is less well studied and its effects are not fully known. IL-18 is constitutively expressed in its precursor pro-form in many cells and can be rapidly cleaved upon inflammasome activation. IL-18 can also be processed by other proteins such as

8


Page 9 of 38

9 proteinase 3 (92) which is a serine protease expressed primarily in neutrophil granulocytes. IL-18 can also be cleaved by caspase-3 resulting in its inactivation. Like IL-1β, IL-18 has also been shown to promote collagen synthesis (82, 108, 113) (Figure 2).

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) and their roles in NLRP3 inflammasome activation and control Under normal non-stressed conditions chaperones assist in the folding of proteins and prevent aggregation of proteins in the ER. Only properly folded proteins are transported to the Golgi for additional processing. Immunoglobulin heavy chain-binding protein/glucose regulated protein 78 (BiP/GRP78) is a critical chaperone that becomes upregulated when ER stress is encountered. Indeed, GRP78 levels have been used to measure ER stress. GRP78 binds PERK (transmembrane sensor protein PKR-like ER kinase), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE-1). When GRP78 is bound to PERK, ATF6, and IRE-1 are inactive. However under ER stress, GRP78 is released from the transmembrane proteins and binds to the newly translated proteins and PERK, ATF6 and IRE-1, which then become active and promote the UPR. The role of PERK, ATF6 and IRE-1 is to downregulate protein translation returning the ER to homeostasis and normal functioning (Figure 3). Growth arrest and DNA damage gene 153 (GADD153) also called CCAAT-enhancerbinding protein homologous protein is another protein involved in ER-stress and is thought to mediate apoptosis (74). Lipopolysaccharide specifically activates the IRE-1 pathway, but this response is delayed compared to thapsigargin which induces depletion of the ER Ca2+ store. Moreover, lipopolysaccharide does not activate the PERK-ATF4 pathway that is responsible for the induction of GADD153 and subsequent cellular apoptosis (74). Under ER stress the role of

9


Page 10 of 38

10 the transcription factor X-box binding protein-1 (XBP1) has recently been shown to be a modulator of the down-stream signaling cascade and the UPR. XBP1 has two isoforms, spliced (XBP1s) and unspliced (XBP1u). The unspliced isoform is constitutively expressed in quiescent cells. However, upon ER stress, XBP1 mRNA has a 29 base intron unit removed by IRE-1 and this causes a reading frame shift. The result of this splicing event causes the translation of a larger isoform of XBP-1 protein known as the spliced isoform (XBP1s). Once spliced, XBP1s is a known gene transcription regulator and inducer of the UPR. It is involved in transiently regulating many important innate immune signaling genes and is able to regulate its own expression (112). Nishotoh et al., postulated that the delayed time course activation of the PERK-ATF4 vs. IRE-1 pathway differentiates cellular fate in response to ER stress. Such that IRE-1/XBP1 mediates UPR and cell survival or that PERK/ATF4/GADD153 mediates cellular apoptosis (74). Interestingly, the actions of XBP1s have been shown to be negatively regulated by XBP1u (111). In that report, Yoshida et al. showed that XBP1u engaged XBP1s to form an XBP1u/XBP1s heterodimer that was rapidly degraded, thus demonstrating a possible mechanism for the negative feedback control of XBP1s gene transcription and a sustained pro-inflammatory response or UPR. Each of the three arms of the ER-stress response has been interrogated separately for inflammasome activation. Tunicamycin inhibits glycoprotein synthesis through the inhibition of GlcNAc-1 phosphate transferase inducing ER stress. Thapsigargin promotes ER stress by blocking ER sarco/endoplasmic reticulum Ca2+-ATPase pump. Brefeldin A blocks protein transport from the ER to the Golgi. The different modes of action of these three compounds inducing ER stress promotes the activation of caspase-1 and the maturation of IL-1β; thus,

10


Page 11 of 38

11 confirming that all arms of the ER stress response activates the NLRP3 inflammasome in a mechanism that is classically independent of the UPR (67). In addition to the ER, it has been shown that mitochondria also sense various danger signals resulting in NLRP3 inflammasome activation in a mechanism that is yet to be fully defined. Increased ROS and/or potassium efflux appear to be universal signals that mediate the activation of the inflammasome and inhibition of ROS or potassium efflux abrogates IL-1β release (116). The NLRP3 inflammasome is one of the most extensively studied inflammasomes and is capable of sensing a wide variety of alarm signals (63). Recently, it was found that the assembly of the NLRP3 inflammasome requires the presence of ROS (16). The direct interaction between the ER and the mitochondria was also shown to be very important in this signaling (119). Quiescent NLRP3 is localized to ER structures; however, once the inflammasome is activated, both NLRP3 and ASC redistribute to the perinuclear region of the cell where they colocalize with the ER and mitochondria organelles (119). These data suggests that ROS and mitochondrial signaling play a significant role in the assembly and activation of the NLRP3 inflammasome. Other inflammasomes such as AIM2 and IPAF do not appear to require the ER or mitochondria for their activation (119). The role of the mitochondria in the innate immune response has been well characterized and has been reviewed by West et al. (104). Recent studies have demonstrated that the source of ROS appears to be important for inflammasome activation. Mitochondrial ROS is necessary for mitochondrial membrane permeability transition (54). Inhibition of mitochondrial membrane permeability transition with cyclosporine A prevents the secretion of IL-1β. This suggests that caspase-1 activation is dependent on the integrity of the mitochondrial membrane and on mitochondrial ROS (72). Loss of mitochondrial membrane integrity and cell death appear to be dependent on the NLRP3

11


Page 12 of 38

12 inflammasome and the release of IL-1β precedes cell death (32). Furthermore, mitochondrial DNA has been reported to be a damage-associated molecular pattern capable of driving inflammasome activation (114). Intriguingly, the NLRP3 inflammasome appears to be involved in the release of mitochondrial DNA into the cytosol as NLRP3 deficiency reduced cytosolic mitochondrial DNA (72). However the NLRP3 inflammasome does not appear to be required at all for the generation of mitochondrial ROS (72).

NAD(P)H oxidases, ROS, and fibrosis Other sources of ROS occur within the cell and can alter cellular function and fate. Nicotinamide adenine dinucleotide phosphate/NAD(P)H-oxidase, also known as NOX, plays an essential role in ROS-mediated innate immunity and host defense against pathogens. Like inflammasomes this class of proteins is highly conserved from plant phyla to mammals (43). There are 4 isoforms and each isoform functions differently to induce ROS and mediate downstream signaling in the cell (39). Fibroblasts express a number of NOX enzymes and their expression and regulation of their downstream signaling is dependent on the activation state of the fibroblast (60). NOX4 has been implicated in myofibroblast differentiation and fibrosis (1, 11, 31). Furthermore, NOX4 mediates TGF-β1 signaling by regulating the phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (109). This protein functions to repress protein translation during UPR by inhibiting GRP78 and ATF4 (66) and is a major enzymatic component of ROS production during UPR. Activation of NOX enzymes may also induce the production of ROS by mitochondria (27). However, increased ROS can activate NOX as both mitochondria and NOX enzymes are in close association with the ER (80, 87, 93). Significant cross-talk between ROS from the mitochondria and NOX has been found and this has

12


Page 13 of 38

13 been associated with numerous pathologies. NOX4 may promote the differentiation of alternative signaling pathways such as activating p38 leading to cell stress and increased ROS (59, 60). Moreover, NOX4 has been found to play a critical role in myofibroblast differentiation and NOX4 levels specifically correlate with the myofibroblast phenotype leading to fibrosis (85). There is more evidence to suggest that NOX4 derived ROS plays a significant role in renal, lung and liver fibrosis (26, 31, 40). ROS generated via NAD(P)H oxidase pathways is apparent in many fibrotic diseases and we hypothesize that this pathway contributes to the overall level of oxidative stress leading to chronic activation of the NLRP3 inflammasome. Oxidative stress is apparent in systemic sclerosis (SSc) a diseases where the skin and organs becomes fibrotic. ROS has been extensively studied and reports suggest that there is constant intracellular production of ROS derived from NOX. Further exacerbating this, IL-1β, TGF-β1 platelet-derived growth factor-BB, IL-4, IL-6, tumor necrosis factor-α, and connective tissue growth factor all induce a positive feedback signals for ROS production (84). Further studies have elucidated that NOX4 is involved in TGFβ1 induced differentiation of cardiac fibroblasts into myofibroblasts and that the superoxide production is required for extracellular matrix proteins (11). Furthermore, this study demonstrates that TGF-β1-mediated phosphorylation of Smad2/3 is dependent on NOX4 (11). NOX derivatives are involved in multiple pathologies including cardiovascular disease, fibrosis, and cardiomyopathy signifying the important role of this enzyme in ROS production. In addition, NOX4 is able to promote ER stress (60) that may further promote NLRP3 inflammasome activation, leading to more IL-1β secretion, TGF-β1 synthesis and NOX4 activation. One recent study suggested that NLRP3 inflammasome activation is independent of NOX (98). Thioredoxin-interacting protein (TXNIP) is upregulated by ROS and plays an important role in

13


Page 14 of 38

14 inflammation mediated by oxidative stress (76). TXNIP has been shown to be involved in NOX4 expression, increased levels of mitochondrial ROS and NADPH oxidase signaling (88). Moreover, TXNIP has been recognized as a critical regulator of the NLRP3 inflammasome (Figure 4) and the direct interaction between TXNIP and NLRP3 has been documented (76, 118) but this study did not hold up to other investigations (65).

Are intracellular calcium levels a universal switch for NLRP3 inflammasome control? The ER is a major site for Ca2+ storage and the ER controls intracellular Ca2+ homeostasis (49). Changes in the ER environment and in ER related Ca2+ levels can result in ER stress and ROS production. Ca2+ mobilization controls a diverse range of cellular processes including cell proliferation and differentiation, transcription, metabolism and apoptosis (9). Ca2+ mediated signaling has also been found to be crucial during NLRP3 inflammasome activation (70). Mitochondria also take up Ca2+ from the ER or cytosol to regulate cellular signaling (62). Murakami et al. (70), demonstrated that extracellular ATP activates the NLRP3 inflammasome via P2X7 receptor and this process requires ER Ca2+ release and influx of extracellular calcium. ER Ca2+ depletion causes the influx of extracellular Ca2+ via store operated Ca2+ entry. Thus it was found that inhibition of Ca2+-mediated signaling blocked the assembly of the NLRP3 inflammasome suggesting that Ca2+ signaling is crucial for NLRP3 inflammasome activation. Furthermore, this process was shown to upregulate mitochondrial ROS production leading to inflammasome activation (70). Murakami et al., elegantly demonstrated that Ca2+-mediated signaling induced mitochondrial damage and that this resulted in NLRP3 inflammasome activation (70).

14


Page 15 of 38

15 ATP stimulation leads to mitochondrial ROS production and loss of membrane potential and the release of mitochondria contents into the cytosol. Inhibiting any of these processes prevents NLRP3 inflammasome activation. It has been shown that GADD153 regulates ER Ca2+ release (70). In GADD153-deficient bone marrow-derived macrophages there was an attenuated response to ATP and reduced NLRP3 activation. This was not characterized by an overall defect in inflammasome signaling as the NLRC4 inflammasome activation was normal (70). This finding is intriguing as GADD153 is involved in many chronic diseases and thus ER stress may amplify inflammation via NLRP3 inflammasome activation. Lee et al. (52), recently demonstrated that a Ca2+/cAMP control mechanism can also regulate the NLRP3 inflammasome. Brough et al. (7), made the first observation that Ca2+ from intracellular stores might be involved in ATP-induced caspase-1 activation and IL-1β release. They observed that the chelation of cellular calcium abrogated IL-1β release and that this was not found to be dependent on extracellular calcium. Furthermore, thapsigargin and nigericin which are known to increase intracellular Ca2+ via depletion of the ER Ca2+ stores induced IL-1β release (7). In contrast, Lee et al. (52), found that extracellular calcium could promote IL-1β release. Under normal physiological conditions, cAMP blocks NLRP3 inflammasome activation of caspase-1 by binding to the NACHT domain. However, when cells are stressed or injured, there is increased extracellular and intracellular Ca2+ and this decreases cAMP levels allowing for the activation of the inflammasome (52). Intracellular Ca2+ have been shown to be an important signaling molecule in fibroblasts and can affect proliferation (3, 42), cell differentiation (47), and collagen synthesis (91). Moreover, ATP-induced Ca2+ waves have been reported to promote TGF-β1, collagen and fibronectin expression during wound healing (37). In the context of normal cellular function, this

15


Page 16 of 38

16 signaling may not be sufficient to continually activate the inflammasome; however, if other cellular stressors are involved this may tip the balance towards chronically active TGF-β1 and fibrosis. We have recently shown that thapsigargin induced IRE-1 mediated ER stress, a unique 18 amino acid peptide is excised from the third extra-membrane loop of ER embedded transient receptor protein channel-1 (TRPC1) and is post-translationally modified by the replacement of the N-terminal aspartate with N-acetyl alanine to form a new signaling peptide (acALY18). TRPC1 is a store operated calcium channel protein that is involved in cellular Ca2+ flux. We found that upon release, acALY18 engages XBP1 within minutes of cellular injury or infection to induce an innate immune response that is NLRP3 dependent (78, 94, 95). The in vitro administration of acALY18 to quiescent cells promotes NLRP3 inflammasome activation and transient gene transcription peaking at 48 h and returning to baseline by 72 h, leading to increased wound healing (94). acALY18 alone does not induce an ER stress response (78). However, in already stressed cells, we found that acALY18 preferentially binds XBP1s (78) and down regulates collagen synthesis leading to resolution of fibrosis (78) (Figure 5). We speculate that acALY18 engagement of XBP1s prevents XBP1u/XBP1s heterodimerization and subsequent degradation to regulate the UPR. This then results in a decrease in collagen synthesis. Thus, a picture of the cellular response to injury or infection is beginning to emerge that involves enzymatic cleavage of the third extra-membrane loop on the lumenal side of ER embedded TRPC1 (95) that can be modulated to alter the ER stress response and inflammasome activation. We propose that the balance between the profibrotic inflammasome activation and inflammasome down regulation may, therefore, be modulated by the XBP1u/XBP1s balance (Figure 6).

16


Page 17 of 38

17

Inflammasome Activation in Fibrotic Diseases The involvement of the inflammasome in fibrosis is slowly being elucidated. The conundrum is that the inflammasome appears to be involved in many responses to pathogens or cellular alarm signals and yet fibrosis does not result in every case. This suggests that inflammasome driven fibrosis could be dependent on specifically activated inflammasome(s), genetic variation(s) that affect the response by cells to inflammasome by-products or cytokine levels, the cell type in which the inflammasome is activated, ROS, and/or by the XBP1u/XBP1s balance. Fibroblasts are not immunologically inert cells that only become activated when the skin is damaged. Historically, fibroblasts have not been considered central to the immune response. However, fibroblasts are sentinel cells, capable of becoming activated by bacterial products and other injurious signals. Once activated they secrete cytokines and chemokines indicative of this activation (90). Furthermore, when activated, they may have a functional inflammasome mediating increased synthesis of profibrotic cytokines leading to myofibroblast activation (2). Gasse et al., (24) investigated the direct connection between inflammasome activation, IL-1 receptor, and MyD88 signaling during fibrosis. Mice deficient in the inflammasome adaptor protein, ASC, had an attenuated response to bleomycin-induced pulmonary fibrosis. This study further demonstrated that the IL-1 receptor signaling and MyD88 adaptor molecule played a role in pulmonary fibrosis and mice deficient in these proteins also had abrogated responses to bleomycin. Furthermore, they found that the direct administration of recombinant mouse IL-1β to the lungs of wild-type mice caused a robust increase in tissue destruction with inflammation and collagen deposition. Inhibition of IL-1 receptor signaling with Anakinra was more effective

17


Page 18 of 38

18 at limiting fibrosis than IL-1β neutralizing antibodies. Gasse et al., showed that IL-1β production was dependent on the inflammasome rather than toll-like receptor signaling (24). These studies add to the crucial role of an activated inflammasome in fibrotic pathologies. Bleomycin induces the release of uric acid in the lungs and this acts as a danger signal (25). It is currently thought that the localization of uric acid causes the deposition of crystals resulting in membrane damage and activation of NLRP3 with release of IL-1β. The inflammatory signaling mediated by uric acid is dependent on IL-1 receptor, MyD88, and the NLRP3 inflammasome suggesting that this is promoting an autocrine signaling loop that mediates fibrosis (25). Couillin et al. (10), found that alveolar breakdown and subsequent pulmonary fibrosis, a pathology that is observed during elastase-induced emphysema, was dependent on ASC inflammasome-mediated signaling and release of IL-1β. They also found that this pathology was attenuated Anakinra (10). The authors demonstrated that administration of elastase induced the release of uric acid by dying cells causing the crystals in murine airways. This results in a transient increase in tissue inhibitor of metalloproteinases (25). Importantly, the release of IL-1β in this model was not found to be cause by elastase cleavage of pro-IL-1β but was mediated via the direct activity of caspase-1 (10) suggesting that the inflammasome plays a significant role in this pathology. Silicosis is also driven by the NLRP3 inflammasome resulting in the excessive deposition of collagen, recruitment of inflammatory cells mediating damaged to the tissues (8). Silica crystals were found to induce ROS in macrophages. Blocking the generation of ROS inhibited the activation of caspase-1, release of IL-1β, and collagen synthesis (8). These studies further

18


Page 19 of 38

19 highlight the role of oxidative stress in inflammasome signaling and suggest that ROS may promote a profibrotic phenotype. We recently reported the role of the inflammasome in the fibrotic disease SSc (2). We found that the inflammasome mediated the increased synthesis of collagen and induced myofibroblast differentiation. To confirm this, we found that caspase-1 activity was significantly upregulated in SSc fibroblasts and secreted more IL-1β and IL-18 protein. We also found that we could abrogate this secretion if chemical or siRNA inhibition of caspase-1 was employed (2). This data suggests that inflammasome activation mediating the release of IL-1β and IL-18 might be driving fibrosis. Our studies focused on myofibroblasts which are the prime cell that synthesizes excessive amounts of collagen in SSc lesions and found that inhibition of caspase-1 (chemically or by siRNA) reduced the expression of α-smooth muscle actin and decreased collagen synthesis. Alpha-smooth muscle actin stress fibers contained less protein and were thinner in diameter in myofibroblasts treated with caspase-1 inhibition, suggesting that the myofibroblasts were dedifferentiating. However, in quiescent fibroblasts, endogenous levels of α-smooth muscle actin were unaltered. In vivo studies using bleomycin in ASC-deficient or NLRP3-deficient mice had abrogated collagen deposition in the dermis. Our findings implicate that caspase-1 activity is wholly involved in SSc fibrosis and suggests that there may be autocrine signaling mediated by IL-1β and/or IL-18 that promotes the profibrotic phenotype in these patients.

Are other inflammasomes involved in fibrosis? The role of other inflammasomes in fibrosis has been less well defined. As yet there are few studies that specifically investigate the role of any of the other inflammasomes in fibrosis,

19


Page 20 of 38

20 Currently, the literature is inconsistent at this early stage. Fernandez-Velasco et al. (19), reported that NOD1 expression in the heart modulates cardiac fibrosis. Caveats to this study are that NOD1-deficient mice were not used and these studies were based on the intraperitoneal administration of the NOD1 agonist C12-iE-DAP into wild-type mice that could have off targets effects. Intriguingly, even thought the authors report NOD1 expression in other organs, they do not study whether fibrosis also occurs in those organs in the mice. Further, collagen expression of siRNA knockdown of NOD1 in cardiac fibroblasts was not performed clouding the results. It has been suggested that bacterial products such as muramly dipeptide activating NOD2 could be involved in the development of fibrosis in Crohn’s disease (55). Mice deficient in NOD2 were protected against some forms of fibrosis, such that NOD2-deficient mice did not get cholestatic-mediated fibrosis but they were susceptible to liver toxin-induced fibrosis (101). However, in another study, NOD2-deficiency was shown to promote cardiac hypertrophy and fibrosis (120).

Is there an association between ER stress and calcium signaling in fibrotic diseases? As previously discussed, the ER is an organelle found in all eukaryotic cells. Recent studies investigating ER stress have found that it can enhance collagen synthesis leading to fibrosis. There are numerous conditions that cause ER stress including calcium depletion, viral infections, environmental agents, glucose, and expression of mutated proteins can alter the normal functioning of the ER resulting in ER stress. However, when ER stress is severe or chronic, injury to the cell can occur, causing cellular dysfunction resulting in inflammatory signaling, cell death, or phenotype transition. Pathogens are able to directly induce ER stress and cause the increased synthesis and release of TGF-β1. For example, in patients who experience

20


Page 21 of 38

21 chronic H1N1 infections approximately 7% develop diffuse alveolar damage and pulmonary fibrosis (13). Influenza virus induces ER stress and activates ATF6 and ER p57 but not GADD153 and this results in the upregulation of TGF-β1 synthesis in a JNK dependent manner (13). Further evidence to suggest that ER stress can play a role in fibrosis has been documented. Mu et al. (69), were able to demonstrate that ER stress can promote liver fibrosis by activating hepatic stellate cells. Using a methionine and choline-deficient diet which induces ER stress, significant levels of fat was found to accumulate in the livers of rats along with increased collagen deposition. ER stress markers were significantly elevated in this group. Hepatic stellate cells were found to play a crucial role in this fibrosis by transdifferentiating into myofibroblasts and increasing TIMP-1, α-smooth muscle actin, and collagens Furthermore, the ER stress and liver fibrosis could be resolved by feeding rats with a diet rich in methionine and choline. Hypoxia and reoxygenation mediates ROS and potassium efflux leading to inflammasome activation has been found to promote fibrosis in cardiac fibroblasts but not in cardiomyocytes (57). Because cardiac fibroblasts can proliferate in the heart and produce extracellular matrix proteins, cytokines and growth factors, this investigation further underscores the fact that these cells can act as sentinel cells able to sense danger signals such as ROS resulting from ischemia/reperfusion to enhance the inflammatory response in the heart inducing deposition of collagens (44). Our work had been focusing on the molecular signaling pathways that are responsible for maintaining fibrosis in SSc. The etiology of SSc is elusive and numerous chemicals and pathogens have been previously reported to be associated with this disease (6, 29, 30). One recently revived hypothesis is that ROS precedes fibrosis in SSc tissues further implicating the

21


Page 22 of 38

22 role for ROS in tissue damage and inflammasome activation. This hypothesis was originally proposed by Murrell in 1993 (71). Studies have confirmed the role of free radicals in SSc pathology on numerous levels suggesting that ROS is a significant player in disease. Maslen et al. (64), reported increased oxidative levels in SSc patient neutrophils and SSc patients have increased hydrogen peroxide production (84) in peripheral blood cells implicating the involvement of NOX2. In contrast, the activation of NOX4 and ROS production can take place when fibroblasts sense the extracellular matrix in the microenvironment; and depending on the signal, collagen synthesis can be induced or decreased (60, 86). Further confirming the role of ROS, it was found that SSc patients have increased levels of different free radical species and that these levels could be reduced with N-acetylcysteine (17).

Therapeutic potential It is now becoming quite apparent that ER stress and the UPR, mitochondrial production of ROS, Ca2+ signaling, NAD(P)H, and the NLRP3 inflammasome are linked and are at the heart of the pathology driving most, if not all, fibrotic diseases. The new knowledge gained from interrogating these signaling pathways has already shown great promise for the development of drugs that target key proteins driving fibrosis. Disrupting this signaling milieu has demonstrated some therapeutic value on several levels discussed herein. The compound Azelnipidine, which is a calcium blocker, has been shown to effectively abrogate liver fibrosis via a mechanism that is thought to increase antioxidant defense (75). Nifepidine down regulates NOX4 induced ROS, also shown to ameliorate fibrosis (38). Furthermore, we have found that our signaling peptide alters the ER stress response, and, as a result, decreases collagen synthesis (78). Finally, pirfenidone has been shown to inhibit NLRP3 activation in an ROS-dependent and -independent

22

Antioxidants & Redox Signaling Molecular Activation of the NLRP3 Inflammasome in Fibrosis: Common Threads Linking Divergent Fibrogenic Diseases (doi: 10.1089/ars.2014.6148) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 23 of 38

23

manner resulting in a decrease in collagen synthesis in a model of cardiac fibrosis (102).

Ultimately, this line of investigation will lead to more effective treatments that significantly

modify the progression of this devastating pathology in patients with fibrosis.

23


Page 24 of 38

24 Abbreviations: ASC: apoptosis speck-like protein containing a CARD ATF: activating transcription factor CARD: caspase recruitment domain Ca2+: calcium ER: endoplasmic reticulum GADD153: growth arrest and DNA damage gene 153 GRP78: glucose regulated protein 78 IL: interleukin IRE-1: inositol-requiring enzyme 1 NLRP: NOD like receptor protein NOX: Nicotinamide adenine dinucleotide phosphate/NAD(P)H-oxidase PERK: PKR-like ER kinase ROS: reactive oxygen species SSc: systemic sclerosis TGF-β1: transforming growth factor-β1 TRPC1: transient receptor protein channel-1 TXNIP: thioredoxin-interacting protein UPR: unfolded protein response XBP1: X-box binding protein-1

24


Page 25 of 38

25 References 1. Amara N, Goven D, Prost F, Muloway R, Crestani B, and Boczkowski J. NOX4/NADPH oxidase expression is increased in pulmonary fibroblasts from patients with idiopathic pulmonary fibrosis and mediates TGFbeta1-induced fibroblast differentiation into myofibroblasts. Thorax 65: 733-738, 2010. 2. Artlett CM, Sassi-Gaha S, Rieger JL, Boesteanu AC, Feghali-Bostwick CA, and Katsikis PD. The inflammasome activating caspase-1 mediates fibrosis and myofibroblast differentiation in systemic sclerosis. Arthritis Rheum 63: 3563-3574, 2011. 3. Barbiero G, Munaron L, Antoniotti S, Baccino FM, Bonelli G, and Lovisolo D. Role of mitogeninduced calcium influx in the control of the cell cycle in Balb-c 3T3 fibroblasts. Cell Calcium 18: 542-556, 1995. 4. Bell JK, Botos I, Hall PR, Askins J, Shiloach J, Segal DM, and Davies DR. The molecular structure of the Toll-like receptor 3 ligand-binding domain. Proc Natl Acad Sci, USA 102: 10976-10980, 2005. 5. Bella J, Hindle KL, McEwan PA, and Lovell SC. The leucine-rich repeat structure. Cell Mol Life Sci 65: 2307-2333, 2008. 6. Black CM, Welsh KI, Walker AE, Bernstein RM, Catoggio LJ, McGregor AR, and Lloyd Jones JK. Genetic susceptibility to scleroderma-like syndrome induced by vinyl chloride. Lancet 1: 53-55, 1983. 7. Brough D, Le Feuvre RA, Wheeler RD, Solovyova N, Hilfiker S, Rothwell NJ, and Verkhratsky A. Ca2+ stores and Ca2+ entry differentially contribute to the release of IL-1 beta and IL-1 alpha from murine macrophages. J Immunol 170: 3029-3036, 2003. 8. Cassel SL, Eisenbarth SC, Lyer SS, Sadler JJ, Colegio OR, Tephly LA, Carter AB, Rothman PB, Flavell RA, and Sutterwala FS. The NALP3 inflammasome is essential for the development of silicosis. Proc Natl Acad Sci, USA 105: 9035-9040, 2008. 9. Clapham DE. Calcium signaling. Cell 131: 1047-1058, 2007. 10. Couillin I, Vasseur V, Charron S, Gasse P, Tavernier M, Guillet J, Lagente V, Fick L, Jacobs M, Coelho FR, Moser R, and Ryffel B. IL-1R1/MyD88 signaling is critical for elastase-induced lung inflammation and emphysema. J Immunol 183: 8195-8202, 2009. 11. Cucoranu I, Clempus R, Dikalova A, Phelan PJ, Ariyan S, Dikalov S, and Sorescu D. NAD(P)H oxidase 4 mediates transforming growth factor-á1-induced differentiation of cardiac fibroblasts into myofibroblasts. Circ Res 97: 900-907, 2005. 12. Dai X, Sayama K, Tohyama M, Shirakata Y, Hanakawa Y, Tokumaru S, Yang L, Hirakawa S, and Hashimoto K. Mite allergen is a danger signal for the skin via activation of inflammasome in keratinocytes. J Allergy Clin Immunol 127: 806-814, 2011. 13. Dhainaut JF, Charpentier J, and Chiche JD. Transforming growth factor-beta: a mediator of cell regulation in acute respiratory distress syndrome. Critical Care M 31: S258-264, 2003. 14. Dinarello CA. Interleukin-1 beta, interleukin-18, and the interleukin-1 beta converting enzyme. Ann NY Acad Sci 856: 1-11, 1998. 15. Dinarello CA. Interleukin-18. Methods 19: 121-132, 1999. 16. Dostert C, Petrilli V, van Bruggen R, Steele C, Mossman BT, and Tschopp J. Innate immune activation through NALP3 inflammasome sensing of asbestos and silica. Science 320: 674-677, 2008. 17. Failli P, Palmieri L, D'Alfonso C, Giovannelli L, Generini S, Rosso AD, Pignone A, Stanflin N, Orsi S, Zilletti L, and Matucci-Cerinic M. Effect of N-acetyl-L-cysteine on peroxynitrite and superoxide anion production of lung alveolar macrophages in systemic sclerosis. Nitric Oxide 7: 277-282, 2002. 18. Feldmeyer L, Keller M, Niklaus G, Hohl D, Werner S, and Beer HD. The inflammasome mediates UVB-induced activation and secretion of interleukin-1beta by keratinocytes. Curr Biol 17: 1140-1145, 2007.

25


Page 26 of 38

26 19. Fernandez-Velasco M, Prieto P, Terron V, Benito G, Flores JM, Delgado C, Zaragoza C, Lavin B, Gomez-Parrizas M, Lopez-Collazo E, Martin-Sanz P, and Bosca L. NOD1 activation induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis. PLoS One 7: e45260, 2012. 20. Ferrari D, Pizzirani C, Adinolfi E, Lemoli RM, Curti A, Idzko M, Panther E, and Di VF. The P2X7 receptor: a key player in IL-1 processing and release. J Immunol 176: 3877-3883, 2006. 21. Fiaschi T, Magherini F, Gamberi T, Lucchese G, Faggian G, Modesti A, and Modesti PA. Hyperglycemia and angiotensin II cooperate to enhance collagen I deposition by cardiac fibroblasts through a ROS-STAT3-dependent mechanism. Biochim Biophys Acta 1843: 2603-2610, 2014. 22. Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev 88: 125-172, 2008. 23. Gadek-Michalska A and Bugajski J. Interleukin-1 (IL-1) in stress-induced activation of limbichypothalamic-pituitary adrenal axis. Pharmacol Rep : PR 62: 969-982, 2010. 24. Gasse P, Mary C, Guenon I, Noulin N, Charron S, Schnyder-Candrian S, Schnyder B, Akira S, Quesniaux VFJ, Lagente V, Ryffel B, and Couillin I. IL-1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice. J Clin Invest 117: 3786-3799, 2007. 25. Gasse P, Riteau N, Charron S, Girre S, Fick L, Petrilli V, Tschopp J, Lagente V, Quesniaux VF, Ryffel B, and Couillin I. Uric acid is a danger signal activating NALP3 inflammasome in lung injury inflammation and fibrosis. Am J Respir Crit Care Med 179: 903-913, 2009. 26. Gorin Y, Block K, Hernandez J, Bhandari B, Wagner B, Barnes JL, and Abboud HE. Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney. J Biol Chem 280: 39616-39626, 2005. 27. Goy C, Czypiorski P, Altschmied J, Jakob S, Rabanter LL, Brewer AC, Ale-Agha N, Dyballa-Rukes N, Shah AM, and Haendeler J. The imbalanced redox status in senescent endothelial cells is due to dysregulated Thioredoxin-1 and NADPH oxidase 4. Exp Gerontol, 2014. 28. Greenhalgh SN, Iredale JP, and Henderson NC. Origins of fibrosis: pericytes take centre stage. F1000 Prime Rep 5: 37, 2013. 29. Haustein UF and Herrmann K. Environmental scleroderma. Clin Dermatol 12: 467-473, 1994. 30. Haustein UF, Ziegler V, and Herrmann K. Chemically-induced scleroderma. Hautarzt 43: 469474, 1992. 31. Hecker L, Vittal R, Jones T, Jagirdar R, Luckhardt TR, Horowitz JC, Pennathur S, Martinez FJ, and Thannickal VJ. NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury. Nat Med 15: 1077-1081, 2009. 32. Heid ME, Keyel PA, Kamga C, Shiva S, Watkins SC, and Salter RD. Mitochondrial reactive oxygen species induces NLRP3-dependent lysosomal damage and inflammasome activation. J Immunol 191: 5230-5238, 2013. 33. Hindle KL, Bella J, and Lovell SC. Quantitative analysis and prediction of curvature in leucine-rich repeat proteins. Proteins 77: 342-358, 2009. 34. Horowitz JC, Rogers DS, Sharma V, Vittal R, White ES, Cui Z, and Thannickal VJ. Combinatorial activation of FAK and AKT by transforming growth factor-beta1 confers an anoikis-resistant phenotype to myofibroblasts. Cell Signal 19: 761-771, 2007. 35. Inohara N and Nunez G. The NOD: a signaling module that regulates apoptosis and host defense against pathogens. Oncogene 20: 6473-6481, 2001. 36. Inohara N and Nunez G. NODS: intracellualr proteins involved in inflammation and apoptosis. Nat Rev 3: 371-382, 2003. 37. Janssen LJ, Farkas L, Rahman T, and Kolb MR. ATP stimulates Ca(2+)-waves and gene expression in cultured human pulmonary fibroblasts. Int J Biochem Cell Biol 41: 2477-2484, 2009.

26


Page 27 of 38

27 38. Jia Y, Xu J, Yu Y, Guo J, Liu P, Chen S, and Jiang J. Nifedipine inhibits angiotensin II-induced cardiac fibrosis via downregulating Nox4-derived ROS generation and suppressing ERK1/2, JNK signaling pathways. Die Pharmazie 68: 435-441, 2013. 39. Jiang F, Liu GS, Dusting GJ, and Chan EC. NADPH oxidase-dependent redox signaling in TGFbeta-mediated fibrotic responses. Redox Biol 2: 267-272, 2014. 40. Jiang JX, Chen X, Serizawa N, Szyndralewiez C, Page P, Schroder K, Brandes RP, Devaraj S, and Torok NJ. Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831, a novel NOX4/NOX1 inhibitor in vivo. Free Rad Biol Med 53: 289-296, 2012. 41. Kanneganti TD, Lamkanfi M, and Nunez G. Intracellular NOD-like receptors in host defense and disease. Immunity 27: 549-559, 2007. 42. Kao JP, Alderton JM, Tsien RY, and Steinhardt RA. Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts. J Cell Biol 111: 183-196, 1990. 43. Kaur G, Sharma A, Guruprasad K, and Pati PK. Versatile roles of plant NADPH oxidases and emerging concepts. Biotechnol Adv, 32: 551-563, 2014. 44. Kawaguchi M, Takahashi M, Hata T, Kashima Y, Usui F, Morimoto H, Izawa A, Takahashi Y, Masumoto J, Koyama J, Hongo M, Noda T, Nakayama J, Sagara J, Taniguchi S, and Ikeda U. Inflammasome activation of cardiac fibroblasts is essential for myocardial ischemia/reperfusion injury. Circulation 123: 594-604, 2011. 45. Keller M, Ruegg A, Werner S, and Beer HD. Active caspase-1 is a regulator of unconventional protein secretion. Cell 132: 818-831, 2008. 46. Kisseleva T and Brenner DA. The phenotypic fate and functional role for bone marrow-derived stem cells in liver fibrosis. Journal of hepatology 56: 965-972, 2012. 47. Kojima N, Hori M, Murata T, Morizane Y, and Ozaki H. Different profiles of Ca2+ responses to endothelin-1 and PDGF in liver myofibroblasts during the process of cell differentiation. Br J Pharmacol 151: 816-827, 2007. 48. Kolb M, Margetts PJ, Anthony DC, Pitossi F, and Gauldie J. Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest 107: 1529-1536, 2001. 49. Krebs J, Groenendyk J, and Michalak M. Ca2+-signaling, alternative splicing and endoplasmic reticulum stress responses. Neurochem Res 36: 1198-1211, 2011. 50. Lalor SJ, Dungan LS, Sutton CE, Basdeo SA, Fletcher JM, and Mills KH. Caspase-1-processed cytokines IL-1beta and IL-18 promote IL-17 production by gammadelta and CD4 T cells that mediate autoimmunity. J Immunol 186: 5738-5748, 2011. 51. LeBleu VS, Taduri G, O'Connell J, Teng Y, Cooke VG, Woda C, Sugimoto H, and Kalluri R. Origin and function of myofibroblasts in kidney fibrosis. Nat Med 19: 1047-1053, 2013. 52. Lee GS, Subramanian N, Kim AI, Aksentijevich I, Goldbach-Mansky R, Sacks DB, Germain RN, Kastner DL, and Chae JJ. The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP. Nature 492: 123-127, 2012. 53. Lekkerkerker AN, Aarbiou J, van Es T, and Janssen RA. Cellular players in lung fibrosis. Curr Pharm Des 18: 4093-4102, 2012. 54. Lemasters JJ, Theruvath TP, Zhong Z, and Nieminen AL. Mitochondrial calcium and the permeability transition in cell death. Biochim Biophys Acta 1787: 1395-1401, 2009. 55. Li C and Kuemmerle JF. Mechanisms that mediate the development of fibrosis in patients with Crohn's disease. Inflamm Bowel Dis 20: 1250-1258, 2014. 56. Li F, Thiele I, Jamshidi N, and Palsson BO. Identification of potential pathway mediation targets in Toll-like receptor signaling. PLoS Comp Biol 5: e1000292, 2009. 57. Liaudet L and Rosenblatt-Velin N. Role of innate immunity in cardiac inflammation after myocardial infarction. Front Biosci 5: 86-104, 2013. 27


Page 28 of 38

28 58. Liu W, Yin Y, Zhou Z, He M, and Dai Y. OxLDL-induced IL-1 beta secretion promoting foam cells formation was mainly via CD36 mediated ROS production leading to NLRP3 inflammasome activation. Inflamm Res 63: 33-43, 2014. 59. Loughlin DT and Artlett CM. 3-Deoxyglucosone-Collagen Alters Human Dermal Fibroblast Migration and Adhesion: Implications for Impaired Wound Healing in Patients with Diabetes. Wound Repair Regen 17: 739-749, 2009. 60. Loughlin DT and Artlett CM. Precursor of Advanced Glycation End Products Mediates ER-stressInduced Caspase-3 Activation of Human Dermal Fibroblasts through NAD(P)H Oxidase 4. Plos One 5: e11093, 2010. 61. Lua I, James D, Wang J, Wang KS, and Asahina K. Mesodermal mesenchymal cells give rise to myofibroblasts, but not epithelial cells, in mouse liver injury. Hepatology, 2014. 62. Marchi S, Patergnani S, and Pinton P. The endoplasmic reticulum-mitochondria connection: one touch, multiple functions. Biochim Biophys Acta 1837: 461-469, 2014. 63. Martinon F and Tschopp J. NLRs join TLRs as innate sensors of pathogens. Trends Immunol 26: 447-454, 2005. 64. Maslen CL, Hall ND, Woolf AD, and Maddison PJ. Enhanced oxidative metabolism of neutrophils from patients with systemic sclerosis. Br J Rheumatol 26: 113-117, 1987. 65. Masters SL, Dunne A, Subramanian SL, Hull RL, Tannahill GM, Sharp FA, Becker C, Franchi L, Yoshihara E, Chen Z, Mullooly N, Mielke LA, Harris J, Coll RC, Mills KH, Mok KH, Newsholme P, Nunez G, Yodoi J, Kahn SE, Lavelle EC, and O'Neill LA. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1beta in type 2 diabetes. Nat Immunol 11: 897-904, 2010. 66. Matsuo J, Tsukumo Y, Sakurai J, Tsukahara S, Park HR, Shin-ya K, Watanabe T, Tsuruo T, and Tomida A. Preventing the unfolded protein response via aberrant activation of 4E-binding protein 1 by versipelostatin. Cancer Sci 100: 327-333, 2009. 67. Menu P, Mayor A, Zhou R, Tardivel A, Ichijo H, Mori K, and Tschopp J. ER stress activates the NLRP3 inflammasome via an UPR-independent pathway. Cell Death Dis 3: e261, 2012. 68. Montorfano I, Becerra A, Cerro R, Echeverria C, Saez E, Morales MG, Fernandez R, CabelloVerrugio C, and Simon F. Oxidative stress mediates the conversion of endothelial cells into myofibroblasts via a TGF-beta1 and TGF-beta2-dependent pathway. Lab Invest, 2014. 69. Mu YP, Ogawa T, and Kawada N. Reversibility of fibrosis, inflammation, and endoplasmic reticulum stress in the liver of rats fed a methionine-choline-deficient diet. Lab Invest 90: 245-256, 2010. 70. Murakami T, Ockinger J, Yu J, Byles V, McColl A, Hofer AM, and Horng T. Critical role for calcium mobilization in activation of the NLRP3 inflammasome. Proc Natl Acad Sci, USA 109: 1128211287, 2012. 71. Murrell DF. A radical proposal for the pathogenesis of scleroderma. J Am Acad Dermatol 28: 7885, 1993. 72. Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, Englert JA, Rabinovitch M, Cernadas M, Kim HP, Fitzgerald KA, Ryter SW, and Choi AM. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol 12: 222-230, 2011. 73. Ngo MA, Muller A, Li Y, Neumann S, Tian G, Dixon IM, Arora RC, and Freed DH. Human mesenchymal stem cells express a myofibroblastic phenotype in vitro: comparison to human cardiac myofibroblasts. Molec Cell Biochem, 2014. 74. Nishitoh H. CHOP is a multifunctional transcription factor in the ER stress response. J Biochem 151: 217-219, 2012.

28


Page 29 of 38

29 75. Ohyama T, Sato K, Kishimoto K, Yamazaki Y, Horiguchi N, Ichikawa T, Kakizaki S, Takagi H, Izumi T, and Mori M. Azelnidipine is a calcium blocker that attenuates liver fibrosis and may increase antioxidant defence. Br J Pharmacol 165: 1173-1187, 2012. 76. Oslowski CM, Hara T, O'Sullivan-Murphy B, Kanekura K, Lu S, Hara M, Ishigaki S, Zhu LJ, Hayashi E, Hui ST, Greiner D, Kaufman RJ, Bortell R, and Urano F. Thioredoxin-Interacting Protein Mediates ER Stress-Induced beta Cell Death through Initiation of the Inflammasome. Cell Metab 16: 265273, 2012. 77. Osuka A, Hanschen M, Stoecklein V, and Lederer JA. A protective role for inflammasome activation following injury. Shock 37: 47-55, 2012. 78. Overley-Adamson B, Artlett CM, Stephens C, Sassi-Gaha S, Weis RD, and Thacker JD. Targeting the unfolded protein response, XBP1, and the NLRP3 inflammasome in fibrosis and cancer. Cancer Biol Ther 15, 2014. 79. Pan SY, Chang YT, and Lin SL. Microvascular pericytes in healthy and diseased kidneys. Int J Nephrol Renovas Dis 7: 39-48, 2014. 80. Parajuli B, Sonobe Y, Horiuchi H, Takeuchi H, Mizuno T, and Suzumura A. Oligomeric amyloid beta induces IL-1beta processing via production of ROS: implication in Alzheimer's disease. Cell Death Dis 4: e975, 2013. 81. Phan SH, Zhang K, Zhang HY, and Gharaee-Kermani M. The myofibroblast as an inflammatory cell in pulmonary fibrosis. Curr Top Pathol 93: 173-182, 1999. 82. Platis A, Yu Q, Moore D, Khojeini E, Tsau P, and Larson D. The effect of daily administration of IL-18 on cardiac structure and function. Perfusion 23: 237-242, 2008. 83. Ramm GA, Carr SC, Bridle KR, Li L, Britton RS, Crawford DH, Vogler CA, Bacon BR, and Tracy TF. Morphology of liver repair following cholestatic liver injury: resolution of ductal hyperplasia, matrix deposition and regression of myofibroblasts. Liver 20: 387-396, 2000. 84. Sambo P, Baroni SS, Luchetti M, Paroncini P, Dusi S, Orlandini G, and Gabrielli A. Oxidative stress in scleroderma: maintenance of scleroderma fibroblast phenotype by the constitutive upregulation of reactive oxygen species generation through the NADPH oxidase complex pathway. Arthritis Rheum 44: 2653-2664, 2001. 85. Sampson N, Berger P, and Zenzmaier C. Redox Signaling as a Therapeutic Target to Inhibit Myofibroblast Activation in Degenerative Fibrotic Disease. BioMed Res Int 2014: 131737, 2014. 86. Sassi-Gaha S, Loughlin DT, Kappler F, Schwartz ML, Su B, Tobia AM, and Artlett CM. Two Dicarbonyl compounds, 3-deoxyglucosone and methylglyoxal, differentially modulate dermal fibroblasts. Matrix Biol 29: 127-134, 2010. 87. Sciarretta S, Volpe M, and Sadoshima J. NOX4 regulates autophagy during energy deprivation. Autophagy 10: 699-701, 2014. 88. Shah A, Xia L, Goldberg H, Lee KW, Quaggin SE, and Fantus IG. Thioredoxin-interacting protein mediates high glucose-induced reactive oxygen species generation by mitochondria and the NADPH oxidase, Nox4, in mesangial cells. J Biol Chem 288: 6835-6848, 2013. 89. Shalhoub J, Falck-Hansen MA, Davies AH, and Monaco C. Innate immunity and monocytemacrophage activation in atherosclerosis. J Inflamm 8: 9, 2011. 90. Smith RS, Smith TJ, Blieden TM, and Phipps RP. Fibroblasts as sentinel cells. Synthesis of chemokines and regulation of inflammation. Am J Pathol 151: 317-322, 1997. 91. Stefanovic B, Stefanovic L, Schnabl B, Bataller R, and Brenner DA. TRAM2 protein interacts with endoplasmic reticulum Ca2+ pump Serca2b and is necessary for collagen type I synthesis. Mol Cell Biol 24: 1758-1768, 2004. 92. Sugawara S, Uehara A, Nochi T, Yamaguchi T, Ueda H, Sugiyama A, Hanzawa K, Kumagai K, Okamura H, and Takada H. Neutrophil proteinase 3-mediated induction of bioactive IL-18 secretion by human oral epithelial cells. J Immunol 167: 6568-6575, 2001. 29


Page 30 of 38

30 93. Teshima Y, Takahashi N, Nishio S, Saito S, Kondo H, Fukui A, Aoki K, Yufu K, Nakagawa M, and Saikawa T. Production of reactive oxygen species in the diabetic heart. Roles of mitochondria and NADPH oxidase. Circ J 78: 300-306, 2014. 94. Thacker JD, Balin BJ, Appelt DM, Sassi-Gaha S, Purohit M, Rest RF, and Artlett CM. NLRP3 inflammasome is a target for development of broad-spectrum anti-infective drugs. Antimicrobial Agents Chemother 56: 1921-1930, 2012. 95. Thacker JD, Brown MA, Rest RF, Purohit M, Sassi-Gaha S, and Artlett CM. 1-peptidyl-2arachidonoyl-3-stearoyl-sn-glyceride: an immunologically active lipopeptide from goat serum (Capra hircus) is an endogenous damage associated molecular pattern. J Nat Prod 72: 1993-1999, 2009. 96. Thannickal VJ, Lee DY, White ES, Cui Z, Larios JM, Chacon R, Horowitz JC, Day RM, and Thomas PE. Myofibroblast differentiation by transforming growth factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase. J Biol Chem 278: 12384-12389, 2003. 97. Thomay AA, Daley JM, Sabo E, Worth PJ, Shelton LJ, Harty MW, Reichner JS, and Albina JE. Disruption of interleukin-1 signaling improves the quality of wound healing. Am J Pathol 174: 2129-2136, 2009. 98. van Bruggen R, Koker MY, Jansen M, van Houdt M, Roos D, Kuijpers TW, and van den Berg TK. Human NLRP3 inflammasome activation is Nox1-4 independent. Blood 115: 5398-5400, 2010. 99. Van De Water L, Varney S, and Tomasek JJ. Mechanoregulation of the Myofibroblast in Wound Contraction, Scarring, and Fibrosis: Opportunities for New Therapeutic Intervention. Adv Wound Care 2: 122-141, 2013. 100. Vyas-Read S, Wang W, Kato S, Colvocoresses-Dodds J, Fifadara NH, Gauthier TW, Helms MN, Carlton DP, and Brown LA. Hyperoxia induces alveolar epithelial-to-mesenchymal cell transition. Am J Physiol Lung Cell Molec Physiol 306: L326-340, 2014. 101. Wang L, Hartmann P, Haimerl M, Bathena SP, Sjowall C, Almer S, Alnouti Y, Hofmann AF, and Schnabl B. Nod2 deficiency protects mice from cholestatic liver disease by increasing renal excretion of bile acids. J Hepatol 60: 1259-1267, 2014. 102. Wang Y, Wu Y, Chen J, Zhao S, and Li H. Pirfenidone attenuates cardiac fibrosis in a mouse model of TAC-induced left ventricular remodeling by suppressing NLRP3 inflammasome formation. Cardiology 126: 1-11, 2013. 103. Watanabe A, Sohail MA, Gomes DA, Hashmi A, Nagata J, Sutterwala FS, Mahmood S, Jhandier MN, Shi Y, Flavell RA, and Mehal WZ. Inflammasome-mediated regulation of hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol 296: G1248-G1257, 2009. 104. West AP, Shadel GS, and Ghosh S. Mitochondria in innate immune responses. Nat Rev Immunol 11: 389-402, 2011. 105. Wilson KP, Black JA, Thomson JA, Kim EE, Griffith JP, Navia MA, Murcko MA, Chambers SP, Aldape RA, and Raybuck SA. Structure and mechanism of interleukin-1 beta converting enzyme. Nature 370: 270-275, 1994. 106. Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol 214: 199-210, 2008. 107. Wynn TA. Integrating mechanisms of pulmonary fibrosis. J Exp Med 208: 1339-1350, 2011. 108. Xing SS, Tan HW, Bi XP, Zhong M, Zhang Y, and Zhang W. Felodipine reduces cardiac expression of IL-18 and perivascular fibrosis in fructose-fed rats. Mol Med 14: 395-402, 2008. 109. Yang HC, Cheng ML, Ho HY, and Chiu DT. The microbicidal and cytoregulatory roles of NADPH oxidases. Microbes Infect 13: 109-120, 2011. 110. Yin Y, Yan Y, Jiang X, Mai J, Chen NC, Wang H, and Yang XF. Inflammasomes are differentially expressed in cardiovascular and other tissues. Int J Immunopathol Pharmacol 22: 311-322, 2009. 111. Yoshida H, Matsui T, Yamamoto A, Okada T, and Mori K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107: 881-891, 2001. 30


Page 31 of 38

31 112. Yoshida H, Oku M, Suzuki M, and Mori K. pXBP1(U) encoded in XBP1 pre-mRNA negatively regulates unfolded protein response activator pXBP1(S) in mammalian ER stress response. J Cell Biol 172: 565-575, 2006. 113. Yu Q, Vazquez R, Khojeini EV, Patel C, Venkataramani R, and Larson DF. IL-18 induction of osteopontin mediates cardiac fibrosis and diastolic dysfunction in mice. Am J Physiol Heart Circ Physiol 297: H76-H85, 2009. 114. Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, Brohi K, Itagaki K, and Hauser CJ. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 464: 104-107, 2010. 115. Zhang Y, Yang J, Jiang S, Fang C, Xiong L, Cheng H, and Xia Y. The lupus-derived anti-doublestranded DNA IgG contributes to myofibroblast-like phenotype in mesangial cells. J Clin Immunol 32: 1270-1278, 2012. 116. Zhong Z, Zhai Y, Liang S, Mori Y, Han R, Sutterwala FS, and Qiao L. TRPM2 links oxidative stress to NLRP3 inflammasome activation. Nat Commun 4: 1611, 2013. 117. Zhou CF, Zhou DC, Zhang JX, Wang F, Cha WS, Wu CH, and Zhu QX. Bleomycin-induced epithelial-mesenchymal transition in sclerotic skin of mice: Possible role of oxidative stress in the pathogenesis. Toxicol Appl Pharmacol, 2014. 118. Zhou R, Tardivel A, Thorens B, Choi I, and Tschopp J. Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat Immunol 11: 136-140, 2010. 119. Zhou R, Yazdi AS, Menu P, and Tschopp J. A role for mitochondria in NLRP3 inflammasome activation. Nature 469: 221-225, 2011. 120. Zong J, Salim M, Zhou H, Bian ZY, Dai J, Yuan Y, Deng W, Zhang JY, Zhang R, Wu QQ, and Tang QZ. NOD2 deletion promotes cardiac hypertrophy and fibrosis induced by pressure overload. Lab Invest 93: 1128-1136, 2013.

31


Page 32 of 38

32

32


Page 33 of 38

33

Figure 1: Assembly of the inflammasome activates caspase-1 leading to processing of IL-1β and IL-18. The inflammasome can be activated by damage associated molecular patterns (DAMPs), pathogen associated molecular patterns (PAMPs) and reactive oxygen species (ROS). It assembles into a large complex leading to the autocatalytic cleavage of pro-caspase-1 to active caspase-1. Active caspase-1 is then able to cleave a wide variety of precursors including IL-1β and IL-18.

33


Page 34 of 38

34

Figure 2. Activation of the inflammasome can result in a feed forward mechanism in fibroblasts that does not resolve mediating the differentiation to myofibroblasts. Assembly of the inflammasome in fibroblasts can result in the normal processing and secretion of IL-1β and IL-18 resulting in the upregulation of transcription factors leading to increased expression of IL-1β and IL-8, increased TGF-β1 and collagen synthesis causing myofibroblast differentiation. Autocrine signaling by TGF-β1 can produce reactive oxygen species (ROS) providing a positive feedback mechanism than further promotes inflammasome activation and IL-1β and IL-18 activation, TGF-β1 and collagen synthesis.

34


Page 35 of 38

35

Figure 3. Three arms of the ER stress response. ATF6, PERK, and IRE1 sense the proteinfolding conditions in the ER lumen. (Left Panel) ATF6/BiP migrates to the Golgi where BiP is cleaved releasing ATF6 to translocates to the nucleus and induces UPR associated target gene transcription. (Middle Panel) Unfolded proteins induce PERK dimerization and phosphorylation of eIF2a and ATF4 translocation to the nucleus to induce transcription of apoptosis and autophagy associated genes as well as GADD34 and REDOX enzymes. (Right Panel) Unfolded proteins induce IRE1 dimerization and the subsequent splicing of XBP1 mRNA. XBP1s translocates to the nucleus to up-regulate chaperone proteins, lipid biosynthesis, and ER associated degradation proteins.

35


Page 36 of 38

36

Figure 4. Perturbation in calcium signaling is central to reactive oxygen species induction of inflammasome activation. Influx of calcium (Ca2+) due to cellular damage or other mechanisms results in the increase of reactive oxygen species produced by the endoplasmic reticulum and mitochondria. Thioredoxin-interacting protein (TXNIP) is upregulated by the increased levels of ROS and as a result induces the expression of NAD(P)H oxidase 4 (NOX4). TXNIP is also recruited into the inflammasome complex. The increase in NOX4 levels promotes mitochondrial ROS further enhancing the cytosolic ROS pool that leads to inflammasome activation.

36


Page 37 of 38

37

Figure 5. Resolution of chronic endoplasmic reticulum stress (ER) stress by acALY18. During ER stress, acALY18 is cleaved from transient receptor protein channel-1 in the ER in a rate limiting manner. acALY18 binds to XBP1 to regulate the unfolded protein response, however under chronic ER stress it becomes quickly exhausted and inflammation and fibrosis ensue (left panel). However, the endogenous addition of synthetic acALY18 binds XBP1s to regulate this transcription factor and this mediates in resolution of ER stress, inflammation, and fibrosis.

37


Page 38 of 38

38

Figure 6. Two arms of the XBP1 mediated ER stress response. In resting cells XBP1u is the dominant isoform. Exogenously added acALY18 induces IL-1b secretion and a down-stream innate immune response that is NLRP3 dependent (left). In the IRE1 mediated ER stress response, TRPC1 derived acALY18 engages XBP1s to down regulate the NLRP3 inflammasome and translates to the nucleus to up-regulate ERAD and UPR associated genes.

38

Molecular mechanisms regulating NLRP3 inflammasome activation.

Mechanism of NLRP3 inflammasome activation.

Vimentin regulates activation of the NLRP3 inflammasome.

Methylsulfonylmethane inhibits NLRP3 inflammasome activation.

The role of the NLRP3 inflammasome in pulmonary diseases.

Dimethyl sulfoxide inhibits NLRP3 inflammasome activation.

Linking cancer-induced Nlrp3 inflammasome activation to efficient NK cell-mediated immunosurveillance.

Defective mitochondrial fission augments NLRP3 inflammasome activation.

NLRP3 inflammasome activation by Paracoccidioides brasiliensis.

Activation of the NLRP3 inflammasome in Porphyromonas gingivalis-accelerated atherosclerosis.

Activation of the NLRP3 inflammasome by cellular labile iron.

Age-Dependent Susceptibility to Pulmonary Fibrosis Is Associated with NLRP3 Inflammasome Activation.

Posttranscriptional control of NLRP3 inflammasome activation in colonic macrophages.

TRPM2 links oxidative stress to the NLRP3 inflammasome activation (P1268).

NLRP3 inflammasome: activation and regulation in age-related macular degeneration.

NLRP3 inflammasome activation in dialyzed chronic kidney disease patients.

NLRP3 Inflammasome Activation by Viroporins of Animal Viruses.

Characterization of porcine NLRP3 inflammasome activation and its upstream mechanism.

3,4-methylenedioxy-β-nitrostyrene inhibits NLRP3 inflammasome activation by blocking assembly of the inflammasome.

Mitophagy: a balance regulator of NLRP3 inflammasome activation.

Celastrol ameliorates inflammation through inhibition of NLRP3 inflammasome activation.

NLRP3 Inflammasome as a Molecular Marker in Diabetic Cardiomyopathy.

Cutting edge: SHARPIN is required for optimal NLRP3 inflammasome activation.

Uropathogenic Escherichia coli strain CFT073 disrupts NLRP3 inflammasome activation.