An inherited mutation in NLRC4 causes autoinflammation in human and mice

Autoinflammatory syndromes cause sterile inflammation in the absence of any signs of autoimmune responses. Familial cold autoinflammatory syndrome (FCAS) is characterized by intermittent episodes of rash, arthralgia, and fever after exposure to cold stimuli. We have identified a missense mutation in the NLRC4 gene in patients with FCAS. NLRC4 has been known as a crucial sensor for several Gram-negative intracellular bacteria. The mutation in NLRC4 in FCAS patients promoted the formation of NLRC4-containing inflammasomes that cleave procaspase-1 and increase production of IL-1β. Transgenic mice that expressed mutant Nlrc4 under the invariant chain promoter developed dermatitis and arthritis. Inflammation within tissues depended on IL-1β–mediated production of IL-17A from neutrophils but not from T cells. Our findings reveal a previously unrecognized link between NLRC4 and a hereditary autoinflammatory disease and highlight the importance of NLRC4 not only in the innate immune response to bacterial infections but also in the genesis of inflammatory diseases.Autoinflammatory syndrome is characterized by inflammatory responses in the absence of autoimmunity or infections, and it is generally caused by hyperactivation of innate immune cells (Chen and Nuñez, 2010; Park et al., 2012). Several studies, including those from our group, have identified the causative genes for familial autoinflammatory syndromes (McDermott et al., 1999; Jéru et al., 2008; Masters et al., 2009; Agarwal et al., 2010; Kitamura et al., 2011; Liu et al., 2012; Park et al., 2012). Among these genes, mutations in NLRP3 cause autoinflammatory syndromes, including familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal onset multisystem inflammatory disease (NOMID; Hoffman et al., 2001; Jéru et al., 2008; Masters et al., 2009; Aksentijevich and Kastner, 2011; Park et al., 2012). These diseases are named cryopyrin-associated periodic syndromes (CAPS). FCAS, the mildest of the CAPS, is characterized by rash, fever, and arthralgia by exposure to cold stimuli. Patients with MWS have more frequent inflammatory episodes and they frequently develop progressive sensorineural hearing loss and systemic amyloidosis. NOMID is the most severe of the three syndromes and is characterized by severe chronic inflammation involving the joints and nervous system. However, there are still significant numbers of CAPS without any mutations in NLRP3 (Aksentijevich et al., 2007).Heterozygous mutations in NLRP3 result in overactivation of caspase 1. This enzyme cleaves the precursors of IL-1β and IL-18 (members of the IL-1 family of cytokines) into their active forms (Masters et al., 2009; Aksentijevich and Kastner, 2011). The recombinant IL-1 receptor antagonist anakinra, canakinumab, and the IL-1 receptor type I fusion protein rilonacept have induced clinical response in CAPS, demonstrating that signaling via the IL-1 receptor is crucial for the pathogenesis of CAPS (Aksentijevich and Kastner, 2011; Dinarello and van der Meer, 2013). Recent studies have provided evidence that heterozygous mutations in NLRP12 cause FCAS-like symptoms (Jéru et al., 2008). The mutations are reported to inhibit NF-κB or activate caspase 1, depending on the genetic variation (Jéru et al., 2008; Jéru et al., 2011).In the current study, we used exome resequencing to analyze candidate genes of patients in one Japanese family with cold-induced urticaria and arthritis but without mutations in NLRP3 or NLRP12. We identified a heterozygous missense mutation in NLRC4. The mutant NLRC4 activates caspase-1 and this activation results in increased production of IL-1β. Expression of the mutant Nlrc4 in mice causes severe dermatitis, arthritis, and splenomegaly with augmented infiltration of neutrophils as well as cold-induced exanthema. The inflammation depended on IL-1β and IL-17A produced by neutrophils but not T cells. These data indicate that NLRC4 is a causative gene for this disease and highlight the crucial roles of NLRC4 not only in the innate immune response to bacterial infections but also in the pathogenesis of human inflammatory diseases.     

In vivo regulation of interleukin 1β in patients with cryopyrin-associated periodic syndromes

The investigation of interleukin 1β (IL-1β) in human inflammatory diseases is hampered by the fact that it is virtually undetectable in human plasma. We demonstrate that by administering the anti–human IL-1β antibody canakinumab (ACZ885) to humans, the resulting formation of IL-1β–antibody complexes allowed the detection of in vivo–produced IL-1β. A two-compartment mathematical model was generated that predicted a constitutive production rate of 6 ng/d IL-1β in healthy subjects. In contrast, patients with cryopyrin-associated periodic syndromes (CAPS), a rare monogenetic disease driven by uncontrolled caspase-1 activity and IL-1 production, produced a mean of 31 ng/d. Treatment with canakinumab not only induced long-lasting complete clinical response but also reduced the production rate of IL-1β to normal levels within 8 wk of treatment, suggesting that IL-1β production in these patients was mainly IL-1β driven. The model further indicated that IL-1β is the only cytokine driving disease severity and duration of response to canakinumab. A correction for natural IL-1 antagonists was not required to fit the data. Together, the study allowed new insights into the production and regulation of IL-1β in man. It also indicated that CAPS is entirely mediated by IL-1β and that canakinumab treatment restores physiological IL-1β production.IL-1α and β, which were originally described as leukocytic pyrogens (1), are important regulators of the response to tissue damage and infections and mediate symptoms of fever, fatigue, pain, arthritis, and the hepatic acute phase responses including synthesis of C-reactive protein (CRP) and serum amyloid A protein (SAA) (2). Although studies using recombinant IL-1 in cancer patients confirmed the causative role of IL-1 for many of these symptoms (3), its direct investigation in man is hampered by the inability to detect IL-1 in biological fluids. cryopyrin-associated periodic syndromes (CAPS) is a clinical disease syndrome resulting from heterozygous gain-of-function mutations in NLRP3, the gene encoding cryopyrin. These mutations are supposed to promote release of IL-1, thereby providing an excellent paradigm for studying human IL-1 in vivo (4, 5). CAPS patients present with a spectrum of autoinflammatory diseases (6–11) involving almost all organ systems. NLRP3 mutations result in overactivation of caspase 1, the enzyme which cleaves the precursors of IL-1β, IL-18, and IL-33, members of the IL-1 family of cytokines, into their active forms (12). Although pro–IL-1α is not a substrate of caspase 1, recent studies in mice indicate that secretion of bioactive IL-1α requires functional NLRP3 (13) and activated caspase-1 (14). The recombinant IL-1 receptor antagonist (IL-1Ra) anakinra and the IL-1 receptor type I (IL-1RI) fusion protein rilonacept (IL-1 trap) have both induced clinical response in CAPS, demonstrating that signaling via the IL-1RI is crucial for the pathogenesis of CAPS (15–17). This strongly implies that neither IL-18 nor IL-33 plays significant roles in the disease, as neither of these two cytokines signals via the IL-1RI, and suggests that the disease is caused by overproduction of IL-1. By administering the human anti–IL-1β antibody canakinumab to CAPS patients, we provide evidence in this paper that IL-1β is pivotal in the pathogenesis of CAPS. Treatment with the antibody allowed the detection of IL-β and the creation of a mathematical model, which indicates that the in vivo production rate of IL-1β is fivefold higher in CAPS as compared with healthy subjects. Furthermore, in vivo IL-β production could be completely restored in these patients after canakinumab treatment.     

Aberrant actin depolymerization triggers the pyrin inflammasome and autoinflammatory disease that is dependent on IL-18, not IL-1β

Gain-of-function mutations that activate the innate immune system can cause systemic autoinflammatory diseases associated with increased IL-1β production. This cytokine is activated identically to IL-18 by an intracellular protein complex known as the inflammasome; however, IL-18 has not yet been specifically implicated in the pathogenesis of hereditary autoinflammatory disorders. We have now identified an autoinflammatory disease in mice driven by IL-18, but not IL-1β, resulting from an inactivating mutation of the actin-depolymerizing cofactor Wdr1. This perturbation of actin polymerization leads to systemic autoinflammation that is reduced when IL-18 is deleted but not when IL-1 signaling is removed. Remarkably, inflammasome activation in mature macrophages is unaltered, but IL-18 production from monocytes is greatly exaggerated, and depletion of monocytes in vivo prevents the disease. Small-molecule inhibition of actin polymerization can remove potential danger signals from the system and prevents monocyte IL-18 production. Finally, we show that the inflammasome sensor of actin dynamics in this system requires caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain, and the innate immune receptor pyrin. Previously, perturbation of actin polymerization by pathogens was shown to activate the pyrin inflammasome, so our data now extend this guard hypothesis to host-regulated actin-dependent processes and autoinflammatory disease.Autoinflammatory syndromes are caused by dysregulation of the innate immune system, frequently affecting the inflammasome or other pathogen recognition pathways and leading to the overproduction of active IL-1β and IL-18 (Masters et al., 2009). To date, there are at least 12 known genetic causes of autoinflammatory disease, including familial Mediterranean fever (FMF), hyper-IgD syndrome, and cryopyrin-associated periodic syndrome. Therapeutic options for these diseases include nonsteroidal antiinflammatory drugs, corticosteroids, colchicine (for FMF), anti-TNF, and direct blockade of IL-1, which can be highly efficacious (Masters et al., 2009; Caso et al., 2013). IL-18 and IL-1β are produced in many cells, including monocytes and macrophages (Okamura et al., 1995; Ushio et al., 1996). IL-18 and IL-1β are produced as precursors and do not have a signal peptide to facilitate their secretion; instead, they are activated and released extracellularly as mature proteins after cleavage by caspase-1 (Li et al., 1995; Ghayur et al., 1997; Gu et al., 1997). Despite these similarities, there is no known hereditary autoinflammatory disease where the pathology is caused exclusively by IL-18.The inflammasome is an intracellular molecular platform that forms in response to pathogen- or danger-associated molecular patterns (DAMPs), leading to recruitment and activation of caspase-1 (Martinon et al., 2002; Schroder and Tschopp, 2010). A growing number of inflammasomes have been reported, each nucleated by a different innate immune receptor, such as NLRP1 (Martinon et al., 2000; Boyden and Dietrich, 2006), NLRP3 (Agostini et al., 2004), NLRC4 (Franchi et al., 2006), pyrin (Chae et al., 2011), and AIM2 (Hornung et al., 2009). Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is a key adaptor used by most of these innate immune receptors to interact with and recruit caspase-1 (Srinivasula et al., 2002). Activating mutations in NLRP3 result in increased IL-1β and IL-18 production, which can be prevented in mice by deleting caspase-1 or ASC. Furthermore, deleting either the IL-18R or the IL-1R can both independently protect mice from this NLRP3-mediated autoinflammatory disease (Brydges et al., 2013). For the FMF protein, pyrin, activating mutations induce ASC-dependent but NLRP3-independent IL-1β activation and cause severe autoinflammation in mice (Chae et al., 2011). Interestingly, pyrin interacts with ASC, microtubules, and actin filaments (Mansfield et al., 2001; Richards et al., 2001; Waite et al., 2009), and it has recently been shown that modification of RhoGTPases by bacterial toxins can trigger the pyrin inflammasome, perhaps via modulation of actin dynamics (Xu et al., 2014). This raises the fascinating prospect of a link between perturbations in the actin cytoskeleton and autoinflammatory disease.Wdr1 is required for disassembly of actin filaments in conjunction with the actin-depolymerizing factor/cofilin family of proteins. Mice homozygous for a hypomorphic allele of Wdr1 (Wdr1^rd/rd) exhibit spontaneous autoinflammatory disease and thrombocytopenia (Kile et al., 2007). Both defects have been suggested to result from a disruption in actin dynamics. Thrombocytopenia results from defects in megakaryocytes, a cell type that is entirely dependent on a functional cytoskeleton to shed platelets (Patel et al., 2005). Wdr1 mutant mice also exhibit neutrophilia; however, the critical inflammatory mediators and cell types important for the development of inflammation in this genetic condition are unclear (Kile et al., 2007). Intriguingly, Wdr1 was found to be secreted after caspase-1 activation (Keller et al., 2008).We examined the role of key inflammatory mediators that drive autoinflammation in Wdr1^rd/rd mice and demonstrated that this disease is IL-18 dependent, but IL-1 independent. As expected, this IL-18 is produced by the inflammasome; however, it is not produced from neutrophils or macrophages, but instead only from monocytes. Finally, we found that the autoinflammatory disease was mediated by pyrin, providing evidence that this innate immune receptor recognizes alterations in the actin polymerization pathway.     

Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

Nitric oxide (NO) is a ubiquitous mediator of inflammation and immunity, involved in the pathogenesis and control of infectious diseases, autoimmunity, and cancer. We observed that the expression of nitric oxide synthase-2 (NOS2/iNOS) positively correlates with Th17 responses in patients with ovarian cancer (OvCa). Although high concentrations of exogenous NO indiscriminately suppress the proliferation and differentiation of Th1, Th2, and Th17 cells, the physiological NO concentrations produced by patients’ myeloid-derived suppressor cells (MDSCs) support the development of RORγt(Rorc)⁺IL-23R⁺IL-17⁺ Th17 cells. Moreover, the development of Th17 cells from naive-, memory-, or tumor-infiltrating CD4⁺ T cells, driven by IL-1β/IL-6/IL-23/NO-producing MDSCs or by recombinant cytokines (IL-1β/IL-6/IL-23), is associated with the induction of endogenous NOS2 and NO production, and critically depends on NOS2 activity and the canonical cyclic guanosine monophosphate (cGMP)–cGMP-dependent protein kinase (cGK) pathway of NO signaling within CD4⁺ T cells. Inhibition of NOS2 or cGMP–cGK signaling abolishes the de novo induction of Th17 cells and selectively suppresses IL-17 production by established Th17 cells isolated from OvCa patients. Our data indicate that, apart from its previously recognized role as an effector mediator of Th17-associated inflammation, NO is also critically required for the induction and stability of human Th17 responses, providing new targets to manipulate Th17 responses in cancer, autoimmunity, and inflammatory diseases.Nitric oxide (NO; a product of nitrite reduction or the NO synthases NOS1, NOS2, and NOS3; Culotta and Koshland, 1992), is a pleiotropic regulator of neurotransmission, inflammation, and autoimmunity (Culotta and Koshland, 1992; Bogdan, 1998, 2001; Kolb and Kolb-Bachofen, 1998) implicated both in cancer progression and its immune-mediated elimination (Culotta and Koshland, 1992; Coussens and Werb, 2002; Hussain et al., 2003; Mantovani et al., 2008). In different mouse models, NO has been paradoxically shown to both promote inflammation (Farrell et al., 1992; Boughton-Smith et al., 1993; McCartney-Francis et al., 1993; Weinberg et al., 1994; Hooper et al., 1997) and to suppress autoimmune tissue damage through nonselective suppression of immune cell activation (Bogdan, 2001; Bogdan, 2011), especially at high concentrations (Mahidhara et al., 2003; Thomas et al., 2004; Niedbala et al., 2011). Although previous studies demonstrated a positive impact of NO on the induction of Th1 cells (Niedbala et al., 2002) and forkhead box P3–positive (FoxP3⁺) regulatory T (T reg) cells (Feng et al., 2008) in murine models, the regulation and function of the NO synthase (NOS)–NO system have shown profound differences between mice and humans (Schneemann and Schoedon, 2002, Schneemann and Schoedon, 2007; Fang, 2004), complicating the translation of these findings from mouse models to human disease.In cancer, NOS2-derived NO plays both cytotoxic and immunoregulatory functions (Bogdan, 2001). It can exert distinct effects on different subsets of tumor-infiltrating T cells (TILs), capable of blocking the development of cytotoxic T lymphocytes (CTLs; Bronte et al., 2003), suppressing Th1 and Th2 cytokine production, and modulating the development of FoxP3⁺ T reg cells (Brahmachari and Pahan, 2010; Lee et al., 2011). NOS2-driven NO production is a prominent feature of cancer-associated myeloid-derived suppressor cells (MDSCs; Mazzoni et al., 2002; Kusmartsev et al., 2004; Vuk-Pavlović et al., 2010; Bronte and Zanovello, 2005), which in the human system are characterized by a CD11b⁺CD33⁺HLA-DR^low/neg phenotype consisting of CD14⁺ monocytic (Serafini et al., 2006; Filipazzi et al., 2007; Hoechst et al., 2008; Obermajer et al., 2011) and CD15⁺ granulocytic (Zea et al., 2005; Mandruzzato et al., 2009; Rodriguez et al., 2009) subsets (Dolcetti et al., 2010; Nagaraj and Gabrilovich, 2010).Production of NO in chronic inflammation is supported by IFN-γ and IL-17 (Mazzoni et al., 2002; Miljkovic and Trajkovic, 2004), the cytokines produced by human Th17 cells (Veldhoen et al., 2006; Acosta-Rodriguez et al., 2007a,b; van Beelen et al., 2007; Wilson et al., 2007). Human Th17 cells secrete varying levels of IFN-γ (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; Kryczek et al., 2009; Miyahara et al., 2008; van Beelen et al., 2007; Wilson et al., 2007) and have been implicated both in tumor surveillance and tumor progression (Miyahara et al., 2008; Kryczek et al., 2009; Martin-Orozco and Dong, 2009). Induction of Th17 cells typically involves IL-1β, IL-6, and IL-23 (Bettelli et al., 2006; Acosta-Rodriguez et al., 2007a,b; Ivanov et al., 2006; van Beelen et al., 2007; Veldhoen et al., 2006; Wilson et al., 2007; Zhou et al., 2007), with the additional involvement of TGF-β in most mouse models (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006; Zhou et al., 2007; Ghoreschi et al., 2010), but not in the human system (Acosta-Rodriguez et al., 2007a; Wilson et al., 2007). IL-1β₁, IL-6, and IL-23 production by monocytes and DCs, and the resulting development of human Th17 cells, can be induced by bacterial products, such as LPS or peptidoglycan (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; van Beelen et al., 2007). However, the mechanisms driving Th17 responses in noninfectious settings, such as autoimmunity or cancer, remain unclear.Here, we report that the development of human Th17 cells from naive, effector, and memory CD4⁺ T cell precursors induced by the previously identified Th17-driving cytokines (IL-1β, IL-6, and IL-23) or by IL-1β/IL-6/IL-23-producing MDSCs, is promoted by exogenous NO (or NO produced by human MDSCs) and critically depends on the induction of endogenous NOS2 in differentiating CD4⁺ T cells.     

Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella

Intracellular pathogens and endogenous danger signals in the cytosol engage NOD-like receptors (NLRs), which assemble inflammasome complexes to activate caspase-1 and promote the release of proinflammatory cytokines IL-1β and IL-18. However, the NLRs that respond to microbial pathogens in vivo are poorly defined. We show that the NLRs NLRP3 and NLRC4 both activate caspase-1 in response to Salmonella typhimurium. Responding to distinct bacterial triggers, NLRP3 and NLRC4 recruited ASC and caspase-1 into a single cytoplasmic focus, which served as the site of pro–IL-1β processing. Consistent with an important role for both NLRP3 and NLRC4 in innate immune defense against S. typhimurium, mice lacking both NLRs were markedly more susceptible to infection. These results reveal unexpected redundancy among NLRs in host defense against intracellular pathogens in vivo.IL-1β is a central orchestrator of immunity against various classes of pathogens and a key trigger of inflammatory diseases. It is produced by activated macrophages as a proprotein, which is proteolytically processed to its active form by the cysteine protease caspase-1. Caspase-1 itself is synthesized as a zymogen and must be activated by autocatalytic cleavage. This activation involves recruitment of pro–caspase-1 into multiprotein complexes called inflammasomes, which are comprised of the adapter protein ASC and a NOD-like receptor (NLR), with the latter conferring specificity for microbial and endogenous products (Mariathasan and Monack, 2007).The NLR family of cytosolic receptors responds to a variety of pathogens and endogenous danger signals (Brodsky and Monack, 2009). For example, NLRC4 (also known as Ipaf) is required by cultured primary macrophages to activate caspase-1 after infection with Salmonella spp., Pseudomonas aeruginosa, Listeria monocytogenes, and Legionella pneumophila. NLRC4 is thought to recognize bacterial flagellin that is injected accidentally into the cytoplasm by bacterial virulence-associated secretion systems (Amer et al., 2006; Franchi et al., 2006; Miao et al., 2006; Ren et al., 2006). Recently, it has been reported that NLRC4 also detects the rod subunit of certain type 3 secretion systems (T3SSs; Miao et al., 2010), which explains how NLRC4 can detect nonflagellated bacteria such as Shigella flexneri and some strains of P. aeruginosa (Sutterwala et al., 2007; Suzuki et al., 2007). In contrast to NLRC4, NLRP3 (also known as Nalp3) is activated by diverse molecules originating from viruses (Sendai, influenza, and adenoviral strains; Kanneganti et al., 2006; Muruve et al., 2008), fungi (Candida albicans and Saccharomyces cerevisiae; Gross et al., 2009; Hise et al., 2009), and bacteria (Staphylococcus aureus, Neisseria gonorrhoeae, and L. monocytogenes; Mariathasan et al., 2006; Warren et al., 2008; Duncan et al., 2009). The precise signal that is detected by NLRP3 remains unclear. Common terminal signals appear to involve damage to membranes and/or changes in potassium levels (Brodsky and Monack, 2009). Other NLRs include NLRP1, which is activated by anthrax lethal toxin, and Aim2, which appears to recognize double-stranded DNA in the cytoplasm (Fernandes-Alnemri et al., 2009; Hornung et al., 2009).Although several different inflammasomes have been described, the precise molecular structure and composition of inflammasomes remains largely unknown. In vitro studies with purified inflammasome components suggest that NLRs bind their ligands and oligomerize to form a platform for dimerization and autoproteolytic cleavage of pro–caspase-1 (Faustin et al., 2007). Under certain lysis conditions, inflammasome components can form structures between 500 and 700 kD in size (Martinon et al., 2004; Hsu et al., 2008). Overexpressed ASC also forms large aggregates (Masumoto et al., 1999; Richards et al., 2001; Fernandes-Alnemri et al., 2007), although the physiological significance of these aggregates is unclear.Caspase-1 is important for innate immune defense against many pathogens, but the contributions of specific NLRs to caspase-1 activation during animal infections are less clear. Previous in vivo studies indicated a role for NLRC4 in defense against L. pneumophila and P. aeruginosa but not Salmonella (Amer et al., 2006; Lara-Tejero et al., 2006; Sutterwala et al., 2007). This last finding was surprising, given that NLRC4 was critical for caspase-1 activation by macrophages infected with Salmonella in culture (Mariathasan et al., 2004). Thus far, the only described roles for NLRP3 in vivo during microbial infections are in defense against C. albicans, Plasmodium, and influenza A virus infections (Allen et al., 2009; Dostert et al., 2009; Gross et al., 2009; Hise et al., 2009; Joly et al., 2009; Shio et al., 2009; Thomas et al., 2009), whereas no role has been described in bacterial infections so far.Salmonella enterica serovar typhimurium (Stm) is an intracellular Gram-negative bacterium that causes infections in human and animal hosts. After initial colonization of the gastrointestinal tract, Stm persists in tissue macrophages. Salmonella virulence requires two T3SSs encoded on Salmonella pathogenicity islands: SPI-1 and SPI-2. SPI-1 is necessary to invade epithelial cells in the gut, whereas SPI-2 is necessary for replication in macrophages (Beuzón et al., 2000). Inflammasomes play an important role in innate immune defense against Stm because mice lacking caspase-1, IL-1β, or IL-18 succumb earlier to infection and have higher bacterial loads (Raupach et al., 2006). Stm expressing the SPI-1 T3SS induces caspase-1– and NLRC4–dependent cell death and IL-1β processing in cultured macrophages. Because NLRC4 is not essential for Stm clearance in mice (Lara-Tejero et al., 2006), we investigated whether multiple NLRs respond to Stm infection in the whole animal.     

IL-9–mediated survival of type 2 innate lymphoid cells promotes damage control in helminth-induced lung inflammation

IL-9 fate reporter mice established type 2 innate lymphoid cells (ILC2s) as major producers of this cytokine in vivo. Here we focus on the role of IL-9 and ILC2s during the lung stage of infection with Nippostrongylus brasiliensis, which results in substantial tissue damage. IL-9 receptor (IL-9R)–deficient mice displayed reduced numbers of ILC2s in the lung after infection, resulting in impaired IL-5, IL-13, and amphiregulin levels, despite undiminished numbers of Th2 cells. As a consequence, the restoration of tissue integrity and lung function was strongly impaired in the absence of IL-9 signaling. ILC2s, in contrast to Th2 cells, expressed high levels of the IL-9R, and IL-9 signaling was crucial for the survival of activated ILC2s in vitro. Furthermore, ILC2s in the lungs of infected mice required the IL-9R to up-regulate the antiapoptotic protein BCL-3 in vivo. This highlights a unique role for IL-9 as an autocrine amplifier of ILC2 function, promoting tissue repair in the recovery phase after helminth-induced lung inflammation.The cytokine IL-9 was discovered more than 20 yr ago and described as a T cell and mast cell growth factor produced by T cell clones (Uyttenhove et al., 1988; Hültner et al., 1989; Schmitt et al., 1989). Subsequently, IL-9 was shown to promote the survival of a variety of different cell types in addition to T cells (Hültner et al., 1990; Gounni et al., 2000; Fontaine et al., 2008; Elyaman et al., 2009). Until recently, Th2 cells were thought to be the dominant source of IL-9 and the function of IL-9 was mainly studied in the context of Th2 type responses in airway inflammation and helminth infections (Godfraind et al., 1998; Townsend et al., 2000; McMillan et al., 2002; Temann et al., 2002). IL-9 blocking antibodies were shown to ameliorate lung inflammation (Cheng et al., 2002; Kearley et al., 2011) and are currently in clinical trials for the treatment of patients with asthma (Parker et al., 2011). The paradigm that Th2 cells are the dominant source of IL-9 was challenged when it became apparent that naive CD4⁺ T cells cultured in the presence of TGF-β and IL-4 initiate high IL-9 expression without coexpression of IL-4, suggesting the existence of a dedicated subset of IL-9–producing T cells (Dardalhon et al., 2008; Veldhoen et al., 2008; Angkasekwinai et al., 2010; Chang et al., 2010; Staudt et al., 2010). Subsequently, the generation of an IL-9–specific reporter mouse strain enabled the study of IL-9–producing cell types in vivo and revealed that in a model of lung inflammation IL-9 is produced by innate lymphoid cells (ILCs) and not T cells (Wilhelm et al., 2011). IL-9 production in ILCs was transient but important for the maintenance of IL-5 and IL-13 in ILCs. Such type 2 cytokine-producing ILCs (ILC2s; Spits and Di Santo, 2011) were first described as a population of IL-5– and IL-13–producing non-B/non-T cells (Fort et al., 2001; Hurst et al., 2002; Fallon et al., 2006; Voehringer et al., 2006) and later shown to play a role in helminth infection via IL-13 expression (Moro et al., 2010; Neill et al., 2010; Price et al., 2010; Saenz et al., 2010). In addition, important functions were ascribed to such cells in the context of influenza infection (Chang et al., 2011; Monticelli et al., 2011) and airway hyperactivity in mice (Barlow et al., 2012) and humans (Mjösberg et al., 2011). However, although the contribution of ILC2s to host immunity against helminths in the gut is well established (Moro et al., 2010; Neill et al., 2010; Price et al., 2010; Saenz et al., 2010), the function of ILC2s in helminth-related immune responses in the lung remains unknown. ILC2s are marked by expression of the IL-33R (Moro et al., 2010; Neill et al., 2010; Price et al., 2010), as well as the common γ chain (γ_c) cytokine receptors for IL-2 and IL-7 (Moro et al., 2010; Neill et al., 2010). Interestingly, gene expression array analyses have demonstrated that the receptor for IL-9, another member of the γ_c receptor family, is also expressed in ILC2s and differentiates them from Th2 cells (Price et al., 2010) and ROR-γt⁺ ILCs (Hoyler et al., 2012). However, the function of IL-9R expression for ILC2 biology has not been addressed so far.Here we show that the production of IL-5, IL-13, and amphiregulin during infection with Nippostrongylus brasiliensis in the lung depends on ILC2s and their expression of IL-9R. The ability to signal via the IL-9R was crucial for the survival of ILC2s, but not Th2 cells. The absence of IL-9 signaling in IL-9R–deficient mice resulted in reduced lung ILC2 numbers and, consequently, diminished repair of lung damage in the chronic phase after helminth-induced lung injury despite the presence of an intact Th2 cell response. Thus, we identify IL-9 as a crucial autocrine amplifier of ILC2 function and survival.     

Interleukin (IL)-23 mediates Toxoplasma gondii–induced immunopathology in the gut via matrixmetalloproteinase-2 and IL-22 but independent of IL-17

Peroral infection with Toxoplasma gondii leads to the development of small intestinal inflammation dependent on Th1 cytokines. The role of Th17 cells in ileitis is unknown. We report interleukin (IL)-23–mediated gelatinase A (matrixmetalloproteinase [MMP]-2) up-regulation in the ileum of infected mice. MMP-2 deficiency as well as therapeutic or prophylactic selective gelatinase blockage protected mice from the development of T. gondii–induced immunopathology. Moreover, IL-23–dependent up-regulation of IL-22 was essential for the development of ileitis, whereas IL-17 was down-regulated and dispensable. CD4⁺ T cells were the main source of IL-22 in the small intestinal lamina propria. Thus, IL-23 regulates small intestinal inflammation via IL-22 but independent of IL-17. Gelatinases may be useful targets for treatment of intestinal inflammation.Within 8 d after peroral infection with Toxoplasma gondii, susceptible C57BL/6 mice develop massive necrosis in the ileum, leading to death (Liesenfeld et al., 1996). T. gondii–induced ileitis is characterized by a CD4 T cell–dependent overproduction of proinflammatory mediators, including IFN-γ, TNF, and NO (Khan et al., 1997; Mennechet et al., 2002). Activation of CD4⁺ T cells by IL-12 and IL-18 is critical for the development of small intestinal pathology (Vossenkämper et al., 2004). Recently, we showed that LPS derived from gut flora via Toll-like receptor (TLR)–4 mediates T. gondii–induced immunopathology (Heimesaat et al., 2006). Thus, the immunopathogenesis of T. gondii–induced small intestinal pathology resembles key features of the inflammatory responses in inflammatory bowel disease (IBD) in humans and in models of experimental colitis in rodents (Liesenfeld, 2002). However, most animal models of IBD assessed inflammatory responses in the large intestine, and models of small intestinal pathology are scarce (Kosiewicz et al., 2001; Strober et al., 2002; Olson et al., 2004; Heimesaat et al., 2006).IL-12 shares the p40 subunit, IL-12Rβ1, and components of the signaling transduction pathways with IL-23 (Parham et al., 2002). There is strong evidence that IL-23, rather than IL-12, is important in the development of colitis (Yen et al., 2006). The association of IL-23R encoding variant Arg381Gln with IBD (Duerr et al., 2006) and the up-regulation of IL-23p19 in colon biopsies from patients with Crohn''s disease (Schmidt et al., 2005) underline the importance of IL-23 in intestinal inflammation. Effector mechanisms of IL-23 include the up-regulation of matrixmetalloproteinases (MMPs; Langowski et al., 2006), a large family of endopeptidases that mediate homeostasis of the extracellular matrix. MMPs were significantly up-regulated in experimental models of colitis (Tarlton et al., 2000; Medina et al., 2003) and in colonic tissues of IBD patients (von Lampe et al., 2000).Studies in mouse models of autoimmune diseases have associated the pathogenic role of IL-23 with the accumulation of CD4⁺ T cells secreting IL-17, termed Th17 cells (Aggarwal et al., 2003; Cua et al., 2003). Moreover, increased IL-17 expression was reported in the intestinal mucosa of patients with IBD (Fujino et al., 2003; Nielsen et al., 2003; Kleinschek et al., 2009).In addition to IL-17, Th17 cells also produce IL-22, a member of the IL-10 family (Dumoutier et al., 2000). IL-22, although secreted by certain immune cell populations, does not have any effects on immune cells in vitro or in vivo but regulates functions of some tissue cells (Wolk et al., 2009). Interestingly, IL-22 has been proposed to possess both protective as well as pathogenic roles. In fact, IL-22 mediated psoriasis-like skin alterations (Zheng et al., 2007; Ma et al., 2008; Wolk et al., 2009). In contrast, IL-22 played a protective role in experimental models of colitis (Satoh-Takayama et al., 2008; Sugimoto et al., 2008; Zenewicz et al., 2008; Zheng et al., 2008), in a model of Klebsiella pneumoniae infection in the lung (Aujla et al., 2007), and against liver damage caused by concanavalin A administration (Radaeva et al., 2004; Zenewicz et al., 2007). IL-22 has been reported to be produced by CD4⁺ T cells (Wolk et al., 2002; Zheng et al., 2007), γδ cells (Zheng et al., 2007), CD11c⁺ cells (Zheng et al., 2008), and natural killer cells (Cella et al., 2008; Luci et al., 2008; Sanos et al., 2009; Satoh-Takayama et al., 2008; Zheng et al., 2008). The role of IL-22 in small intestinal inflammation remains to be determined.In the present study, we investigated the role of the IL-23–IL-17 axis in T. gondii–induced small intestinal immunopathology. We show that IL-23 is essential in the development of small intestinal immunopathology by inducing local MMP-2 up-regulation that could be inhibited by prophylactic or therapeutic chemical blockage. Interestingly, IL-23–dependent IL-22 production was markedly up-regulated and essential for the development of ileal inflammation, whereas IL-17 production was down-regulated after T. gondii infection. IL-22 was mostly produced by CD4⁺ T cells in the small intestinal lamina propria.     

Inherited IL-17RC deficiency in patients with chronic mucocutaneous candidiasis

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent or persistent infections of the skin, nail, oral, and genital mucosae with Candida species, mainly C. albicans. Autosomal-recessive (AR) IL-17RA and ACT1 deficiencies and autosomal-dominant IL-17F deficiency, each reported in a single kindred, underlie CMC in otherwise healthy patients. We report three patients from unrelated kindreds, aged 8, 12, and 37 yr with isolated CMC, who display AR IL-17RC deficiency. The patients are homozygous for different nonsense alleles that prevent the expression of IL-17RC on the cell surface. The defect is complete, abolishing cellular responses to IL-17A and IL-17F homo- and heterodimers. However, in contrast to what is observed for the IL-17RA– and ACT1-deficient patients tested, the response to IL-17E (IL-25) is maintained in these IL-17RC–deficient patients. These experiments of nature indicate that human IL-17RC is essential for mucocutaneous immunity to C. albicans but is otherwise largely redundant.In humans, chronic mucocutaneous candidiasis (CMC) is characterized by infections of the skin, nail, digestive, and genital mucosae with Candida species, mainly C. albicans, a commensal of the gastrointestinal tract in healthy individuals (Puel et al., 2012). CMC is frequent in acquired or inherited disorders involving profound T cell defects (Puel et al., 2010b; Vinh, 2011; Lionakis, 2012). Human IL-17 immunity has recently been shown to be essential for mucocutaneous protection against C. albicans (Puel et al., 2010b, 2012; Cypowyj et al., 2012; Engelhardt and Grimbacher, 2012; Huppler et al., 2012; Ling and Puel, 2014). Indeed, patients with primary immunodeficiencies and syndromic CMC have been shown to display impaired IL-17 immunity (Puel et al., 2010b). Most patients with autosomal-dominant (AD) hyper-IgE syndrome (AD-HIES) and STAT3 deficiency (de Beaucoudrey et al., 2008; Ma et al., 2008; Milner et al., 2008; Renner et al., 2008; Chandesris et al., 2012) and some patients with invasive fungal infection and autosomal-recessive (AR) CARD9 deficiency (Glocker et al., 2009; Lanternier et al., 2013) or Mendelian susceptibility to mycobacterial diseases (MSMD) and AR IL-12p40 or IL-12Rβ1 deficiency (de Beaucoudrey et al., 2008, 2010; Prando et al., 2013; Ouederni et al., 2014) have low proportions of IL-17A–producing T cells and CMC (Cypowyj et al., 2012; Puel et al., 2012). Patients with AR autoimmune polyendocrine syndrome type 1 (APS-1) and AIRE deficiency display CMC and high levels of neutralizing autoantibodies against IL-17A, IL-17F, and/or IL-22 (Browne and Holland, 2010; Husebye and Anderson, 2010; Kisand et al., 2010, 2011; Puel et al., 2010a).These findings paved the way for the discovery of the first genetic etiologies of CMC disease (CMCD), an inherited condition affecting individuals with none of the aforementioned primary immunodeficiencies (Puel et al., 2011; Casanova and Abel, 2013; Casanova et al., 2013, 2014). AR IL-17RA deficiency, AR ACT1 deficiency, and AD IL-17F deficiency were described, each in a single kindred (Puel et al., 2011; Boisson et al., 2013). A fourth genetic etiology of CMCD, which currently appears to be the most frequent, has also been reported: heterozygous gain-of-function (GOF) mutations of STAT1 impairing the development of IL-17–producing T cells (Liu et al., 2011; Smeekens et al., 2011; van de Veerdonk et al., 2011; Hori et al., 2012; Takezaki et al., 2012; Tóth et al., 2012; Al Rushood et al., 2013; Aldave et al., 2013; Romberg et al., 2013; Sampaio et al., 2013; Soltész et al., 2013; Uzel et al., 2013; Wildbaum et al., 2013; Frans et al., 2014; Kilic et al., 2014; Lee et al., 2014; Mekki et al., 2014; Mizoguchi et al., 2014; Sharfe et al., 2014; Yamazaki et al., 2014). We studied three unrelated patients with CMCD without mutations of IL17F, IL17RA, ACT1, or STAT1. We used a genome-wide approach based on whole-exome sequencing (WES). We found AR complete IL-17RC deficiency in all three patients.     

The G protein βγ subunit mediates reannealing of adherens junctions to reverse endothelial permeability increase by thrombin

The inflammatory mediator thrombin proteolytically activates protease-activated receptor (PAR1) eliciting a transient, but reversible increase in vascular permeability. PAR1-induced dissociation of Gα subunit from heterotrimeric Gq and G12/G13 proteins is known to signal the increase in endothelial permeability. However, the role of released Gβγ is unknown. We now show that impairment of Gβγ function does not affect the permeability increase induced by PAR1, but prevents reannealing of adherens junctions (AJ), thereby persistently elevating endothelial permeability. We observed that in the naive endothelium Gβ1, the predominant Gβ isoform is sequestered by receptor for activated C kinase 1 (RACK1). Thrombin induced dissociation of Gβ1 from RACK1, resulting in Gβ1 interaction with Fyn and focal adhesion kinase (FAK) required for FAK activation. RACK1 depletion triggered Gβ1 activation of FAK and endothelial barrier recovery, whereas Fyn knockdown interrupted with Gβ1-induced barrier recovery indicating RACK1 negatively regulates Gβ1-Fyn signaling. Activated FAK associated with AJ and stimulated AJ reassembly in a Fyn-dependent manner. Fyn deletion prevented FAK activation and augmented lung vascular permeability increase induced by PAR1 agonist. Rescuing FAK activation in fyn^−/− mice attenuated the rise in lung vascular permeability. Our results demonstrate that Gβ1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.A persistent increase in endothelial permeability during inflammatory conditions such as pneumonia, trauma, and burn leads to the life-threatening illness acute respiratory distress syndrome (Mehta and Malik, 2006; Liu and Matthay, 2008). Increased endothelial permeability occurs because of loss of cell–cell contacts and disruption of cell–extracellular matrix (ECM) adhesions (Yuan, 2002; Mehta and Malik, 2006). Focal adhesion kinase (FAK) and VE-cadherin play a fundamental role in establishing the endothelial barrier to macromolecules and liquid by maintaining intercellular adhesion and cell–ECM adhesivity (Nelson et al., 2004; van Buul et al., 2005; Wu, 2005; Mehta and Malik, 2006; Dejana et al., 2008; Rudini and Dejana, 2008). We have shown that thrombin, a serine protease generated early on during acute respiratory distress syndrome, plays a critical role in increasing endothelial permeability by inducing the loss of VE-cadherin homotypic adhesion and redistribution of focal adhesions dependent on FAK (Mehta et al., 2002; Kouklis et al., 2004; Holinstat et al., 2006). Interestingly, the thrombin-induced increase in endothelial permeability is reversed within 2–3 h, indicating activation of endogenous pathways that limit the persistent increase in endothelial permeability produced by thrombin (Kouklis et al., 2004; Holinstat et al., 2006; Kaneider et al., 2007).Thrombin binds to endothelial cell surface receptor, protease-activating receptor 1 (PAR1) and PAR4 (Coughlin, 2000, 2005; Kataoka et al., 2003). We have shown that the permeability increasing effects of thrombin in lung endothelium are predominantly mediated through PAR1 because thrombin and selective PAR1 peptide agonists failed to induce endothelial contraction and lung microvascular permeability increase in mice lacking PAR1 (Vogel et al. 2000). PAR1 is a seven-transmembrane domain receptor that couples to heterotrimeric G proteins of the Gq and G12/13 families (Hung et al., 1992; Coughlin 1999). Upon ligation by thrombin, PAR1 signals the dissociation of the α-subunits of Gq and G12/13 from the Gβγ dimer. Gαq and Gα12/13 activate myosin light chain kinase and RhoA pathways, which by inducing endothelial cell contraction increase permeability (Goeckeler and Wysolmerski, 1995; Dudek and Garcia, 2001; Holinstat et al., 2003; McLaughlin et al., 2005; Knezevic et al., 2007; Singh et al., 2007; Gavard and Gutkind, 2008; Korhonen et al., 2009). However, the role of Gβγ after its dissociation from these heterotrimeric G proteins in the mechanism of PAR1-induced alteration in endothelial barrier function is unknown.The Gβγ pathway has progressively emerged as a critical element of GPCR signaling (Clapham and Neer, 1997; Cabrera-Vera et al., 2003; Oldham and Hamm, 2008). Gβγ is known to induce cyclic AMP generation (Tang and Gilman, 1992; Taurin et al., 2007), Ca²⁺ signaling (Herlitze et al., 1996; Blackmer et al., 2001), oxidant generation (Niu et al., 2003), neurotransmission (Blackmer et al., 2005), chemotaxis (Neptune and Bourne, 1997; Jin et al., 2000), and caveolae-mediated transcytosis (Shajahan et al., 2004). The β subunit of Gβγ contains WD 40 repeats that are thought to mediate protein–protein interactions (Neer et al., 1994; Chen et al., 2004b). Studies show that Gβ interacts with receptor for activated C kinase 1 (RACK1; Dell et al., 2002; Chen et al., 2004a), p60cSrc (Luttrell et al., 1996; McGarrigle and Huang, 2007), and Fyn (Yaka et al., 2002; Thornton et al., 2004). Fyn, p60cSrc, and RACK1 are known to influence adherens junctions (AJ) and focal adhesions (Xing et al., 1994; Bockholt and Burridge, 1995; Thomas and Brugge, 1997; Roura et al., 1999; Owens et al., 2000; Mourton et al., 2001; Schaller, 2001; Piedra et al., 2003). We tested the hypothesis that, besides restraining Gα subunits, Gβγ orchestrates signaling to terminate endothelial permeability increase through its ability to coordinate intercellular and cell–matrix interactions.To explore the function of Gβγ in regulating endothelial permeability, we interfered with expression of Gβγ, RACK1, or Fyn using small interfering RNA (siRNA) or Fyn knockout mice. We show that Gβγ plays a fundamental role in signaling endothelial barrier recovery. Moreover, we identified Fyn and FAK as the key downstream effectors of Gβγ. Fyn-mediated activation of FAK facilitated the association of FAK with p120-catenin and reannealing of AJ, which reversed the increased endothelial permeability responses produced by G protein–coupled receptor agonists.     

IL-12 and type I interferon prolong the division of activated CD8 T cells by maintaining high-affinity IL-2 signaling in vivo

TCR ligation and co-stimulation induce cellular division; however, optimal accumulation of effector CD8 T cells requires direct inflammatory signaling by signal 3 cytokines, such as IL-12 or type I IFNs. Although in vitro studies suggest that IL-12/type I IFN may enhance T cell survival or early proliferation, the mechanisms underlying optimal accumulation of CD8 T cells in vivo are unknown. In particular, it is unclear if disparate signal 3 cytokines optimize effector CD8 T cell accumulation by the same mechanism and how these inflammatory cytokines, which are transiently produced early after infection, affect T cell accumulation many days later at the peak of the immune response. Here, we show that transient exposure of CD8 T cells to IL-12 or type I IFN does not promote survival or confer an early proliferative advantage in vivo, but rather sustains surface expression of CD25, the high-affinity IL-2 receptor. This prolongs division of CD8 T cells in response to basal IL-2, through activation of the PI3K pathway and expression of FoxM1, a positive regulator of cell cycle progression genes. Thus, signal 3 cytokines use a common pathway to optimize effector CD8 T cell accumulation through a temporally orchestrated sequence of cytokine signals that sustain division rather than survival.The magnitude of the effector CD8 T cell response is critical for eliminating intracellular pathogens and for regulating the size of the memory pool after resolution of infection or vaccination (Hou et al., 1994; Badovinac and Harty, 2006; Schmidt et al., 2008). TCR stimulation by mature DCs expressing cognate antigen in the context of MHC I initiates activation of naive, pathogen-specific CD8 T cells. Additional signals from co-stimulatory molecules amplify the magnitude and/or duration of the TCR signaling, thereby enhancing activation and functionality (Cronin and Penninger, 2007). Although these two signals are sufficient to induce the division of naive CD8 T cells, pathogen-, or adjuvant-induced inflammatory cytokines act as third signals to promote optimal accumulation of effector CD8 T cells (Curtsinger and Mescher, 2010). Because the clearance of intracellular pathogens is often dependent on the total number of responding effector CD8 T cells (Badovinac and Harty, 2006; Hikono et al., 2006; Lefrançois, 2006), it is important to understand how the magnitude of CD8 T cell responses are regulated to effectively control pathogen burden.Using short-term (∼3 d) in vitro experiments, an early study by Curtsinger et al. (1999) clearly established that addition of a specific inflammatory cytokine (IL-12) during T cell activation in response to artificial APCs expressing signal 1 and signal 2 and with exogenous addition of IL-2 increased the accumulation of responding CD8 T cells. The importance of signal 3 cytokines for the optimal accumulation of effector CD8 T cells has also been established in vivo (Gately et al., 1992; Trinchieri, 1998). For example, direct IL-12 signaling is essential for optimal accumulation of antigen-specific CD8 T cells after Listeria monocytogenes (LM) infection (Keppler et al., 2009; Xiao et al., 2009; Keppler et al., 2012). Direct IFN-α/β receptor signaling has also been shown to be critical for the optimal accumulation of CD8 T cells in some in vitro studies (Curtsinger et al., 2005) and for P14 TCR-transgenic CD8 T cells responding to lymphocytic choriomeningitis virus (LCMV) infection (Aichele et al., 2006; Kolumam et al., 2005). Together, these studies highlighted the impact of IL-12 and IFN α/β on the accumulation of activated CD8 T cells. However, a mechanistic understanding of how inflammatory cytokines such as IL-12 and IFN α/β regulate accumulation of effector CD8 T cells in vivo has yet to be determined.Results from short-term in vitro studies provide two models to explain how the signal 3 cytokine IL-12 promotes the optimal accumulation of activated CD8 T cells. The first model suggests that IL-12 stimulation during activation promotes increased accumulation by conferring a survival advantage to responding CD8 T cells (Mitchell et al., 2001; Valenzuela et al., 2005; Curtsinger and Mescher, 2010). This conclusion was drawn from experiments where IL-12 enhanced accumulation of CD8 T cells over the 3-d culture period, without detectable impact on cellular division. However, validated survival pathways regulated by signal 3 cytokines in vivo have not been identified to date. Alternatively, other data suggest that IL-12 increases the accumulation of activated CD8 T cells by transiently increasing the expression of the high-affinity IL-2 receptor subunit (IL-2Rα; CD25; Pipkin et al., 2010; Valenzuela et al., 2002) and IL-2Rβ (CD122; Valenzuela et al., 2002), providing an early proliferative advantage leading to increased accumulation in short-term in vitro studies (Valenzuela et al., 2002; Curtsinger and Mescher, 2010; Pipkin et al., 2010). Consistent with this notion, the absence of CD25 prevented optimal accumulation of CD8 T cells after LM infection (Obar et al., 2010) or LCMV infection (Williams et al., 2006). However, the IL-12–stimulated increase in CD25 expression in vitro was transient, peaking 2 d after cognate-antigen stimulation (Valenzuela et al., 2002). Thus, mechanistic assessment of signal 3 activities to date are limited to short-term in vitro studies focused on IL-12 and the mechanisms by which IL-12 or other signal 3 cytokines (e.g., type I IFN) regulate CD8 T cell accumulation in vivo are not established. For example, it remains unknown if signal 3 cytokines function by common or distinct mechanisms, if these mechanisms regulate survival pathways in vivo or confer an early proliferative advantage, or if both mechanisms account for signal 3-dependent optimal accumulation of effector CD8 T cells in vivo.In addition, the temporal disconnection between early and transient production of signal 3 cytokines (Pham et al., 2009; Keppler et al., 2012) and optimal accumulation of effector CD8 T cells at the peak of the response, many days later, has not been addressed (Harty and Badovinac, 2008). For example, most in vivo experiments used gene KO mouse strains or TCR-transgenic T cells (Kolumam et al., 2005; Aichele et al., 2006; Keppler et al., 2009; Pham et al., 2011; Keppler et al., 2012) that constitutively lack the receptors for inflammatory cytokines, and most in vitro studies were analyzed within a short window (∼3 d) after CD8 T cell activation (Curtsinger et al., 1999, 2003a, Curtsinger et al., a2005; Valenzuela et al., 2002, 2005). These are important considerations given that acute infections (as well as adjuvant-coupled immunizations) induce transient elevations of inflammatory cytokines, often peaking within hours of stimulation, and then returning to baseline within 1–2 d (Pham et al., 2009; Keppler et al., 2012), whereas corresponding CD8 T cell responses generally peak in numbers between day 7 and 9 after immunization/infection (Harty and Badovinac, 2008). Here, we address this temporal conundrum and-dissect the mechanisms by which signal 3 cytokines IL-12 and type I IFN guide the optimal accumulation of CD8 T cells in response to in vivo activation. To address these issues, we use an immunization model with mature, peptide-loaded DCs, wherein antigen concentrations are fixed but the inflammatory milieu can be manipulated by co-administration of Toll-like receptor ligands (Badovinac et al., 2005; Boyman et al., 2006; Pham et al., 2009). Using this model, we uncover a molecular pathway whereby signal 3 cytokines (both IL-12 and IFN-α/β) promote optimal CD8 T cell accumulation after in vivo activation, not by improving survival or early proliferation, but rather by a common mechanism regulating cytokine signaling pathways that maintain cellular division at late time points.     

Oxidative metabolism enables Salmonella evasion of the NLRP3 inflammasome

Microbial infection triggers assembly of inflammasome complexes that promote caspase-1–dependent antimicrobial responses. Inflammasome assembly is mediated by members of the nucleotide binding domain leucine-rich repeat (NLR) protein family that respond to cytosolic bacterial products or disruption of cellular processes. Flagellin injected into host cells by invading Salmonella induces inflammasome activation through NLRC4, whereas NLRP3 is required for inflammasome activation in response to multiple stimuli, including microbial infection, tissue damage, and metabolic dysregulation, through mechanisms that remain poorly understood. During systemic infection, Salmonella avoids NLRC4 inflammasome activation by down-regulating flagellin expression. Macrophages exhibit delayed NLRP3 inflammasome activation after Salmonella infection, suggesting that Salmonella may evade or prevent the rapid activation of the NLRP3 inflammasome. We therefore screened a Salmonella Typhimurium transposon library to identify bacterial factors that limit NLRP3 inflammasome activation. Surprisingly, absence of the Salmonella TCA enzyme aconitase induced rapid NLRP3 inflammasome activation. This inflammasome activation correlated with elevated levels of bacterial citrate, and required mitochondrial reactive oxygen species and bacterial citrate synthase. Importantly, Salmonella lacking aconitase displayed NLRP3- and caspase-1/11–dependent attenuation of virulence, and induced elevated serum IL-18 in wild-type mice. Together, our data link Salmonella genes controlling oxidative metabolism to inflammasome activation and suggest that NLRP3 inflammasome evasion promotes systemic Salmonella virulence.Pattern recognition receptors (PRRs) that detect and respond to evolutionarily conserved microbial structures such as LPS or peptidoglycan, as well as pathogen-specific virulence activities, are critical for host immune defense (Medzhitov, 2007; Vance et al., 2009). To promote infection, microbial pathogens inject virulence factors into the cytosol of infected cells to disrupt or modulate critical host physiological processes (Cornelis, 2006). During this process, contamination of the target cell cytosol by microbial components triggers cytosolic PRRs of the nucleotide binding domain leucine-rich repeat (NLR) family (Lamkanfi and Dixit, 2009). Diverse NLRs respond to a variety of endogenous and exogenous signals associated with infection, tissue stress, or damage. For example, NLRC4 responds to microbial products such as bacterial flagellin or structurally related specialized secretion system components that are injected into the cytosol of infected cells during infection by bacterial pathogens including Pseudomonas, Legionella, and Salmonella spp. (Miao et al., 2006; Molofsky et al., 2006; Sutterwala et al., 2007). NLRs recruit pro–caspase-1 to multiprotein complexes termed inflammasomes, where pro–caspase-1 is processed and activated, leading to cleavage and secretion of caspase-1–dependent cytokines (Martinon et al., 2002, 2007), as well as pyroptosis, a caspase-1–dependent pro-inflammatory cell death (Bergsbaken et al., 2009).Inflammasome activation and subsequent production of caspase-1–dependent cytokines is important for both innate and adaptive antimicrobial responses (Mariathasan and Monack, 2007), as IL-1 family cytokines released upon inflammasome activation promote neutrophil migration to infected tissues and drive T_H17 and T_H1 responses against mucosal pathogens (Chung et al., 2009; Ichinohe et al., 2009). How pathogens evade inflammasome activation, and whether persistent bacterial pathogens evade or suppress inflammasome activation to establish or maintain persistence remains poorly understood.Salmonella enterica species cause a range of disease from severe gastroenteritis to persistent systemic infection (Bäumler et al., 1998). Salmonella enterica serovar Typhimurium (Stm) invades host cells by means of a type III secretion system (T3SS) encoded on Salmonella pathogenicity island I (SPI-1; Lee, 1996; Collazo and Galán, 1997). Salmonella subsequently replicates within a Salmonella-containing vacuole (SCV) that is established and maintained by the activity of a second T3SS, encoded on a second pathogenicity island, SPI-2 (Cirillo et al., 1998; Hensel et al., 1998). Intestinal inflammation during Stm infection is triggered by NLRC4-dependent responses to Stm flagellin, accompanied by caspase-1–dependent cytokine secretion and pyroptosis (Franchi et al., 2012). Activity of a SPI-1 effector protein, SopE, also contributes to SPI-1–dependent inflammasome activation in intestinal epithelial cells (Müller et al., 2009). Within the inflamed intestine, specialized adaptations allow Stm to resist mucosal antimicrobial defenses (Raffatellu et al., 2009; Winter et al., 2010; Thiennimitr et al., 2011). However, flagellin expression is down-regulated at systemic sites (Cummings et al., 2005, 2006), and enforced flagellin expression enhances NLRC4 activation and bacterial clearance, indicating that inflammasome activation in response to bacterial flagellin is detrimental for Stm replication during systemic infection (Miao et al., 2010a; Stewart et al., 2011).NLRP3 responds to a wide variety of structurally unrelated molecules and activities, including extracellular ATP, bacterial pore-forming proteins, bacterial nucleic acids, crystals, and unsaturated fatty acids (Kanneganti et al., 2006; Mariathasan et al., 2006; Martinon et al., 2006; Hornung et al., 2008; Wen et al., 2011). While ATP, crystals, and the Yersinia T3SS all induce rapid formation of an NLRP3 inflammasome that leads to caspase-1 activation within 1–2 h (Mariathasan et al., 2006; Martinon et al., 2006; Brodsky et al., 2010), Stm induces delayed activation of a noncanonical NLRP3 inflammasome 12–16 h after infection (Broz et al., 2010). This noncanonical NLRP3 inflammasome is independent of the activities of the SPI-1 T3SS and instead is regulated by caspase-11 and TLR4-dependent production of type I interferon (Broz et al., 2012; Gurung et al., 2012; Rathinam et al., 2012). We therefore hypothesized that Stm might evade or prevent rapid activation of a canonical NLRP3 inflammasome, and that this evasion might contribute to systemic bacterial virulence. Several bacterial and viral pathogens evade NLRP3 inflammasome activation (Taxman et al., 2010; Gregory et al., 2011), but whether Salmonella is capable of doing so is unknown.To identify potential negative regulators of NLRP3 inflammasome activation, we generated and screened a transposon library of flagellin-deficient Stm mutants for elevated inflammasome activation in NLRC4-deficient BM-derived macrophages (BMDMs). Sequencing of candidate hits identified acnB, the gene encoding the TCA cycle enzyme aconitase, which converts citrate to isocitrate, as well as several other genes that had been previously isolated in a screen for Salmonella genes that are required for persistent Salmonella infection in vivo (Lawley et al., 2006). Intriguingly, isocitrate lyase (encoded by aceA), which generates glyoxylate from isocitrate in the glyoxylate cycle, contributes to persistent but not acute infection by Salmonella as well as Mycobacterium tuberculosis (McKinney et al., 2000; Fang et al., 2005).To test the potential role of Salmonella TCA cycle metabolism in inflammasome modulation, we generated targeted deletions in acnB as well as genes encoding other TCA cycle enzymes. Notably, deletion of aconitase, isocitrate lyase, or isocitrate dehydrogenase (icdA), but not other TCA enzymes, induced rapid NLRP3-dependent inflammasome activation in Stm-infected macrophages, suggesting that activity of these enzymes limits NLRP3 inflammasome activation by intracellular Salmonella. Moreover, aconitase-deficient Salmonella exhibited a defect in acute systemic virulence after oral administration and were deficient in their ability to persist in a chronic infection. These findings define the first genes that mediate NLRP3 inflammasome evasion by Salmonella and suggest that inflammasome evasion contributes to persistence of bacterial pathogens. Our data further suggest that sensing of bacterial metabolites may provide an additional level of innate immune recognition, and that regulation of metabolite production by intracellular pathogens represents a pathogen immune evasion strategy.