NLRC4 inflammasomes distinguish friend from foe

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For gut’s sake: NLRC4 inflammasomes distinguish friend from foe Bernardo S Franklin & Eicke Latz

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© 2012 Nature America, Inc. All rights reserved.

In the gut, cells of the immune system must tolerate commensal bacteria but also detect pathogens. To achieve this, intestinal phagocytes are hyporesponsive to Toll-like receptor stimulants released from commensals, but can detect invasion of pathogens via the intracellular NLRC4 inflammasome.

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he intestine is the largest compartment of the immune system and is an envi ronment crowded by trillions of bacteria. Commensal microorganisms are tolerated by the gut immune system because of their beneficial effects on the host1. However, cells of the immune system must detect and kill a large number of pathogenic microorganisms to protect the host from their invasive intentions. Hence, a fundamental question in immunol ogy is how the immune system discriminates commensal bacteria from pathogenic bacteria. In this issue of Nature Immunology, Franchi et al. provide new mechanistic insights into how this selective recognition of invading pathogenic bacteria occurs2. Cells of the innate immune system are equipped with an array of signaling receptors that recognize microbes and are involved in detecting tissue damage. Some of these receptors, such as the Toll-like receptors (TLRs), are triggered by direct contact with the microbe during phagocytosis. TLRs can even sense the mere presence of microbes in otherwise sterile environments by detecting signature molecules shed by microorgan isms. In either case, signaling receptors of the innate immune response trigger various

Bernardo S. Franklin is with the Institute of Innate Immunity, University Hospitals Bonn, University of Bonn, Germany. Eicke Latz is with the Institute of Innate Immunity, University Hospitals Bonn, University of Bonn, Germany; the Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, USA; and the German Center for Neurodegenerative Diseases, Bonn, Germany. e-mail: [email protected]

defense signaling pathways that culminate in an inflammatory response and the production of antimicrobial molecules. The microenvironment of the gut is one of the least sterile environments in the body. Unsurprisingly, the responsiveness of sensors of the innate immune response needs to be finely adjusted to the particular microenvironment in which the cells of the immune system reside to prevent uncontrolled tissue inflammation. Unrestrained immune responses to commensal microorganisms can, for example, contribute to inflammatory disease of the bowel3. However, inadequate detection of pathogenic micro organisms can result in the development of life-threatening infections or septic shock. Thus, immune homeostasis in the gut depends on a delicate balance between cellular mecha nisms that control harmful immune responses to commensals and those that maintain respon siveness to invading pathogens. The intestinal mononuclear phagocytes (iMPs) that reside in the lamina propria are mainly macrophages and dendritic cells (DCs), and macrophages greatly outnumber DCs4. It is assumed that iMPs are key regulators of gut homeostasis, as they continuously clear apop totic cells and cellular debris and act to promote tolerance to oral antigens. In stark contrast to phagocytes in other microenvironments, such as the lymph node or spleen, iMPs are aner gic to a broad range of stimuli, including TLR agonists and cytokines, and they constitu tively express anti-inflammatory cytokines5–8. Thus, iMPs have been thought to serve mainly regulatory and homeostatic functions. Given their TLR-hyporesponsive phenotype, how iMPs are able to induce an appropriate host defense against pathogenic microbes in the intestine has remained mysterious. One theory

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assumes these cells ‘ignore’ microbes and sug gests that the proinflammatory phagocytic cells found in inflammatory pathologies or infec tions of the gut represent a cell population dis tinct from resident iMPs4. The study by Franchi and colleagues, however, identifies a mecha nism by which iMPs themselves can distin guish between ‘friends and foes’ in the gut and suggests that iMPs are centrally involved in the host defense against pathogenic bacteria4. They demonstrate that the cytosolic signaling mole cule NLRC4 (IPAF) recognizes invading patho gens in the gut, whereas commensals remain undetected. NLRC4 is a member of the family of cytosolic Nod-like receptors that, after being activated, form multimolecular complexes called ‘inflammasomes’ that activate caspase-1 and interleukin 1β (IL-1β). NLRC4 is triggered when intracellular sensors of the NAIP family detect the presence of protein components deliv ered to the cytosol through bacterial type III or IV secretion systems9,10. Franchi et al. show that Salmonella or Pseudomonas species, but not the commensal bacteria Lactobacillus plantarum, Bacterioides fragilis or Enterococcus fecalis, elicit substantial amounts of mature IL-1β from iMPs but not from bone marrow– derived macrophages. This release of IL-1β is NLRC4 dependent. As type III or IV secretion systems are essential for the pathogenicity of many bacteria, iMPs seem to detect invasion via the NLRC4 intracellular sensor but are not alerted by the mere presence of bacteria or their soluble components2. In a mouse model of salmonellosis, they find no differences in susceptibility when NLRC4 is lacking on the C57BL/6 background. However, BALB/c mice deficient in NLRC4 (and IL-1R) are more susceptible to orogastric challenge with Salmonella and have higher titers of bacteria in 429

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Commensal bacteria

TLR signaling

Pathogenic bacteria

Blood macrophage

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-κ NF

Type III secretion system Flagellin NLRC4 Inflammasome

NF-κBinduced pro-IL-1β TNF IL-6

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IL-1β

Commensal bacteria Pathogenic bacteria

TLR tolerance

Neutrophil recruitment and activation

Flagellin Type III secretion system

Constitutively expressed pro-IL-1β

iMPs

IL-1β

Figure 1 Phagocytic cells respond to stimulants of the innate immune response and microbes depending on the microenvironment in which they are located. Nonpathogenic or pathogenic bacteria activate phagocytes in the blood or lymph system via the TLR system to induce proinflammatory cytokines and pro-IL-1b in the cytosol (top). In contrast, intestinal phagocytic cells are largely tolerant to such stimuli and do not produce proinflammatory cytokines, yet they constitutively express pro-IL-1b. During invasion by pathogenic bacteria, intestinal phagocytes activate the NLRC4 inflammasome, which leads to cleavage and release of IL-1b (below). These cytokines are important mediators of neutrophil recruitment and the polarization of effector T cells. TNF, tumor-necrosis factor.

several organs than do their wild-type counter parts. The reasons for the observed differences among the mouse strains tested remain unclear. It seems possible that in BALB/c mice, which carry a point mutation in the gene encoding the pathogen-resistance protein Nramp1 and are thus hypersusceptible to infection with Salmonella, NLRC4 deficiency is function ally more important for host defense against pathogenic bacteria, or that supraphysiological doses of bacteria could obscure the phenotype. Indeed, another published study investigating a different Salmonella infection model has shown that loss of NLRC4 on the C57BL/6 background results in greater susceptibility to infection with flagellate Salmonella species but not with aflagellate Salmonella species11. Given such strain- and model-dependent disparities in disease outcome, the studies by Franchi et al. 430

of human colonic macrophages are particularly important. They isolate, from healthy human colons, cells of the immune system that express the LPS receptor CD14 and challenge these cells with Salmonella. They find that human CD14+ intestinal cells mirror the effects noted in iMPs isolated from mouse intestines: they respond to flagellated Salmonella with caspase-1 activation and IL-1β while being similarly hyporespon sive to extracellular TLR stimulation. The iMPs further differ from their phago cytic counterparts that reside outside the gut in that they constitutively express pro-IL-1β. This allows iMPs to respond directly to pathogenic bacteria without needing to be primed through a TLR or other type of innate sensor2 (Fig. 1). However, the mechanisms that selectively regu late pro-IL-1β expression in iMPs remain unde termined. One possibility raised by the authors

is that epithelial and/or stromal cells respond to commensal bacteria by inducing molecules that upregulate pro-IL-1β in resident iMPs. Indeed, an intriguing published report has suggested that a three-way crosstalk among the immune system, commensal bacteria and the intestinal epithelial cells is essential for normal gut function12. B cells are key con tributors to gut immunity through the secre tion of large amounts of immunoglobulin A into the intestine. In the absence of B cells or immunoglobulin A, the intestinal epithelium can adapt and drive alternative defensive functions toward the resident microbiota at the expense of the epithelium’s normal fatabsorbing functions12. It seems that research is just beginning to elucidate the intricate molecular coexistence of cells of the immune system with the many microbes hosted by humans. The proposed mechanism whereby the NLRC4 inflam masome senses pathogens by detecting their secretion systems seems simple and plausible. However, extensive genomic analysis of micro biota in the gut by deep sequencing has also shown the existence of type III and IV secre tion systems in species that are deemed com mensals. Whether other pathogenicity factors are necessary for these commensals to become pathogenic or whether their secretion systems differ is not understood. Furthermore, not all pathogens are dependent on the expression of secretion systems, which therefore necessitates the existence of additional host mechanisms for the detection of a microbe’s pathogenicity. Conceivably, other cell types in the intestinal barrier, such as M cells or DCs, which were not explicitly studied by Franchi et al., might contribute to the discrimination of harmless microorganisms versus potentially pathogenic microorganisms. Exciting work has been pub lished on yet another apparently intestinespecific immune-defense mechanism. Gut epithelial cells have been found to express components of the NLRP6 inflammasome, and deficiency in NLRP6 leads to profound changes in gut microbiota13. Intriguingly, the dysbiosis of NLRP6-deficient mice results in exacerbation of chemically induced coli tis, which suggests that inflammasomes also act to shape gut microbiota into a ‘tolerated’ state13. The concept that inflammasomes sense pathogenic bacteria is not restricted to cells of the gut immune system. In the lung, another organ continuously exposed to microbes, only bacteria with translocation-competent type III secretion systems activate a vicious immune response via a caspase-1- and IL-1dependent pathways14. Such studies high light the crucial role of inflammasomes in the detection of microbes and suggest that

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n e w s a nd v i e w s downstream IL-1β- or IL-18-dependent effector mechanisms, such as the recruitment of neutrophils and the induction of particular T cell subpopulations, contribute to effective antimicrobial defense (Fig. 1). There is a clear need for better understanding of the exact role of the various inflammasomes in maintaining microbial homeostasis and in the defense against pathogens. This is of particular importance as pharmacological strategies are now being explored clinically for the blockade of inflammasomes and the IL-1β family.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Lebeer, S., Vanderleyden, J. & De Keersmaecker, S.C. Nat. Rev. Microbiol. 8, 171–184 (2010). 2. Franchi., L. et al. Nat. Immunol. 13, 449–456 (2012). 3. Xavier, R.J. & Podolsky, D.K. Nature 448, 427–434 (2007). 4. Bain, C.C. & Mowat, A.M. Eur. J. Immunol. 41, 2494–2498 (2011). 5. Manicassamy, S. et al. Science 329, 849–853 (2010). 6. Denning, T.L., Wang, Y.C., Patel, S.R., Williams, I.R. & Pulendran, B. Nat. Immunol. 8, 1086–1094 (2007).

7. Smythies, L.E. et al. J. Clin. Invest. 115, 66–75 (2005). 8. Lotz, M. et al. J. Exp. Med. 203, 973–984 (2006). 9. Franchi, L. et al. Nat. Immunol. 7, 576–582 (2006). 10. Kofoed, E.M. & Vance, R.E. Nature 477, 592–595 (2011). 11. Carvalho, F.A. et al. Mucosal Immunol. advance online publication, doi:10.1038/mi.2012.8 (8 February 2012). 12. Shulzhenko, N. et al. Nat. Med. 17, 1585–1593 (2011). 13. Elinav, E. et al. Cell 145, 745–757 (2011). 14. Wangdi, T., Mijares, L.A. & Kazmierczak, B.I. Infect. Immun. 78, 4744 (2010).

Thymic signatures of tailored peripheral functions

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Marc Bonneville Gene profiling by the Immunological Genome Project consortium has identified distinct transcriptional programs that determine the migratory and functional fates of emerging gd thymocytes.

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or many years, major histocompatibility complex–restricted αβ T cells were thought to be the only participants in cellular adaptive immunity, responsible for all beneficial and harmful effects, so the discovery in the 1980s of T cells that express a different class of T cell antigen receptors (TCRs) with γδ subunits came as a surprise. What might their physiological functions be? It is now known that many γδ T cells survey the tissues in which they reside for early signs of infection or cell alterations1. They express discrete sets of TCR variable (V) regions and have pre-established effector functions that differ from one tissue location to another1,2. For some γδ T cell subsets, these functions are programmed during their intrathymic development3,4. It is not known how general this developmental programming is or when it occurs. In this issue of Nature Immunology, Kang and colleagues suggest that most emerg ing γδ thymocytes are already engaged in transcriptional programs that impose the coordinated acquisition of restricted migratory properties and effector functions5. Their work also identifies similarities between the develop mental programs of γδ T cells and those of other tissue-resident lymphoid subsets with ‘innate’ or ‘innate-like’ characteristics. Barrier tissues such as skin and mucosae are under constant assault by physicochemical Marc Bonneville is with the Cancer Research Center Nantes-Angers, UMR 892 Institut National de la Santé et de la Recherche Médicale, Nantes University, and UMR6299 Centre National de la Recherche Scientifique, Nantes, France. e-mail: [email protected]

and microbial stress agents. Epithelial cells have developed several weapons to fight local infection rapidly, but the maintenance of tissue integrity requires the extra help of resident effector cells of the immune system. Most circulating T cells in young mice and humans express αβ TCRs and display mark ers of antigen-naive lymphocytes. In contrast, many tissue-resident lymphoid cells express γδ TCRs, show hallmarks of effector memory T cells and carry receptors specific for con served stress-induced ligands1,2. This allows swift activation in response to epithelial stress and the early release of effector molecules with bactericidal, cytotoxic, inflammatory or epithe lial trophic properties. Although γδ T cells as a whole have broad functional potential, they generally serve a discrete set of effector func tions in a given tissue. For example, γδ T cells in liver tend to produce both interleukin 4 (IL-4) and interferon-γ (IFN-γ)6, cytokines involved in the generation of antibody responses by immunoglobulins E and G1 and the control of various parasitic and intracellular infections. In contrast, γδ T cells in the peritoneal cavity or mucosa of reproductive organs tend to pro duce IL-17A1,2, a proinflammatory molecule that contributes to neutrophil attraction and protection against extracellular bacterial and fungal infections. The tissue location and functional features of peripheral γδ T cells also correlate with the expression of distinct sets of identical or highly related TCRs1,2,7. Indeed, most peripheral γδ T cells exploit a tiny frac tion of the vast TCR repertoire that can be gen erated by somatic recombination of TCR gene segments. Typically, many mouse γδ T cells

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residing in the epidermis, liver and mucosa of reproductive organs express identical, so-called ‘invariant’, TCRs that contain Vγ3Vδ1, Vγ4Vδ1 and Vγ1.1Vδ6.3 variable regions (referred to as the V3, V4 and V6 subsets by Kang and colleagues5). In other locations, such as the intestinal epithelium or spleen, γδ T cells use a greater diversity of TCRs that nevertheless remain very biased in terms of TCR V-region composition2. The dominant expression of invariant V3 TCRs by intraepidermal γδ T cells is accounted for by TCR recombina tion and enzymatic constraints that favor the generation of these particular TCRs in fetal γδ thymocytes7 and by intrathymic selection processes that induce the selective expression of skin-homing receptors on fetal thymocytes after engagement of invariant V3 TCRs by stromal ligands8,9. This process allows skin colonization by the fetal V3 subset early after birth, which is then self-renewed locally during adult life10. Subsequent studies have suggested that TCR signals received by γδ thymocytes also affect their functional fate3,4,11. However, a unifying picture is still lacking for the genera tion of peripheral subsets with pre-established tissue-homing and effector features. To gain more insight into this process, Kang and colleagues have undertaken an in-depth transcriptome analysis of γδ thymocyte subsets, sorted on the basis of expression of the cell sur face marker CD24 (heat-stable antigen) and the use of particular TCR Vγ or Vδ regions5. As CD24 is rapidly down-modulated on both αβ thymo cytes and γδ thymocytes after the engagement of TCRs by selecting thymic ligands, they use this marker to distinguish immature (preselected) 431