Pyroptotic and non‐pyroptotic effector functions of caspase‐11

Abstract Innate immune cells, epithelial cells, and many other cell types are capable of detecting infection or tissue injury, thus mounting regulated immune response. Inflammasomes are highly sophisticated and effective orchestrators of innate immunity. These oligomerized multiprotein complexes are at the center of various innate immune pathways, including modulation of the cytoskeleton, production and maturation of cytokines, and control of bacterial growth and cell death. Inflammasome assembly often results in caspase‐1 activation, which is an inflammatory caspase that is involved in pyroptotic cell death and release of inflammatory cytokines in response to pathogen patterns and endogenous danger stimuli. However, the nature of stimuli and inflammasome components are diverse. Caspase‐1 activation mediated release of mature IL‐1β and IL‐18 in response to canonical stimuli initiated by NOD‐like receptor (NLR), and apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC). On the other hand, caspase‐11 delineates a non‐canonical inflammasome that promotes pyroptotic cell death and non‐pyroptotic functions in response to non‐canonical stimuli. Caspase‐11 in mice and its homologues in humans (caspase‐4/5) belong to caspase‐1 family of cysteine proteases, and play a role in inflammation. Knockout mice provided new genetic tools to study inflammatory caspases and revealed the role of caspase‐11 in mediating septic shock in response to lethal doses of lipopolysaccharide (LPS). Recognition of LPS mediates caspase‐11 activation, which promotes a myriad of downstream effects that include pyroptotic and non‐pyroptotic effector functions. Therefore, the physiological functions of caspase‐11 are much broader than its previously established roles in apoptosis and cytokine maturation. Inflammation induced by exogenous or endogenous agents can be detrimental and, if excessive, can result in organ and tissue damage. Consequently, the existence of sophisticated mechanisms that tightly regulate the specificity and sensitivity of inflammasome pathways provides a fine‐tuning balance between adequate immune response and minimal tissue damage. In this review, we summarize effector functions of caspase‐11.


| INTRODUC TI ON
Inflammasomes assemble when a subset of intracellular receptors that belongs to the NOD-like receptor (NLR) protein family, such as NLRP3 or NLRC4, sense PAMPs or DAMPs. Pattern sensing is followed by nucleation and oligomerization of the adapter protein ASC (apoptosis-associated speck-like protein containing CARD), and engagement of the cysteine protease pro-caspase-1. [1][2][3] Within the oligomerized inflammasome complex, dimers of pro-caspase-1 undergo proteolytic auto-cleavage into the enzymatically active caspase-1, which consequently catalyzes the final processing of the inflammatory cytokines, pro-IL-1β and pro-IL-18 precursors, into their mature and secreted forms. 4 In addition, inflammasome assembly and activation results in pyroptosis, a distinguished programmed cell death that is initiated in response to bacterial infections. It is associated with the release of inflammatory cytokines (IL-1β, IL-18), alarmins such as IL-1α and high mobility group box 1 (HMGB1), and unbound and trapped bacteria within cellular debris of pyroptotic cells. 2,[5][6][7] Assembly and activation of inflammasomes is triggered by a wide array of stimuli such as nucleic acids, bacterial toxins, and flagellin. [8][9][10] Further, inflammasome activation can be mediated via endogenous damage signals such as ATP and uric acid. 11 Activation of caspase-1 by NLRP3/ASC or NLRC4/ASC delineates the canonical inflammasome pathway. However, the non-canonical inflammasome requires caspase-11-mediated activity.
Caspase-11 in mice, or caspase-4/5 in humans, belongs to the family of inflammatory caspases and exhibits (46%) similarities to caspase-1. [12][13][14][15][16][17][18][19] Inflammatory caspases are located on chromosome 9 in mice, and are thought to create an inflammatory cluster due to close proximity and high degree of similarity. Indeed, caspase-11 is located adjacent to caspase-1, only 0.012 centimorgans, or ~1500 bp, on chromosome 9 in mice. 20 Early on, Wang et al showed that caspase-11 induced a pyroptotic cell death in vitro and formed heterocomplex with caspase-1, but inefficiently cleaved IL-1β, suggesting that cytokine maturation might not solely be dependent on caspase-11. 18,21 Generation of Casp1 −/− 22,23 and Casp11 −/− 18 knockout mice provided the scientific community with genetic tools to study inflammatory caspases. For over a decade, it was thought that the functions of caspase-1 and caspase-11 are redundant as both knockout mice were resistant to lethal doses of LPS-mediated septic shock. 18 However, it was shown that the original Casp1 −/− are double knockout and, indeed, lacked both caspase-1 and caspase- 11. 20 The same study showed that Casp1 −/− harbors a spontaneous 5-bp deletion in the exon 7 splice acceptor site of caspase-11, which generated a stop codon, thus creating highly unstable caspase-11 transcripts. 20 To compensate for the loss of caspase-11, Casp1 −/− mice were engineered to express caspase-11 via an artificial chromosome (Casp1 −/− Casp11 Tg ). 20 This study showed that caspase-11, rather than caspase-1, mediates LPS-induced septic shock in mice, suggesting that the functions of each caspase are distinct. 20 The identification that caspase1 −/− mice are lacking both caspase-1 and caspase- 11 20 promoted further research to dissect the roles of caspase-11. We now know that the physiological function of caspase-11 is much broader than its previously established roles in apoptosis and cytokine maturation. Herein, we discuss induction and activation and review pyroptotic and non-pyroptotic functions of caspase-11 that are required to control pathogens. We further discuss the role of caspase-11 in diseases including asthma and gout. Understanding all the facets of caspase-11 functions will help us decipher its role and contribution to inflammatory diseases and infection. It will greatly enhance our understanding to its complete role in immunity and pave the way for development of new strategies that will enhance physiological roles of caspase-11 during infection and inflammatory diseases.
Until recently, appreciated functions of CASP11 were the recognition of cytosolic LPS followed by the activation of CASP1, cleavage of gasdermin D (GSDMD), pro-inflammatory cytokine secretion, and cell death.

| S TIMULI THAT INDUCE C A S PA S E-11 E XPRE SS I ON AND AC TIVATI ON
Caspase-11 is expressed broadly in immune and non-immune cells, and its expression in resting cells is low. 18,20 In contrast to other caspases that are regulated by proteolytic cleavage, caspase-11 is regulated at both transcriptional and post-translational levels. Genetic analysis revealed that the caspase-11 promoter region contains several putative binding sites for transcriptional factors including nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), signal transducer and activator of transcription-1 (STAT-1), interferon regulatory factor (IRF), nuclear factor of activated T cells (NFAT), and cAMP response element-binding protein (CREB). 24 Further, induction of caspase-11 expression is achieved by activation of p38 mitogen-activated protein kinase (MAPK) in rat glial cells, c-Jun N-terminal kinase (JNK) in mouse embryonic fibroblasts, and C/EBP homologous (CHOP) protein in mice. 7,25,26 Caspase-11 expression is induced through LPS-activated TLR4 signaling via the adapter TIR-domain-containing adapter-inducing interferon-β (TRIF) and TRIF-dependent type I interferon (IFN) production. 6,21,24,27 Physiologically, during Gram-negative bacterial infection, caspase-11 expression and other inflammasome components are initiated concertedly by multiple TLRs in response to bacterial PAMPs.
Induction of caspase-11 and other inflammasome component expression is considered a "priming step," and therefore is not sufficient for caspase-11-mediated activation and downstream effector functions. It was found that the "triggering step," which is initiated by cytosolic recognition of LPS by caspase-11, mediates activation and downstream effector functions of caspase-11. 2,28 It was shown that caspase-11 senses cholera toxin B (CTB) and extracellular bacteria such as Escherichia coli, Citrobacter rodentium and Vibrio cholera. 20 Subsequent studies showed that caspase-11 is activated by other Gram-negative bacterial infection. 20,27,[29][30][31] We have reported the induction of caspase-11 expression in mouse macrophages following infection with Legionella pneumophila. Caspase-11 expression was shown to be independent of bacterial flagellin, ASC and the NLRC4 inflammasome. 30 However, caspase-11 interaction with the Nlrc4 inflammasome members and its activation required bacterial flagellin. 30 Hence, non-canonical stimuli such as cholera toxin or intracellular LPS trigger caspase-11-dependent inflammasome activation in the cytoplasm independently of TLR4. 1,20,32 Recently, we have demonstrated that expression of caspase-11 is induced by IL-1β and IL-1α via the IL-1R/MYD88. 33 Indeed, single myd88 −/− , trif −/− , and myd88/ trif −/− , macrophages, 34 showed reduced caspase-11 expression in response to IL-1α and IL-1β, or in combination, respectively. 33 Additionally, we have revealed that induction of caspase-11 by LPS and HMGB1 is independent of IL-1R. HMGB1 utilizes a variety of receptors to promote its signaling events, and LPS is sensed by TLR4, which uses both adapters to signal. 35,36 We have shown that caspase-11 expression was not significantly hampered in the WT or the single knockout macrophages during stimulation with either HMGB1 or LPS, except when both MYD88 and TRIF were absent, indicating that either adapter was able to countervail for the deficient one in order to induce caspase-11. 33 Certain diseases have been classified as an IL-1β-mediated condition such as gout since neutralizing antibodies to IL-1β or the caspase-1 inhibitor z-YVAD significantly reduce inflammation and the production of other cytokines within the joints. [37][38][39] Induction of caspase-11 expression by released IL-1β and IL-1α could prime immune cells for inflammasome activation by subsequent insults including monosodium urate (MSU).
Thus, preventing the expression of caspase-11 in gout-prone individuals may prevent the instigation of tissue damage. 33 Additionally, we have shown that house dust mites (HDM) induce caspase-11 expression in bone marrow-derived macrophages and in C57BL/6 mice (Abu Khweek et al under review). Intriguingly, HDM contain LPS, proteases, and chitin from the mite exoskeleton. 40,41 Therefore, the expression of caspase-11 is induced not only by microbes but also by specific cytokines and foreign particles such as house dust mites.

| C A S PA S E-11 PROTE A S E AC TIVIT Y AND SUBS TR ATE S PECIFICIT Y
The signaling pathways upstream and downstream of caspase-11 are still unclear, the molecular mechanism by which caspase-11 acquires protease function in response to LPS binding or cytosolic bacterial infection is not fully understood. LPS interaction with the caspase-11 CARD domain facilitates activation of the protease domain. 42 Caspase-11 is composed of an N-terminal recruitment domain and large and small subunits that are linked by linkers. The CARD domain linker (CDL) connects the large subunit with the N-terminal CARD domain, while the interdomain linker (IDL) connects the large and small catalytic subunits. Caspase-11 can be detected by immunoblot into full-length (43 kDa), shorter (38 kDa), which is thought to arise from alternative start codon within the CARD domain, 43 and a shorter fragment generated during non-canonical signaling in macrophages. 20,44 The catalytic cysteine C254 is located within the large subunit. Dimerization of caspase-11 is sufficient for caspase-11 to acquire basal proteolytic activity. However, full spectrum of caspase-11 activities such as cleavage of gasdermin D, macrophage death, NLRP3 inflammasome activation, and IL-1β release requires dimerization of caspase-11 and auto-cleavage. 45 Indeed, caspase-11 harbors multiple candidate sites within CDL and IDL for autocleavage, which could generate fragments of multiple sizes. Ross et al showed that full-length, uncleaved p36 and p32 cleavage fragments are released in the cell culture media in response to LPS transfection. 45 Auto-cleavage within IDL at residue D285, but not CDL, generates fully active P32/P10 species corresponding to CARDlarge/small subunit of caspase-11 dimer. The generated subunits are independent of NLRP3 or caspase-1 activation, which further implies the auto-cleavage of caspase-11. Human caspase-4 is similarly cleaved to generate a p32 fragment upon exposure to cytosolic LPS or Gram-negative bacteria, suggesting that the caspase-11 signaling mechanism is conserved during non-canonical inflammasome signaling in humans. 46 Further, it has been shown that D285 residue within IDL is important for caspase-11 function in vivo. 47 In this study, the authors proposed that the active species of caspase-11 are likely to be P26/P10 rather than P32/P10, wherein large subunits contains P26 generated by cleavage within the D285 and D59 or alternatively, by cleaving p36, a short form of caspase-11 derived from an alternative start site that is lacking most of the CARD domain required for interacting with LPS. However, the precise identity of the large subunit cleavage fragment is not resolved yet. Ross et al suggest that the P32/P10, rather than P26/P10, fragments are more likely to be the cleaved fragments for several reasons. Importantly, the p36 fragment is unable to bind LPS 42 and is unexpected to dimerize and auto-cleave at IDL to generate p26/p10. In addition, the cleavage fragments proposed by Lee et al are unlikely to be the targets for cleavage as caspases prefer to cleave in flexible loop regions. 45 In contrast to caspase-1, caspase-11 requires both dimerization and auto-cleavage to mediate gasdermin D cleavage, which may or may not be associated with cell death depending on the intensity of the stimulus. The authors propose that dimerized P43 subunits of caspase-11 have intrinsic catalytic activity, but could potentially be suboptimal to cleave substrates. 45 Further, the dimer form could be unstable and cleavage within the IDL could potentially induce stability of the active site and substrate binding pocket. 48,49 Moreover, auto-cleavage at IDL site may expose recognition sites for interaction with particular protein substrates, thus altering substrate specificity. Therefore, identification of caspase-11 substrates that can be processed by dimerized but uncleaved caspase-11 would further enhance our understanding of the function of this protease. The mechanisms regulating activation of caspase-4/5/11 protease functions within the non-canonical inflammasome are suggested to follow a distinct mechanism from that of caspase-1 for several reasons. The first reason is associated with the ability of caspase-11 to interact directly with LPS without the requirement for traditional receptor or signaling adapter. 42 Second, non-canonical inflammasome assembly is proposed to generate oligomers rather than dimers associated with the canonical inflammasome. 50  Substrate specificity of caspase-1 and caspase-11 was analyzed by using massive hybrid combinatorial substrate library (HyCoSuL) screens using purified caspase-1 and caspase-11. The substrate preference was correlated with caspase-11 activity on three endogenous substrates (IL-1β, IL-18, and gasdermin D). Caspase-1 rapidly processed IL-1β and IL-18, but caspase-11 poorly cleaved these cytokines. However, both caspase-1 and caspase-11 efficiently cleaved gasdermin D. The authors hypothesized that caspase-11 might exhibit an exosite that is specifically proficient in pyroptosis, but not cleaving IL-1β and IL-18. Exosites are secondary binding sites that are remote from the active site and tend to direct proteases toward specific substrates that are not normally cleaved. Caspase-11 may represent such a case by cleaving peptides and pro-interleukins poorly, but cleaving gasdermin D well, and thus developed highly selective substrate specificity via a yet-to-be-identified exosite. 51 Therefore, caspase-11 has restricted substrate specificity preferring gasdermin D over all the substrates examined. 20,51 Genetic evidence shows that caspase-1 but not caspase-11 is able to cleave IL-1β and IL-18. 20,52 However, both of these caspases are able to cleave gasdermin D and induce pyroptosis. 53,54 Hence, there must be biochemical differences that distinguish these closely related proteases. Using full length or CARD lacking recombinant expressed enzymes showed that the CARD domain does not inhibit the activity of caspase-11 toward endogenous substrates. The authors predict that the specificity and activity of caspase-11 would not be affected by the CARD domain. Until now, there has been no selective substrate for caspase-11 over caspase-1. This makes it difficult to design compounds that interfere with the non-canonical pathway over the canonical one. Characterization of the putative exosite will pave the way for designing compounds that will selectively target caspase-11.

| I L-1α IS A D IREC T SUBS TR ATE OF MOUS E C A S PA S E-11 AND H UMAN C A S PA S E-5 DURING NON -C ANONIC AL INFL AMMA SOME AC TIVATI ON AND DURING S ENE SCEN CE
Although IL-1α and IL-1β are released in response to non-canonical inflammasome activation, the upstream target/s involved in cleaving and activating IL-1α has/have not been characterized until recently. Wiggins et al showed that IL-1α is specifically F I G U R E 1 A, Domain structure of caspase-11 showing caspase cleavage sites, the CDL, IDL, and the catalytic cysteine (C254), and the molecular weights of caspase-11 fragments. Cleavage fragments P32/10 are shown in red lines as indicated by. 45  cleaved and activated by human caspase-5 or mouse caspase-11 at a conserved position adjacent to the calpain site. 55 In human macrophages, caspase-5 promotes cleaved IL-1α release after inflammasome activation. In contrast to IL-1β release that requires caspase-11 and caspase-1, the cleavage and release of IL-1α is exclusively dependent upon caspase-11 in murine macrophages.
Importantly, IL-1α acts in an autocrine/paracrine manner to promote senescence-associated secretory phenotype (SASP), 56,57 where senescent cells develop altered secretory activities. SASP is involved in immune surveillance and clearance of senescent cells. 58 Further, it is associated with the release of pro-inflammatory cytokines, chemokines, proteases, and growth factors, thus driving chronic inflammation leading to diseases and unhealthy aging. 59 Additionally, senescent human cells show an increase in caspase-5 expression and total cleaved IL-1α compared to growing cells. Knockdown of caspase-5 reduces IL-1α release and cytokines associated with SASP such as IL-6, MCP1, and IL-8, suggesting that caspase-5 is required for IL-1α release during senescence in vitro.
We also showed that IL-6, KC, and TNF-α are drastically reduced in the absence of caspase-11. 33 Additionally, caspase-11 −/− mice showed reduction in the level of IL-1α and KC in response to MRSA infection. 60 Consequently, directly targeting caspase-11/-5 may reduce inflammation and limit the deleterious effects of senescent cells that accumulate during disease and aging. 55

| TRP C1 IS A SUBS TR ATE FOR C A S PA S E-11-MED IATED IL-1β RELE A S E
The cationic channel subunit transient receptor potential channel 1 (TRPC1) is a membrane protein that can form channel permeable to Ca 2+ . It was identified via yeast two-hybrid screen and was shown to interact with the catalytic (P10 and P20), but not the CARD domain of caspase-11. Co-expression of the catalytic domains of caspase-11, but not caspase-1 with TRPC1 reduced the level of TRPC1 in HEK293 cells. 61 Furthermore, incubation of purified recombinant caspase-11 p30 with S 35 -labeled TRPC1 in vitro led to appearance of multiple TRPC1 fragments and a reduction of full-length TRPC1, 61 suggesting that the TRPC1 is a substrate for caspase-11.
Since caspase-11 is inducible via LPS, Py et al examined the effect of LPS treatment on the level of ectopically expressed TRPC1. It was shown that HA-TRPC1 protein levels were significantly reduced, but not in the presence of the pan caspase inhibitor z-VAD.fmk or IDUN-6556, indicating that TRPC1 is degraded by inflammatory caspases.

Moreover, TRPC1 degradation was shown in macrophages infected
with E coli (J53 strain), or following prolonged stimulation with LPS (16hrs) followed by ATP stimulation. These stimulators have been shown to induce caspase-11 expression. However, low dose of LPS stimulation followed by ATP, which is sufficient for caspase-1 secretion but insufficient to induce the expression of caspase-11, did not lead to TRPC1 degradation. 61 Thus, caspase-1 activation in the absence of caspase-11 expression does not lead to TRPC1 degradation.
In addition, caspase-11-mediated degradation of TRPC1 following LPS stimulation suggests that the TRPC1 is remodeled at the onset of inflammation and exhibits roles in regulating innate immunity. and remodeling of associated channel complexes independently of caspase-1. 61 These data suggest that cleavage of TRPC1 likely impacts events downstream of caspase-1 activation, including the unconventional secretion pathway that is responsible for mature IL-1β release. 61 The authors speculate that the inflammatory response may alter the gating properties of TRPC1-containing channels in the plasma or intracellular membranes to promote unconventional protein secretion. Therefore, the possibility for the role of the TRPC1associated channelosome in regulating unconventional secretion needs further investigation.

| INTR ACELLUL AR LPS , MODE OF C Y TOSOLI C DELIVERY, AND DE TEC TI ON BY C A SPA SE-11
Specific recognition of Gram-negative bacteria via However, modifying LPS structure by adopting penta-acylated or tetra-acylated LPS allows some pathogenic bacteria to evade the detection by caspase-11 and to avoid provoking inflammation. 2,31 Since caspase-11 was activated by non-cytosolic and extracellular bacteria, the semantic question was how do Gram-negative pathogens deliver their LPS to the cytosol to mediate activation of caspase-11? Some bacteria such as the Burkholderia spp can access the cytosol following invasion of the cell, thus delivering their LPS to the cytosol. 28 Other pathogens have secretion systems to deliver virulence factors into eukaryotic cells. 27,63 It has been demonstrated that clathrin-mediated endocytosis of bacterial outer membrane vesicles (OMVs), which are overloaded with LPS, mediate the delivery of LPS into the host cell cytosol. 64 Intriguingly, caspase-11-mediated activation, pyroptosis, and release of mature inflammatory cytokines were observed with purified OMVs derived from Gramnegative but not Gram-positive bacteria. 63 Another mode of delivering cytosolic LPS was through a family of dynamin-related large GTPases, the so-called guanylate-binding proteins (GBPs). 65 Expression of GBPs is induced in response to interferons and other pro-inflammatory cytokines. 66 The GBPs promote a wide spectrum of innate immune functions against intracellular pathogens. 67 Meunier and his colleagues showed that GBPs are required for activation of caspase-11 in response to infection with vacuolar Gram-negative bacteria. 65 Indeed, induction of caspase-11-dependent pyroptosis by cytoplasmic L pneumophila-derived LPS required GBPs. 68 Further, macrophages lacking GBPs showed impaired caspase-11 activation and attenuated pyroptosis. 68 Recently, it has been shown that GBPs activate caspase-11 and regulate non-canonical NLRP3 inflammasome and IL-1β release in response to T3SS-negative Pseudomonas (P). aeruginosa. 69 The fact that GBPs are exclusively relevant in inducing immune response to T3SS-negative P aeruginosa demonstrates the that these mechanisms have evolved to detect pathogens that escape detection by canonical inflammasomes. 69 Therefore, GBPs promote caspase-11driven, cell-autonomous immune defense against Gram-negative pathogens accessing the cytosol. 68 The cytosolic detection of intracellular LPS by caspase-11 could serve as an extra checkpoint that is established proceeding to initiating irreversible steps in the cell.

| G A S DERMIN D, A SUBS TR ATE FOR C A S PA S E-11-MED IATED S ECRE TI ON OF IL-1β AND IL-18
In cases of high bacterial burdens, caspase-11 mediates pyroptotic cell death, which consequently results in clearing intracellular bacteria. 6 to promote pyroptosis, not only in vitro, but also in vivo. 53,54,76 Moreover, the N-terminal binds to negatively charged phosphoinositides and cardiolipin in the membrane, and lipid binding mediates oligomerization. 75,77 The oligomerized N-terminal domain causes a pore, which dissipates the electrochemical gradient across the membrane and disrupts the osmotic potential, causing cell lysis.
Gasdermin D-deficient cells have impaired processing of caspase-1 and release of IL-1β in cell lysate following LPS electroporation. 78 Further, gasdermin D pore indirectly activates the NLRP3 inflammasome to generate active caspase-1, which cleaves pro-IL-1β to its mature form that is secreted. 79 Plasma membrane pore formation by gasdermin D oligomers results in K + efflux, which is an established trigger of NLRP3 activation. 80

| NON -AP OP TOTIC FUN C TIONS OF C A S PA S E-11 AND G A S DERMIN D
Recent study showed that at sublytic levels of inflammasome activation, and in the absence of pyroptotic cell death, gasdermin D pores mediate the release of IL-1β or IL-18 and other cytosolic proteins. 84 Notably, we have shown that the release of IL-1β from macrophages in response to MSU was not accompanied by cell death. 33 Our findings are corroborated by recent reports describing how gasdermin forms pores within the intact plasma membrane, allowing the release of IL-1β independently of cell death. 85,86 In a state of hyperactivation (truly viable phagocytes that release IL-1), gasdermin D regulates IL-1β release in these viable cells independent of cell lysis. 85 84 So gasdermin D pore exhibits a bifunctional role by mediating cell lysis-dependent and cell lysis-independent release of cytokines. Gasdermin D pores may act as unspecific channels that release cytosolic proteins in size-dependent way, with a possible cutoff between 25 and 50 kDa. 84 Interestingly, proteins released subsequently to inflammasome activation are secreted through the gasdermin D pore. 87 These secreted proteins such as IL-1α and HMBG1 are alarmins or DAMPs that alarm other cells, and therefore modulate the inflammatory responses downstream of inflammasome activation. 20 The unconventional secretion of these endogenous alarmins, following cytosolic LPS recognition, is promoted by caspase-11 and is independent of caspase-1. 20 The pore-forming mechanism shared by the gasdermins could be uti-

| PYROP TOTIC AND NON -PYROP TOTIC ANTI -BAC TERIAL IMMUNE SURVEILL AN CE ROLE OF C A S PA S E-11
Given the extensive number of bacterial pathogens, it is expected that mammalian cells harbor several inflammasomes to sense wide arrays of pathogenic stimuli. Caspase-11 is involved in protecting the host against a wide variety of extracellular and intracellular Gram-negative pathogens. 6  and IL-1β. 92 Together, these data suggest that caspase-11 exhibits additional effector mechanism besides its typical pyroptotic function to control intracellular infections.

| ROLE OF C A S PA S E-IN MODUL ATING AUTOPHAGY IN RE S P ONS E TO BAC TERIAL INFEC TI ON
Autophagy, an intracellular catabolic pathway, is important for cellular homeostasis and recycling of damaged organelles and proteins, as well as clearance of intracellular pathogens. 104  sol. 65 Although it is not known how autophagy regulates caspase-11 activation, it is possible that autophagy removes excessive reactive oxygen species that are suggested to enhance caspase-11 expression. 113 Autophagic removal of intracellular DAMPs, inflammasome components, or cytokines can reduce inflammasome activation.
Similarly, inflammasomes can regulate the autophagic process, allowing for a two-way mutual regulation of inflammation that may hold the key for treatment of multiple diseases. Therefore, mutual regulation of caspase-11 activation and autophagy is important for the maintenance of cellular homeostasis and optimized innate immune response to intracellular bacterial pathogens.

| ROLE OF C A S PA S E-11 IN CELL MIG R ATION
It has been demonstrated that caspase-11 facilitates cell migration of different cell types during inflammation. Intriguingly, splenocytes and macrophages derived from caspase-11 −/− mice are defective in migration toward different chemokines in vitro and in vivo. 114 In vitro actin depolymerization assays demonstrated that caspase-11 interacts physically and functionally with actin-interacting protein 1 (Aip1). 114 The resulting interaction increases the proximity of Aip1

| ROLE OF C A S PA S E-11 IN ALLERGY AND A S THMA
Importantly, caspase-4/11 contributes to allergic airway inflammation, with implications for pathophysiology of asthma. Two independent studies have shown that caspase-11-deficient mice are resistant to developing experimental allergic airway inflammation in response to two different allergens. We used the common aer- to lower KC and IL-17A. Alternatively, it may be due to reduced IL-33 that can lead to reduced IL-4 and IL-5 production by T cells.
Our study offers several intriguing scenarios for the diverse functions of caspase-11. Expression of caspase-11 in innate immune cells following HDM exposure may be required for the antigen presentation by macrophages and dendritic cells to naive T cells.
Subsequently, insufficient antigen presentation leads to reduction in cytokines and chemokines released in the BAL fluids. Our published work and that of others also support the notion for inherent defect in migration of caspase-11 −/− cells due to defect in the actin cytoskeleton. 33

| CON CLUS IONS
Different studies indicate that the inflammatory caspases have functions beyond those associated with pyroptosis and cytokine release.
The canonical inflammasome has been studied extensively including Despite these important advances, several gaps need to be unraveled to increase our understanding of caspase-11-mediated functions. These include (a) the signals that activate caspase-11; (b) why only pathogenic bacteria mediate caspase-11 activation; and (c) how caspase-11 mediates caspase-1 activation. The stoichiometry of the caspase-11-LPS complex is not defined, and it remains unclear whether the higher order caspase-4/5/11 structures induced by LPS are true oligomers, or represent multiple caspase dimers binding to single LPS molecule or LPS aggregate. Understanding the diverse functions of caspase-11 will pave the way for designing therapeutics.

CO N FLI C T O F I NTE R E S T
The authors of the manuscript declare that the submitted work was carried out in the absence of any personal, professional, or financial relationships that could potentially be construed as a conflict of interest.

AUTH O R CO NTR I B UTI O N S
AAK wrote the review. AOA edited the manuscript.