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´ Jos´e M. Gonzalez-Navajas et al.

DOI: 10.1002/eji.201344291

Eur. J. Immunol. 2014. 44: 2536–2549

Review

The immediate protective response to microbial challenge Jos´e M. Gonz´ alez-Navajas1,2 , Mary P. Corr2 and Eyal Raz2 1

Networked Biomedical Research Center for Hepatic and Digestive Diseases (CIBERehd), Hospital General de Alicante, Alicante, Spain 2 Division of Rheumatology, Allergy and Immunology, Department of Medicine, University of California San Diego, La Jolla, CA, USA The innate immune system detects infection and tissue injury through different families of pattern-recognition receptors (PRRs), such as Toll-like receptors. Most PRR-mediated responses initiate elaborate processes of signaling, transcription, translation, and secretion of effector mediators, which together require time to achieve. Therefore, PRRmediated processes are not active in the early phases of infection. These considerations raise the question of how the host limits microbial replication and invasion during this critical period. Here, we examine the crucial defense mechanisms, such as antimicrobial peptides or extracellular traps, typically activated within minutes of the initial infection phase, which we term the “immediate protective response”. Deficiencies in different components of the immediate protective response are often associated with severe and recurrent infectious diseases in humans, highlighting their physiologic importance.

Keywords: Antimicrobial response r Infection

r

Innate immunity

Introduction Infection activates the innate immune response through different families of germline-encoded pattern-recognition receptors (PRRs), among which the Toll-like receptors (TLRs) are the most studied. These PRRs are expressed on the cell membranes or in the cytosol of immune and other cells and respond to microbial signature compounds, known as pathogen-associated molecular patterns (PAMPs). As a result, an acute inflammatory/defense response is initiated that is able to target a wide range of microbial agents and/or infected cells. Interestingly, tissue injury caused by sterile insults, such as trauma or hypoxia, activates a similar early inflammatory response. One potential evolutionary explanation for these similarities is that tissue injury usually results in the disruption of physiological barriers followed by microbial invasion. Another possibility is that microbes can also cause cell necrosis, so responding to tissue injury may be an indirect way of detecting microbial invasion. Thus, the mammalian host has developed a system

´ Correspondence: Dr. Jose´ M. Gonzalez-Navajas e-mail: [email protected]

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of endogenous warning signals that are constitutively available and are rapidly activated in the very early phase of both sterile and nonsterile inflammation. These warning signals are provoked by alarmins or danger-associated molecular patterns. Alarmins are multifunctional endogenous molecules of structural heterogeneity, such as ATP, high-mobility group protein B1, or heat shock proteins, that are passively released following nonapoptotic cell death or actively secreted by leukocytes and epithelial cells [1]. As potent inducers of inflammatory responses, alarmins contribute to the defense against pathogens and promote tissue repair, but excessive alarmin secretion is also associated with acute and chronic inflammatory conditions [2]. Stimulation of immune cells by endogenous alarmins or microbial derived PAMPs evokes similar signaling cascades and activates similar transcription factors [3] that consequently result in the production of protective effector molecules. These molecules include chemokines that recruit leukocytes (e.g., IL-8) to the site of injury or infection [4], cytokines that amplify the inflammatory response (e.g., tumor necrosis factor [TNF-α]) or activate the antiviral pathways (e.g., type 1 interferon) [5], and the activation of killing mechanisms (e.g., reactive oxygen species [ROS]) [6] leading to pathogen clearance. However, de novo production of microbial triggered PRR effector molecules requires the appropriate genes www.eji-journal.eu

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Immediate Protective Response (IPR)

Classical Innate Immunity

Phase 1

Phase 2

- AMPs - Complement - Perforin - Opsonins and crossreacting antibodies

- Phagocytosis - Degranulation - NETs - ROS/RNS production - Processing of procytokines

Constitutive

Post-translational

0 min

De novo cytokine and chemokine production

Adaptive Immunity

Activation of immune cells by cytokines and chemokines

Transcriptional/Translational

60-120 min

Antigen-specific Tand B-cell responses 24 h

days/weeks

Time (postpathogen exposure) Figure 1. How the IPR fits into the classical views of innate and adaptive immunity. Several mechanisms are activated sequentially during the various stages of the immune response to infection. During the first phase of the IPR, effector molecules that are constitutively expressed (e.g., AMPs or complement) have the ability to kill or neutralize microbes in a matter of seconds or minutes after pathogen encounter. Simultaneously, innate immune cells activate posttranslational mechanisms (e.g., degranulation) that result in the rapid release of more AMPs and other effector molecules (e.g., mature cytokines) or structures (e.g., NETs) that further increase the protective repertoire in minutes. These mechanisms restrict the infectious agent and protect the host until the transcriptional activation of PRRs leads to de novo production of cytokines (hours) and the awakening of the adaptive immune response (days to few weeks). AMPs: antimicrobial peptides; NETs: neutrophil extracellular traps; RNS: reactive nitrogen species.

to be transcribed and translated, and the gene product secreted, processes that require time following the initial encounter with infectious agents. How, then, are pathogen invasion and replication limited during the early phases of infection?

ronments. These and other considerations are discussed in more detail below.

The first phase of the IPR: Action of preformed extracellular effectors

What are the major mechanisms of the IPR? AMPs When a pathogen breaches the host’s anatomic barriers, several evolutionarily conserved mechanisms of the immediate protective response (IPR) are activated, as our term implies, within minutes of pathogen exposure (Fig. 1). First, the pathogen encounters preformed structures or molecules in blood, extracellular fluids, and mucosal secretions. These effectors include soluble molecules, such as antimicrobial peptides (AMPs), broadly neutralizing antibodies, and plasma proteins of the complement system (Table 1). These molecules have pleiotropic functions that contribute to both pathogen elimination and the regulation of physiological processes, such as inflammation, angiogenesis, and wound healing. In a second phase of the IPR, cells of the innate immune system activate certain posttranslational mechanisms, including phagocytosis and intracellular killing by ROS, formation of extracellular traps, degranulation of preformed effectors, or processing of proproteins into mature effectors. It should be noted, however, that the activation of the IPR is not totally separated from PRRs, as several of these responses can be also induced by PRRs, such as extracellular trap formation [7]. It should also be mentioned that in some instances, such as in the gut environment, the microbiota are continuously stimulating PRRs expressed on immune and epithelial cells, facilitating, under certain conditions, a faster PRR-mediated response than that occurring in sterile envi C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The mammalian immune system has evolved several families of peptides with rapid and direct microbicidal activities against bacteria, viruses, and fungi. The first AMP was accidentally discovered by Alexander Fleming [8] and was termed lysozyme because of its ability to lyse bacteria. Lysozyme is readily available in secretions, such as saliva and tears, is secreted by Paneth cells at the bottom of the crypts of Lieberk¨ uhn in the intestine, and is also found in cytoplasmic granules in neutrophils. It selectively targets the peptidoglycan component of bacterial cell walls, and therefore is more effective against Gram-positive bacteria [9, 10]. The most abundant human AMPs are the defensins [11], which are small cationic amphipathic peptides with a predominantly β-sheet structure stabilized by three disulfide bonds. They can be further categorized into α-, β-, and θ-defensins. Humans express six α-defensins and up to 31 β-defensins, but lack θ-defensins [12]. In neutrophils, α-defensins are constitutively stored as active peptides in the azurophilic granules where they can be released by degranulation or used as a part of the intracellular killing mechanism of these cells. Intestinal Paneth cells, activated by commensal bacteria, continuously secrete inactive precursors of α-defensins into the lumen where they are cleaved into the active form by trypsin in humans [13] or matrilysin (MMP7) in mice [14]. www.eji-journal.eu

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Epithelial cells, PMNCs

Neutrophils, macrophages, epithelial cells IECs Paneth cells

Mucosal plasma cells

Liver, endothelial cells, epithelial cells, macrophages, others

PMNCs

Phagocytic cells

Innate immune cells

Defensins

Cathelicidins (LL-37)

Neutralizing antibodies

Complement proteins

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Extracellular traps

ROS

Preformed cytokines

Host immune cells (indirect antibacterial actions)

Inflammation. Degranulation of mast cells

Recruitment of phagocytes Inflammation, enhance adaptive immunity

Oxidative damage Induction of bactericidal enzymes

Trapping and exposing microbes to antimicrobial agents

Opsonization, Direct binding to bacterial membranes and formation of MAC

Trap antigens in the mucus layer Reduce motility of bacteria Opsonization

Attachment and disruption of membranes

Attachment and disruption of membranes

Attachment and insertion of hydrophobic regions causing membrane disruption

Binding to peptidoglycans and membrane disruption

Antimicrobial actions

Activation of GPCRs and ion channels (TRPs, ASIC)

(i) Processing of procytokines (IL-1β/IL-18) (ii) Degranulation by PMNCs (TNF-α, IL-17A)

Activation of NADPH oxidase upon phagocytosis

(i) Released after cell death (NETosis) (ii) Recognition of complement receptors and ERK phosphorylation

Extracellular cleavage of inactive peptides

Active secretion at mucosal surfaces

Extracellular cleavage of inactive precursor

Extracellular cleavage of inactive precursor

(i) Extracellular cleavage of precursor (ii) Degranulation of active peptide (iii) Secretion of active peptide by epithelial cells (β-defensin)

Constitutive release as active peptide

Regulation

ASIC: acid-sensing ion channels; GPCR: G protein coupled receptor; IECs: intestinal epithelial cells; MAC: membrane attack complex; NET: neutrophil extracellular trap; PMNCs: polymorphonuclear cells; ROS: reactive nitrogen species; TRP: transient receptor potential channel.

Neuropeptides

Host immune cells (indirect antibacterial actions)

Bacteria, viruses, parasites

Extracellular pathogens

Fungi, Gram-positive and Gram-negative bacteria

Virus, bacteria, toxins

Gram-positive and Gram-negative bacteria

Fungi, viruses, bacteria

Fungi, viruses, bacteria

Gram-positive bacteria

Target

´ Jos´e M. Gonzalez-Navajas et al.

Sensory neurons

Epithelial cells, PMNCs

Lysozyme

C-type lectins

Cellular source

Effector

Table 1. Main effector molecules of the IPR

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Bacterial antigens stimulate Paneth cells to secrete α-defensins by inducing an increase in cytosolic Ca2+ by the sequential mobilization of intracellular and extracellular Ca2+ stores [15]. Human β-defensin 1 is constitutively expressed in many epithelial cells [16, 17] and rapidly released to the extracellular space as an active peptide. Another group of AMPs activated by posttranscriptional processing is the cathelicidin family. Cathelicidins are constitutively expressed in neutrophils, macrophages, and epithelial cells [18]. In response to infection, cathelicidin precursors are cleaved by serine proteases to generate an active peptide of 37 amino acid residues known as LL-37 [18]. Similar to α-defensins, the C-type lectins RegIIIβ and RegIIIγ are constantly produced by Paneth cells and other intestinal epithelial cells upon recognition of luminal bacteria and released into the gut lumen where they are cleaved by trypsin to create the active peptide [19]. In conjunction, C-type lectins recognize a wide range of microorganisms and play a prominent role in the normal immune surveillance and early resistance to infection at skin and mucosal surfaces, such as the intestinal epithelium [20, 21]. It is worth noting that defensins, cathelicidins, and RegIII lectins are normally synthesized as propeptides and then readily activated by proteolytic cleavage either in the extracellular compartment or in phagosomes, which allows targeting of antimicrobial activity to the right place at the right time.

Opsonins and cross-reacting antibodies Antibodies are synthesized by plasma cells derived from antigenspecific clones of B cells that have responded to an antigen. As part of the adaptive immune response, this process takes time, usually days, and therefore the inclusion of immunoglobulin (Ig) synthesis as part of the IPR must be addressed carefully. However, the constant secretion of some Igs at mucosal surfaces and the existence of cross-reacting antibodies are mechanisms that help in the immediate response to pathogens, and therefore we discuss these mechanisms in more detail below. Cross-reacting antibodies have the ability to react against antigens that are different from the ones that induced their production. In some cases, this polyreactivity allows the cross-reacting antibodies to rapidly neutralize strains of related microbes, even if they have never been encountered before, inducing complementmediated lysis, enhancing phagocytosis, and neutralizing the activity of bacterial LPS [22]. Usually, broadly neutralizing antibodies work as opsonins, that is, molecules that bind to foreign antigens and enhance the action of phagocytes, NK cells, and the complement system, and therefore interfere with the biological effects of bacterial toxins and many viruses [23]. In addition to circulating cross-reacting antibodies, antibody-secreting plasma cells in mucosal linings continuously produce large quantities of IgA, IgM, or IgG. In the human intestine, for example, several grams of secretory IgA are released every day [24], which can also be modulated by environmental stimuli, such as stress [25]. Secretory IgA is the major class of antibody present in mucosal tissues of mammals, where it acts as a first-line barrier by entrapping inhaled  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

or ingested antigens in the mucus layer [26], decreasing the virulence of bacteria by reducing their motility [27] or invasiveness [28], or facilitating the uptake of luminal antigens into lymphoid compartments [29]. In addition, mucosal IgM and IgG form antigen complexes that are taken up by dendritic cells (DCs) and drive a host-protective response resulting in pathogen clearance [30]. Shortly after infection, the profile of protein synthesis by the liver is altered, resulting in a rapid increase or decrease in the plasma levels of several proteins [31]. These proteins are collectively termed positive and negative acute phase proteins, respectively. Some of these proteins also function as opsonins and can recruit immune cells modulating the host’s immune response. However, the most rapidly reacting group of these proteins, including C-reactive protein and serum amyloid A, are released by hepatocytes after cytokine stimulation (primarily IL-1β, IL-6, and TNF-α), and therefore their plasma levels increase 4–5 h after the infectious stimulus [32]. Complement factors, such as C3 or C4, are also considered acute phase proteins but they differ in that they are normally circulating as inactive precursors and therefore are readily available for immediate action. These properties of the complement system are discussed below.

Complement and perforin proteins Of the three major pathways that activate the complement system, the alternative pathway provides the earliest response. It can be initiated within seconds after pathogen encounter and is fully activated, resulting in the formation of multiple C3b molecules and the anaphylactic peptides C3a and C5a within 5– 20 min [33]. C3a is not discriminatory and is able to bind both host cells and microbes, including Gram-positive and Gram-negative bacteria and fungi, inducing their disruption in a way similar to cationic AMPs [34, 35]. Analogous to C3a, C4a and C4a-derived C-terminal peptides, which are rapidly generated by C1 during the first step of the classical pathway, possess direct bactericidal and fungicidal activity [36]. Activation of the complement pathway eventually leads to the assembly of the membrane attack complex also in a matter of minutes [37, 38]. The C9 component of the membrane attack complex and the protein perforin, a calciumdependent pore-forming protein released by cytotoxic T cells and NK cells [39], share a common membrane attack complex/perforin (MACPF) domain responsible for their pore-formation activity. MACPF proteins of the complement system freely circulate in the plasma and their main role is the lysis of Gram-negative bacteria and protozoan pathogens, with the accessory help of AMPs [40]. It is worth mentioning that MACPF-containing proteins comprise a large family that also includes pore-forming toxins produced by pathogenic bacteria as virulence factors, such as the cholesteroldependent cytolysins [41]. Perforin is colocalized in acidified secretory granules with granzymes, peptidoglycans, and cytolytic peptides, such as granulysin. Upon contact with a target cell, these cytoplasmic granules move to the immunological synapse and their contents are delivered into the target cell in endosome-like vesicles [42]. www.eji-journal.eu

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The release of the granzymes from these vesicles into the cytoplasm is largely dependent on the action of perforin [39]. Once in the cytoplasm, granzymes cleave and activate proapoptotic proteins, resulting in cell death. Although this pathway is mainly involved in the elimination of virus-infected and tumor cells, perforin is capable of disrupting cell membranes nonspecifically and therefore its function is tightly regulated [43].

The second phase of the IPR: Cellular and posttranslational events Recruitment and activation of phagocytes Leukocyte recruitment into an infected area is paramount for the innate immune response and is dependent on chemotactic factors. Different chemoattractants, including bacterial derived (e.g., N-formylated peptides) and host-derived (e.g., complement C5a fragment) molecules, are able to recruit phagocytes to the site of infection. The overall process of leukocyte extravasation from the bloodstream has been recently reviewed elsewhere [44–46] and will not be further discussed here. Phagocytosis is a receptor-mediated process of actin reorganization and vesicular internalization of microbes and dying cells [47]. This process is initiated upon direct recognition of microorganisms by receptors expressed at the plasma membrane of phagocytes, or through receptors that recognize opsonins bound to the microbes. After internalization, the phagosome undergoes a series of interactions with lysosomes and endosomes in a process known as phagosome maturation, leading to the formation of the phagolysosome and the degradation of microbes [48]. The importance of phagocytosis for the IPR resides mainly in the rapid engulfment and destruction of microbes, as well as the activation of other IPR mechanisms, such as the formation of ROS and reactive nitrogen species (RNS), due to the assembly of the NADPH oxidase at the membrane of the phagosome [49]. However, these are not the only outcomes of phagocytosis. Depending on the type of receptor that is engaged during phagocytosis, the cellular response can vary from phagocytosis alone, initiation of inflammatory signaling, or both [48]. An example of a receptor that initiates phagocytosis alone, probably the most characteristic of the IPR, is the mannose receptor. This receptor is expressed in some populations of human macrophages and DCs, and recognizes mannose residues on the surface of microorganisms inducing their engulfment without inflammatory signaling [50]. Also, important for the IPR is opsonic phagocytosis, where opsonization with complement proteins, Igs, or acute phase proteins induces phagocytosis by binding to Fcγ receptors or complement receptors at the surface of phagocytes [51]. Other important outcomes of phagocytosis not related to the IPR include the processing and presentation of peptides within major histocompatibility complex (MHC) molecules and the activation of intracellular PRRs, such as endosomal TLRs and cytosolic nucleotide oligomerization domain like receptors (NLRs), leading to inflammatory cytokine secretion [48].  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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N-formyl peptides and their receptors In contrast to host-derived chemokines, which need to be synthesized and secreted, natural formylated peptides are chemoattractants derived from bacteria (also from components of disrupted mitochondria) and are therefore capable of rapidly recruiting and activating phagocytes [52, 53]. N-formyl-methionyl-leucylphenylalanine (FMLP), isolated from Escherichia coli, is the most studied member of the formyl peptide family. All chemoattractant receptors are members of the superfamily of G protein coupled receptors (GPCRs). In human phagocytes, formyl peptides bind to three GPCRs, the high affinity formyl peptide receptor (FPR) and its variants FPR like 1 (FPRL1) and FPRL2. The human FPR and FPRL1 are expressed in monocytes, macrophages, neutrophils, immature DCs, platelets (FPR), T cells (FPRL1), and B cells (FPRL1), as well as some nonhematopoietic cells. By contrast, human FPRL2 expression in immune cells is restricted to monocytes, macrophages, and DCs [52, 54]. In mice there are at least seven FPR receptors expressed mostly by neutrophils and DCs [54]. Activation of FPR by FMLP results in the initiation of several signaling pathways, including the mitogen-activated protein kinase, PLC, and phosphatidylinositol 3-kinase (PI3K) pathways, which in turn trigger inositol triphosphate and diacylglycerol production [55]. Inositol triphosphate regulates calcium mobilization, and cytosolic Ca2+ increase is one of the earliest events of phagocyte response to FMLP [56]. In addition to N-formylated peptides, numerous unrelated ligands are capable of interacting with FPR and FPRL1 [54]. These agonists include both pathogenand host-derived molecules. Pathogen-derived agonists of human FPRs include peptides derived from HIV-1 envelope proteins [57], Helicobacter pylori [58], and herpes simplex virus type 2 [59]. Host-derived agonists include lipid mediator lipoxin A4 [60], acute phase proteins such as serum amyloid A [61], neuropeptides [62], and AMPs [63]. Since some of these agonists, such as lipoxin A4 and serum amyloid A, are also released in sterile injury, this family of receptors may establish a link between the immune response against sterile injury and infection. The engagement of the FPR family triggers various cellular responses associated with immunity, including migration, degranulation, cytokine production, secretion of antimicrobial enzymes, and ROS production mainly by cells of the innate immune system [55]. The key role of the FPR in host defense is demonstrated by the observation that FPR deficiencies are associated with increased susceptibility to bacterial infections in mice [64] and humans [65]. Thus, the activation of FPR in response to bacterial stimuli may represent an important mechanism to rapidly boost the host defense.

Extracellular traps The recent identification of neutrophil extracellular traps (NETs) has expanded the repertoire of antimicrobial strategies employed by neutrophils. Studies with human neutrophils showed that upon www.eji-journal.eu

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microbial encounter, activated neutrophils die and release a meshlike structure composed of decondensed chromatin and antimicrobial effectors that are capable of trapping and killing a wide range of microbes extracellularly [66–68]. These effectors include AMPs released from neutrophil granules, myeloperoxidase (MPO), and histones, which are very powerful antimicrobial agents [69]. However, not all microbes are vulnerable to NETs. Some bacterial species, such as Staphylococcus aureus, can express nucleases to facilitate escape from NETs [70, 71]. Others, by the strategy of preventing ROS production, inhibit NET formation [72]. Little is known about the molecular mechanisms that regulate NET formation, but it involves a distinctive cell-death program known as NETosis [73]. Upon stimulation, NETosis triggers nuclear envelope breakdown, followed by expansion and decondensation of chromatin, while the cytoplasmic membrane remains intact. Shortly after that, the plasma membrane breaks, releasing the chromatin together with adhered antimicrobial effectors [73]. This turn of events requires the activation of NADPH oxidase and the production of ROS [73]. ROS activate NET release through a neutrophil elastase mediated mechanism, whereby neutrophil elastase released from azurophilic granules translocates to the nucleus, where it degrades specific histones promoting chromatin decondensation in synergy with MPO [74]. NET release is a rapid process (