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Reassessing the Evolutionary Importance of Inflammasomes Vivien I. Maltez and Edward A. Miao This information is current as of August 9, 2017. Subscription Permissions Email Alerts This article cites 74 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/196/3/956.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 References J Immunol 2016; 196:956-962; ; doi: 10.4049/jimmunol.1502060 http://www.jimmunol.org/content/196/3/956 Brief Reviews The Journal of Immunology Reassessing the Evolutionary Importance of Inflammasomes Vivien I. Maltez and Edward A. Miao S ensors in the innate immune system survey the extracellular/vacuolar space or the cytosol. TLRs and C-type lectin receptors survey the extracellular/ vacuolar compartment. In contrast, RIG-I–like receptors, Nod-like receptors (NLRs), AIM2-like receptors (ALRs), and certain TRIM family proteins survey the cytosolic space. Among these cytosolic sensors, the canonical inflammasomes activate caspase-1. The exception to this paradigm of upstream sensors activating downstream signaling molecules is the so-called “noncanonical inflammasome” pathway, in which murine caspase-11 (and its human orthologs caspase-4 and -5) has the cytosolic sensor and effector functions built into the same protein (1, 2). Caspase-1 activation leads to processing and secretion of the inflammatory cytokines IL-1b and IL-18, as well as to a form of programmed lytic cell death called pyroptosis. Inflammasomes detect a wide range of cytosolic contaminants. AIM2 detects cytosolic DNA (3). NLRP3 responds to diverse stimuli that seem to trigger catastrophic cellular events (3). In a relatively unique mechanism, NLRC4 is activated by an upstream helper NLR in the NAIP family that is the direct sensor for one of three bacterial proteins: T3SS rod, T3SS needle, or flagellin. These components aberrantly enter the host cytosol through T3SS, presumably while the bacteria is injecting effector proteins to alter host cell function(s) (4). NLRP1b in the mouse responds to the anthrax lethal toxin by serving as a “lure” for this pathogen protease (5). Finally, pyrin (also called TRIM20) senses perturbation of Rho GTPases by bacterial toxins (6). In contrast to these canonical inflammasomes, caspase-11/4/5 detect cytosolic LPS by direct interaction between LPS and their CARD domains (2). These noncanonical inflammasomes trigger pyroptosis but do not process IL-1b directly. Inflammasomes are evolutionarily maintained in vertebrates (7), although the repertoire may be expanded or contracted in various species. Inflammasomes are proposed to aid in defense against a variety of infections. However, inflammasome activation can also drive septic shock, leading to death (8, 9). This would exert a selective pressure to lose inflammasomes. With this in mind, does the current published evidence support a positive selective pressure that explains the maintenance of inflammasomes over evolutionary time? In vivo data suggest that inflammasomes delay, but do not eradicate, infection In considering the positive selective pressure to maintain inflammasomes in defense against infection, many infectious studies were performed using knockout (KO) mice, typically on the C57BL/6 background. Although inflammasomedeficient mice have increased susceptibility to many pathogens, the effect of inflammasomes on experimental survival studies is typically incremental. Salmonella enterica serovar Typhimurium (S. typhimurium) was one of the best and earliest examples. S. typhimurium replicates somewhat faster in Casp12/2Casp112/2 mice, resulting in significantly increased burdens at any given time point postinfection; for Department of Microbiology and Immunology, Center for Gastrointestinal Biology and Disease, and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Address correspondence and reprint requests to Dr. Edward A. Miao, University of North Carolina at Chapel Hill, Campus Box 7290, Chapel Hill, NC 27599-7290. E-mail address: [email protected] ORCIDs: 0000-0003-2330-7518 (V.I.M.); 0000-0001-7295-3490 (E.A.M.). Abbreviations used in this article: ALR, AIM2-like receptor; KO, knockout; NLR, Nod-like receptor; WT, wild-type. Received for publication September 21, 2015. Accepted for publication November 6, 2015. This work was supported by National Institutes of Health Grants AI119073, AI097518, and AI097518-02S. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502060 Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 Inflammasomes monitor the cytosol for microbial contamination or perturbation and, thus, are predicted to provide potent defense against infection. However, the compendium of data from murine infection models suggests that inflammasomes merely delay the course of disease, allowing the host time to mount an adaptive response. Interpretations of such results are confounded by inflammasome-evasion strategies of vertebrateadapted pathogens. Conversely, environmental opportunistic pathogens have not evolved in the context of inflammasomes and, therefore, are less likely to evade them. Indeed, opportunistic pathogens do not normally cause disease in wild-type animals. Accordantly, the extreme virulence of two opportunistic bacterial pathogens, Burkholderia thailandensis and Chromobacterium violaceum, is fully counteracted by inflammasomes in murine models. This leads us to propose a new hypothesis: perhaps animals maintain inflammasomes over evolutionary time not to defend against vertebrateadapted pathogens but instead to counteract infection by a plethora of undiscovered opportunistic pathogens residing in the environment. The Journal of Immunology, 2016, 196: 956–962. The Journal of Immunology mice. It should be noted that, although many survival studies were performed with bacteria, fewer were published with viral, fungal, and parasitic infection (discussed further below) (15– 18). After collation of published survival studies (Table I), it was striking that most changes in lethal dose were estimated to be in the 1–5-fold range. Therefore, the combined results of all infection survival studies in inflammasome-deficient mice support our interpretation that inflammasomes delay the course of infection but do not have a dominant role in defense. Conversely, what if inflammasomes could provide rapid and potent defense against infection? This would exert a strong selective pressure for pathogens to evolve inflammasome-evasion strategies, minimizing the usefulness of inflammasomes. The difference between these two interpretations has a profound influence on how we think about the importance of inflammasomes. Vertebrate-adapted pathogens evade inflammasomes Indeed, many pathogens evade inflammasome detection. For example, S. typhimurium induces rapid and profound caspase1 activation in vitro. However, S. typhimurium encodes two T3SSs (SPI-1 and SPI-2), of which only SPI-1 is detected by NLRC4. Therefore, upon host cell entry and during the systemic phase of infection in vivo, S. typhimurium suppresses SPI-1 expression in favor of the “silent” SPI-2 system. Further, S. typhimurium represses flagellin expression (19). The significance of these strategies in vivo cannot be understated, because S. typhimurium engineered to express flagellin or SPI-1 rod protein during systemic infection are completely attenuated in WT mice but are fully virulent in inflammasome-deficient mice (4, 20). L. monocytogenes also represses flagellin and is attenuated when engineered to persistently express it (21, 22). By virtue of being a Grampositive bacterium, L. monocytogenes also naturally evades caspase-11, simply because it lacks LPS. In contrast, cytosolinvasive Francisella spp. have evolved to actively modify their LPS structure to evade caspase-11 detection (9). Another mechanism to evade cytosolic LPS detection by caspase-11 is by replicating in the vacuole, as is the case with numerous vacuolar pathogens, including S. typhimurium (13). In contrast, Yersinia spp. encode the effector YopM, which is capable of inhibiting caspase-1 by directly binding to the active site. This is critically important in vivo, because Y. pseudotuberculosis yopM mutants are attenuated in WT mice but retain full virulence in Casp12/2Casp112/2 mice (23). Similarly, Shigella flexneri encode the OspC3 T3SS effector that inhibits human caspase-4 (24), thus preventing detection of its LPS as the bacterium invades the cytosol. Direct inhibition of inflammasome components is not limited to bacteria. Indeed, poxviruses encode crmA, a serpin that can inhibit several caspases (both inflammatory and apoptotic) and was identified as a caspase-1 inhibitor before the discovery of pyroptosis (25). Further, Kaposi’s sarcoma–associated herpesvirus encodes an NLR analog (the tegument protein Orf63) that inhibits NLRP1 and NLRP3 activation of caspase-1 (26). Another possible strategy for pathogens to cope with inflammasomes is to avoid the consequences downstream of inflammasome detection. B. pseudomallei may be an example of this strategy by resisting the neutrophil killing (27) that occurs after the bacteria are ejected into the extracellular space Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 example, at day 5 postinfection, Casp12/2Casp112/2 mice have 10-fold higher burdens than wild-type (WT) mice (10). This translates into the WT mice surviving 2 d longer than Casp12/2Casp112/2 mice (11); however, it is important to remember that the WT mice still succumb, and the ultimate outcome of the infection remains unchanged. Our interpretation of these results is that inflammasomes alone cannot eradicate S. typhimurium but merely slow its kinetics, which could be beneficial in that the animal may survive long enough to develop an adaptive immune response. In further support of this hypothesis, mice infected with Listeria monocytogenes that survive to days 7–8 postinfection are able to mount a CTL response that combats and clears the infection. It is well established that this CTL response eliminates L. monocytogenes (12). Therefore, slowing the course of infection could be the positive selective pressure that maintains inflammasomes over evolutionary time. Conversely, at first glance, some infectious studies would suggest that inflammasomes have a critical role in defense. For example, we reported that Burkholderia pseudomallei is lethal in Casp12/2Casp112/2 mice, whereas WT mice survive the infection (13). On its surface, such a result seems to have incredible power, leading to the interpretation that inflammasomes fully prevent lethal infection. However, interpretation of this result is confounded by the fact that we chose the dose specifically because it was not lethal in WT mice (i.e., we defined the dose as being sublethal). When a deeper analysis of the dose response is performed, the results lead to a more nuanced interpretation. In this regard, Fabio Re’s group (14) examined three doses of B. pseudomallei infection. They demonstrated that as few as 25 CFU resulted in 100% lethality in Casp12/2Casp112/2 mice, whereas all of the WT mice survived, consistent with our results. However, when Re’s group increased the dose, they found that 100 CFU caused an intermediate lethality in WT mice, and 200 CFU was sufficient to cause 100% lethality in WT mice (14). Therefore, inflammasome detection of B. pseudomallei resulted in an ∼8-fold shift in the lethal infectious dose. Although this is not an insignificant degree of protection, the general conclusion could be that both WT and Casp12/2Casp112/2 mice succumb to extremely low-dose B. pseudomallei challenge. Thus, our interpretation is that, although experiments performed at doses just under the lethal dose can appear fully penetrant, they may actually reflect a more subtle phenotype. We next considered whether this interpretation is consistent with other published data in infectious models. In contrast to Re’s work (14), most studies only use one infectious dose. Although this limits our ability to precisely evaluate the shift in lethal dose, we can estimate the minimum change supported by the data (rubric is described in Table I legend). Because we wished to contrast various infectious models, we realized the difficulty of comparing pathogen burdens, pathology, or cytokine responses at various time points. In contrast, the lethal challenge provides the same readout (survival) for an array of pathogens, routes, and doses; the survival assay also integrates temporal effects. Therefore, we attempted to identify and collate all lethal challenge studies that were performed in inflammasome-KO mice to illustrate the relative importance of inflammasomes in defense against numerous pathogens (Table I). Unless otherwise indicated (Table I, “Notes” column), all KO mice are Casp12/2Casp112/2 957 958 BRIEF REVIEWS: REASSESSING EVOLUTIONARY IMPORTANCE OF INFLAMMASOMES Table I. Inflammasome survival studies reveal incremental inflammasome protection Pathogen Bacteria Bacillus anthracis Bacillus anthracis Ames Bacillus anthracis Sterne Burkholderia cepacia B. pseudomallei WT KO WT KO D Lethal Dosea 105 4 3 102 2.5 3 107 106 200 100 25 100 100 2 3 107 106 105 104 1000 100 106 104 100 1.5 3 105 1.5 3 105 5 3 103 106 7.4 3 104 1000 104 106 s.c. i.p. i.p. i.p. i.n. i.n. i.n. i.n. i.n. i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p. s.c. s.c. s.c. i.p. i.t. i.n. i.n. i.v. ‘ 3d ‘ ‘ 4d 4d ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ 4d 6d 3d ‘ 50 h 5d 4d 5d 80 h 3d 4d ‘ n.d. 4d 4d 2–3 d 3.5 d 1d 2d 2d 2d 3d 3d n.d. 3d 4d 3d 4d 2.5 d ‘ 45 h 5d 6d 3–4 d 0 25 0 100 n.d. 0 0 0 0 0 0 0 0 0 0 n.d. 0 0 0 0 0 100 0 40 15 0 250–350 250–350 50–100 106 2 3 107 7 3 105 100 106 108 2 3 108 1 3 104 105 105 1.5 3 104 1 3 104 1000 1 3 109 i.n. i.n. i.n. i.t. i.n. i.t. i.p. Oral Oral i.n. i.c. i.p. i.n. i.p. i.n. i.p. Oral 200 d 170 d 200 d ‘ 36 h 3d 5d 9d 8d 20 h 20 h ‘ 3d ‘ 72 h 6d 7d 148 d 170 d 110 d 6d 40 h ‘ 5d 6d 5.5 d 45 h 18 h 24 h 2.5 d 24 h 72 h 4d 6d 100 50 100 100 0 65 100 100 100 100 100 100 100 100 100 100 100 100 65 25 75 100 15 75 50 35– 65 0 0 90 0 20 10 0 0 0 75 60 100 87 100 0 0 0 23 LD50 6 3 104 8 3 103 10 2 3 105 100 i.p. i.n. i.n. i.n. i.n. s.c. 5d 8d 11 d ‘ 7d 9d 5d 7d 10 d 11 d 7d 9d No 1 3 105 105 2 3 105 5 3 106 n.s. 2 3 106 i.p. i.v. i.v. Oral Oral i.v. 6d 9d 18 d ‘ ‘ 90 d Yes Yes Yes Yes Yes 10 104 104 5 3 104 106 i.v. i.v. i.v. i.p. i.p. 9d 6.5 d 6.5 d 11 d 6d Yes Yes Yes No Yes C. violaceum No Francisella tularensis subsp. novicida Yes Francisella philomiragia K. pneumoniae No No L. monocytogenes Yes M. tuberculosis Yes Pseudomonas aeruginosa No S. typhimurium Yes S. flexneri S. aureus Streptococcus agalactiae (Group B) Streptococcus pneumoniae V. vulnificus Yersinia pestis Y. pseudotuberculosis Yes Yes Yes Yes No Yes Yes Paracoccidioides brasiliensis Parasites Plasmodium berghei Plasmodium berghei iRBCs Plasmodium berghei sporozites Plasmodium chabaudi adami Plasmodium falciparum Yes Yes Yes Yes No Yes Notes Ref. .5 .1 .5 1 .8 .8 .8 .5 .5 .1,000,000 .1,000,000 .1,000,000 .1,000,000 .1,000,000 .1,000,000 .50,000 .50,000 .50,000 .2 .1 .2 1 .1 .1 .1 .2 b (42) (43) (43) (33) (14) (14) (14) (44) (13) (29) (29) (29) (29) (29) (29) (33) (33) (33) (45) (46) (47) (33) (35) (48) (48) (49) 0 0 0 0 65 0 0 0 0 20 25 40 55 60 0 0 0 1 1 .2 .5 .1 1 1 1 1 .2 .1 .2 .1 .1 1 1 1 d 10 65 65 100 40 80 15 40 35 0 20 50 .1 .1 .1 .5 .1 .1 4d 5d 17 d 3d 5d 75 d 70 40 83 100 97 50 0 0 50 60 60 0 .2 .1 .1 .1 .1 .2 10 d 6.5 d 6.5 d 12 d 6d 40 0 0 0 0 75 0 0 0 0 .1 1 1 1 1 c c d d d d d (50) (50) (51) (52) (53) (54) (55) (10) (11) (56) (57) (58) (59) (36) (60) (23) (61) (62) (63) (64) (65) (62) (66, 67) d d d d (68) (69) (70) (71) (72) (73) (74) (75) (75) (76) (77) (Table continues) Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 Route No Vesicular stomatitis virus West Nile virus Fungi Aspergillus fumigatus Candida albicans Survival (%) Dose Vertebrate Adapted B. thailandensis Viruses Encephalomyocarditis virus Influenza A virus Time to Death The Journal of Immunology Table I. 959 (Continued ) Pathogen Toxoplasma gondii Trypanosoma cruzi Vertebrate Adapted Yes Yes Dose 4 10 103 103 Time to Death Survival (%) Route WT KO WT KO D Lethal Dosea i.p. i.p. s.c. 10 d 20 d 22 d 9d 20 d 28 d 75 70 80 10 10 90 .2 .2 .1 Notes Ref. (78) (79) (80) a Change in 100% lethality between WT and KO mice. Values based on Ref. 3 because difference was 8-fold between WT 100% lethality and lowest dose listed. However, because there was no dose at which Casp12/2Casp112/2 mice did not die, the difference was listed as .8-fold. Thus, a difference in survival percentages ,50% was estimated to be a .1-fold increase in the infectious dose, .50% was .2-fold, and .100% was .5-fold. b Mice encoding NLRP1b that could detect anthrax lethal toxin, leading to caspase-1 activation. KO mice have this sensitive NLRP1b but lack caspase-1 and caspase-11. c KO are transgenic mice expressing a 129S1/SvImJ(129S1)-derived lethal toxin-sensitive allele of Nlrp1b on a B6 background. WT mice are normal B6. d Aim22/2, Nlrp32/2, or Nlrc42/2 mice. ‘, WT mice that were tested but did not die from the infection during the period presented in the published literature; i.c., intracranial; i.n., intranasal; iRBC, infected RBC; i.t., intratracheal; n.d., not determined. Inflammasomes defend against opportunistic pathogens Vertebrate-adapted pathogens have evolved in the context of selective pressure exerted by the host immune system and have accordingly potent virulence traits. In contrast, for opportunistic pathogens for which humans are dead-end accidental hosts, there is no selective pressure to evade the human immune response. However, such microbes could also encode an impressive array of virulence factors, which can enable extreme pathogenicity. For example, Burkholderia thailandensis encodes cytosol-invasive T3SS but almost never causes infection in people (29). As such, it has been used as an experimental surrogate for its close relative B. pseudomallei (a BSL3 pathogen); in this capacity, our laboratory studied B. thailandensis infection in vivo in mice. Shockingly, during systemic infection with B. thailandensis there is a 1,000,000-fold change between Casp12/2Casp112/2 and WT mice (Table I). The resistance conferred by inflammasomes is extremely efficient; WT mice fully sterilize even high-dose B. thailandensis infection (2 3 107 CFU) within just 1 d (29). Clearly, inflammasomes can provide potent protection when presented with this specific pathogen. The strength of this phenotype was extremely surprising, rivaled only by the effect of inflammasomes upon pathogens that were engineered to remove inflammasome-evasion strategies. This made us re-evaluate the nature of B. thailandensis; we had simplistically considered it to be a surrogate for B. pseudomallei. However, it is important to keep in mind that B. thailandensis occupies a specific environmental niche as a soil microbe, where its T3SS is presumably used to invade the cytosol of an unknown eukaryotic host (30). Is B. thailandensis an isolated unique case? With these thoughts in mind, we began to screen for other environmental opportunistic pathogens that primarily cause disease in immunocompromised individuals, hypothesizing that healthy individuals would competently clear them via the activity of inflammasomes. In this endeavor, we discovered another ubiquitous environmental opportunistic pathogen against which inflammasomes play a similarly striking role, conferring a .50,000-fold shift in the lethal dose (Table I). Chromobacterium violaceum is a Gram-negative bacterium that lives in the sediment of freshwater rivers and lakes. Although C. violaceum encodes two T3SSs that are similar to Salmonella SPI-1 and SPI-2 and can be detected by NLRC4 (31), it virtually never causes disease in immunocompetent people (32). A confounding factor in the identification of these examples is that there may be redundant host defense pathways that independently confer sterilizing innate immunity. Indeed, it is quite surprising that defense against C. violaceum and B. thailandensis is monoallellic. However, our studies with C. violaceum revealed that pyroptosis is partially redundant with NK cell cytotoxicity, although both are dependent on caspase-1 activation (33). Further, if an opportunistic pathogen does not kill the host within 1 wk, the adaptive-immune response could compensate for the loss of inflammasomes. Specific virulence traits are required for inflammasome detection and defense As evidenced by the vertebrate-adapted column in Table I, several opportunistic pathogens have been examined in Casp12/2 Casp112/2 mice, yet none demonstrated such large changes in the lethal dose as seen with C. violaceum and B. thailandensis. C. violaceum encodes two T3SSs (34) and, concomitantly, defense is conferred by NLRC4 in vivo (33). B. thailandensis invades the host cytosol, and its LPS is detected by caspase-11 (13, 29), which confers defense in vivo. In both cases, it is the specific virulence traits that are detected by the inflammasomes. The evasion strategies used by vertebrate-adapted pathogens are likely not present in these environmental bacteria, because they did not evolve in the presence of potential inflammasome detection. For example, Klebsiella pneumoniae and Vibrio vulnificus are opportunistic pathogens that typically cause disease in immunocompromised patients (35, 36), which fits our model that healthy individuals are protected by inflammasomes. However, neither has a strong in vivo phenotype in inflammasome-deficient mice (Table I). Additionally, neither encodes a T3SS or is cytosol invasive, as is the case for B. thailandensis and C. violaceum. Thus, they may lack inflammasome agonists. Hypothesis The predominance of evidence suggests that vertebrateadapted pathogens evolved to minimize the effectiveness of Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 by pyroptosis (4). Staphylococcus aureus may be another example, because they are highly resistant to killing by neutrophils and have numerous toxins that inhibit neutrophil chemotaxis (28). These pathogens are striking examples that demonstrate the importance of evading inflammasome detection (Fig. 1). They show that inflammasomes could provide extremely potent defense against infection in vivo but fail to do so because many pathogens minimize detection. 960 BRIEF REVIEWS: REASSESSING EVOLUTIONARY IMPORTANCE OF INFLAMMASOMES inflammasomes; in vivo phenotypes are typically incremental, and evasion strategies are prevalent. Why, then, are inflammasomes maintained in the human genome? The extreme virulence of B. thailandensis and C. violaceum in inflammasome-deficient mice leads us to propose a new hypothesis regarding the importance of inflammasomes in the immune system. We hypothesize that inflammasomes defend against ubiquitous environmental microbes with specific virulence traits. Further, we propose that the inflammasomedirected defense is so efficient that the infection never progresses to clinically apparent symptoms. It is interesting to note that inflammasome-deficient patients have not been identified. We speculate that a survey of apparently immunocompetent people with susceptibility to specific opportunistic pathogens, such as C. violaceum, could identify patients with inflammasome mutations. For example, in a case series of 106 C. violaceum infections, only 15% were directly attributed to an immunocompromising comorbidity (such as chronic granulomatous disease) (32). Caveats to the hypothesis There are several caveats that temper interpretation of the extreme virulence of B. thailandensis and C. violaceum in Casp12/2Casp112/2 mice. One consideration is that Casp12/2 Casp112/2 animals are inbred and typically maintained on a C57BL/6 background that naturally lacks NRAMP1 (37). Mice lacking NRAMP1 have increased susceptibility to numerous intracellular pathogens, including S. typhimurium, L. monocytogenes, and Mycobacterium tuberculosis. This caveat has not gone unnoticed. In 2006, Lara-Tejero et al. (10) investigated the roles of inflammasomes in Nramp1-sufficient mice during S. typhimurium infection. However, regardless of the presence of a functional Nramp1 gene, Casp12/2Casp112/2 mice still showed an incremental susceptibility to S. typhimurium infection in comparison with their WT counterparts. Further, 129/SvEv mice are Nramp1 sufficient but carry a spontaneous mutation in caspase-11 (38). Aachoui et al. (29) infected 129/SvEv (Casp112/2Nramp1+/+) mice and found that they have extreme susceptibility to B. thailandensis that is comparable to C57BL/6 Casp112/2 (Nramp12/2) mice, whereas BALB/c (Casp11+/+Nramp12/2) mice remained resistant. Thus, the potency of inflammasome defense against this environmental bacterium was not significantly altered by the presence or absence of Nramp1. In addition to the influence of Nramp1 on different inbred mouse strains, there is the fact that mice are not men; although this may seem to be an obvious caveat, it is one worth expanding upon. There are species barriers that can influence the virulence of pathogens. There are several examples of bacteria (Shigella spp., Salmonella typhi) that fail to establish mouse infections despite infectivity in humans. However, there are far more examples of viruses that have humanspecific strains that do not infect mice: human CMV, HIV, hepatitis B virus, hepatitis C virus, EBV, measles, mumps, and many more (39). Therefore, there could be more stringent noninflammasome barriers restricting interspecies dissemination of viruses in comparison with bacteria. Therefore, our hypothesis that inflammasomes defend against environmental microbes might not hold true for viruses. Certain inflammasomes, but not all, are conserved between mice and humans. NLRC4, NLRP3, caspase-1, and AIM2 are highly conserved over evolutionary time from sharks to humans (based on searches of the Basic Local Alignment Search Tool by V.I. Maltez and E.A. Miao of inflammasome com- Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 FIGURE 1. Opportunistic microbes are readily detected by inflammasomes, whereas vertebrate-adapted pathogens evade detection. Numerous pathogens evade inflammasome detection by repression or modification of ligands (yellow lines) or by direct inhibition with specific virulence factors (red lines). Concomitantly, the beneficial effect of inflammasomes against such pathogens is typically blunted in vivo (see Table I). In contrast, two environmental bacteria (C. violaceum and B. thailandensis) are potently detected by inflammasomes (green arrows), and inflammasome defense in vivo is accordingly robust. Thus, we hypothesize that inflammasomes defend against environmental pathogens with specific virulence traits and may have limited usefulness against vertebrate-adapted bacterial pathogens, which have evolved to evade and/or inhibit them. The Journal of Immunology ponents). Yet, despite the high conservation of the ALR family member AIM2, there are 12 additional ALRs in mice but only 3 more in humans, suggesting active evolution of this gene family (40). Because AIM2/ALRs are often attributed to recognizing viral infections, the diversity within this family over evolutionary time could argue against the hypothesis that they defend against specific environmental pathogens. Similarly, mice encode three NLRP1 genes that are highly polymorphic among inbred mouse strains, and there is only one human NLRP1 (5). This again suggests active evolution of the NLRP1 locus. Thus, our hypothesis might only hold true for certain highly conserved inflammasomes and not for the more divergent or polymorphic inflammasomes. Conclusions Acknowledgments We apologize if we missed any publications in our table. We attempted to be as thorough as possible and only cited studies with live infections and both WT and inflammasome-deficient mice. 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