NLRP6 negatively regulates innate immunity and host defence against bacterial pathogens

Members of the intracellular nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family contribute to immune responses through activation of nuclear factor-κB (NF-κB), type I interferon and inflammasome signalling. Mice lacking the NLR family member NLRP6 were recently shown to be susceptible to colitis and colorectal tumorigenesis, but the role of NLRP6 in microbial infections and the nature of the inflammatory signalling pathways regulated by NLRP6 remain unclear. Here we show that Nlrp6-deficient mice are highly resistant to infection with the bacterial pathogens Listeria monocytogenes, Salmonella typhimurium and Escherichia coli. Infected Nlrp6-deficient mice had increased numbers of monocytes and neutrophils in circulation, and NLRP6 signalling in both haematopoietic and radioresistant cells contributed to increased susceptibility. Nlrp6 deficiency enhanced activation of mitogen-activated protein kinase (MAPK) and the canonical NF-κB pathway after Toll-like receptor ligation, but not cytosolic NOD1/2 ligation, in vitro. Consequently, infected Nlrp6-deficient cells produced increased levels of NF-κB- and MAPK-dependent cytokines and chemokines. Thus, our results reveal NLRP6 as a negative regulator of inflammatory signalling, and demonstrate a role for this NLR in impeding clearance of both Gram-positive and -negative bacterial pathogens.     

Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis.

The protein Bid is a participant in the pathway that leads to cell death (apoptosis), mediating the release of cytochrome c from mitochondria in response to signals from 'death' receptors known as TNFR1/Fas on the cell surface. It is a member of the proapoptotic Bcd-2 family and is activated as a result of its cleavage by caspase 8, one of a family of proteolytic cell-death proteins. To investigate the role of Bid in vivo, we have generated mice deficient for Bid. We find that when these mice are injected with an antibody directed against Fas, they nearly all survive, whereas wild-type mice die from hepatocellular apoptosis and haemorrhagic necrosis. About half of the Bid-deficient animals had no apparent liver injury and showed no evidence of activation of the effector caspases 3 and 7, although the initiator caspase 8 had been activated. Other Bid-deficient mice survived with only moderate damage: all three caspases (8 and 37) were activated but their cell nuclei were intact and no mitochondrial cytochrome c was released. We also investigated the effects of Bid deficiency in cultured cells treated with anti-Fas antibody (hepatocytes and thymocytes) or with TNFalpha. (fibroblasts). In these Bid-/- cells, mitochondrial dysfunction was delayed, cytochrome c was not released, effector caspase activity was reduced and the cleavage of apoptosis substrates was altered. This loss-of-function model indicates that Bid is a critical substrate in vivo for signalling by death-receptor agonists, which mediates a mitochondrial amplification loop that is essential for the apoptosis of selected cells.     

Functional complementation between FADD and RIP1 in embryos and lymphocytes

FADD is a common adaptor shared by several death receptors for signalling apoptosis through recruitment and activation of caspase 8 (refs 1-3). Death receptors are essential for immune homeostasis, but dispensable during embryogenesis. Surprisingly, Fadd(-/-) mice die in utero and conditional deletion of FADD leads to impaired lymphocyte proliferation. How FADD regulates embryogenesis and lymphocyte responses has been a long-standing enigma. FADD could directly bind to RIP1 (also known as RIPK1), a serine/threonine kinase that mediates both necrosis and NF-κB activation. Here we show that Fadd(-/-) embryos contain raised levels of RIP1 and exhibit massive necrosis. To investigate a potential in vivo functional interaction between RIP1 and FADD, null alleles of RIP1 were crossed into Fadd(-/-) mice. Notably, RIP1 deficiency allowed normal embryogenesis of Fadd(-/-) mice. Conversely, the developmental defect of Rip1(-/-) lymphocytes was partially corrected by FADD deletion. Furthermore, RIP1 deficiency fully restored normal proliferation in Fadd(-/-) T cells but not in Fadd(-/-) B cells. Fadd(-/-)Rip1(-/-) double-knockout T cells are resistant to death induced by Fas or TNF-α and show reduced NF-κB activity. Therefore, our data demonstrate an unexpected cell-type-specific interplay between FADD and RIP1, which is critical for the regulation of apoptosis and necrosis during embryogenesis and lymphocyte function.     

SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis

SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.     

RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems

The immune system consists of two evolutionarily different but closely related responses, innate immunity and adaptive immunity. Each of these responses has characteristic receptors-Toll-like receptors (TLRs) for innate immunity and antigen-specific receptors for adaptive immunity. Here we show that the caspase recruitment domain (CARD)-containing serine/threonine kinase Rip2 (also known as RICK, CARDIAK, CCK and Ripk2) transduces signals from receptors of both immune responses. Rip2 was recruited to TLR2 signalling complexes after ligand stimulation. Moreover, cytokine production in Rip2-deficient cells was reduced on stimulation of TLRs with lipopolysaccharide, peptidoglycan and double-stranded RNA, but not with bacterial DNA, indicating that Rip2 is downstream of TLR2/3/4 but not TLR9. Rip2-deficient cells were also hyporesponsive to signalling through interleukin (IL)-1 and IL-18 receptors, and deficient for signalling through Nod proteins-molecules also implicated in the innate immune response. Furthermore, Rip2-deficient T cells showed severely reduced NF-kappaB activation, IL-2 production and proliferation on T-cell-receptor (TCR) engagement, and impaired differentiation to T-helper subtype 1 (TH1) cells, indicating that Rip2 is required for optimal TCR signalling and T-cell differentiation. Rip2 is therefore a signal transducer and integrator of signals for both the innate and adaptive immune systems.     

Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis

Intestinal homeostasis is critical for efficient energy extraction from food and protection from pathogens. Its disruption can lead to an array of severe illnesses with major impacts on public health, such as inflammatory bowel disease characterized by self-destructive intestinal immunity. However, the mechanisms regulating the equilibrium between the large bacterial flora and the immune system remain unclear. Intestinal lymphoid tissues generate flora-reactive IgA-producing B cells, and include Peyer's patches and mesenteric lymph nodes, as well as numerous isolated lymphoid follicles (ILFs). Here we show that peptidoglycan from Gram-negative bacteria is necessary and sufficient to induce the genesis of ILFs in mice through recognition by the NOD1 (nucleotide-binding oligomerization domain containing 1) innate receptor in epithelial cells, and beta-defensin 3- and CCL20-mediated signalling through the chemokine receptor CCR6. Maturation of ILFs into large B-cell clusters requires subsequent detection of bacteria by toll-like receptors. In the absence of ILFs, the composition of the intestinal bacterial community is profoundly altered. Our results demonstrate that intestinal bacterial commensals and the immune system communicate through an innate detection system to generate adaptive lymphoid tissues and maintain intestinal homeostasis.     

The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4

Macrophages are pivotal constituents of the innate immune system, vital for recognition and elimination of microbial pathogens. Macrophages use Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns--including bacterial cell wall components, such as lipopolysaccharide or lipoteichoic acid, and viral nucleic acids, such as double-stranded (ds)RNA--and in turn activate effector functions, including anti-apoptotic signalling pathways. Certain pathogens, however, such as Salmonella spp., Shigellae spp. and Yersiniae spp., use specialized virulence factors to overcome these protective responses and induce macrophage apoptosis. We found that the anthrax bacterium, Bacillus anthracis, selectively induces apoptosis of activated macrophages through its lethal toxin, which prevents activation of the anti-apoptotic p38 mitogen-activated protein kinase. We now demonstrate that macrophage apoptosis by three different bacterial pathogens depends on activation of TLR4. Dissection of anti- and pro-apoptotic signalling events triggered by TLR4 identified the dsRNA responsive protein kinase PKR as a critical mediator of pathogen-induced macrophage apoptosis. The pro-apoptotic actions of PKR are mediated both through inhibition of protein synthesis and activation of interferon response factor 3.     

Linear ubiquitination prevents inflammation and regulates immune signalling

Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKγ or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpin(cpdm/cpdm)) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1β was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.     

Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands

Reciprocity of inflammation, oxidative stress and neovascularization is emerging as an important mechanism underlying numerous processes from tissue healing and remodelling to cancer progression. Whereas the mechanism of hypoxia-driven angiogenesis is well understood, the link between inflammation-induced oxidation and de novo blood vessel growth remains obscure. Here we show that the end products of lipid oxidation, ω-(2-carboxyethyl)pyrrole (CEP) and other related pyrroles, are generated during inflammation and wound healing and accumulate at high levels in ageing tissues in mice and in highly vascularized tumours in both murine and human melanoma. The molecular patterns of carboxyalkylpyrroles are recognized by Toll-like receptor 2 (TLR2), but not TLR4 or scavenger receptors on endothelial cells, leading to an angiogenic response that is independent of vascular endothelial growth factor. CEP promoted angiogenesis in hindlimb ischaemia and wound healing models through MyD88-dependent TLR2 signalling. Neutralization of endogenous carboxyalkylpyrroles impaired wound healing and tissue revascularization and diminished tumour angiogenesis. Both TLR2 and MyD88 are required for CEP-induced stimulation of Rac1 and endothelial migration. Taken together, these findings establish a new function of TLR2 as a sensor of oxidation-associated molecular patterns, providing a key link connecting inflammation, oxidative stress, innate immunity and angiogenesis.     

Stem-cell-triggered immunity through CLV3p-FLS2 signalling

Stem cells in the shoot apical meristem (SAM) of plants are the self-renewable reservoir for leaf, stem and flower organogenesis. In nature, disease-free plants can be regenerated from SAM despite infections elsewhere, which underlies a horticultural practice for decades. However, the molecular basis of the SAM immunity remains unclear. Here we show that the CLAVATA3 peptide (CLV3p), expressed and secreted from stem cells and functioning as a key regulator of stem-cell homeostasis in the SAM of Arabidopsis, can trigger immune signalling and pathogen resistance via the flagellin receptor kinase FLS2 (refs 5, 6). CLV3p-FLS2 signalling acts independently from the stem-cell signalling pathway mediated through CLV1 and CLV2 receptors, and is uncoupled from FLS2-mediated growth suppression. Endogenous CLV3p perception in the SAM by a pattern recognition receptor for bacterial flagellin, FLS2, breaks the previously defined self and non-self discrimination in innate immunity. The dual perception of CLV3p illustrates co-evolution of plant peptide and receptor kinase signalling for both development and immunity. The enhanced immunity in SAM or germ lines may represent a common strategy towards immortal fate in plants and animals.     

TREM-1 amplifies inflammation and is a crucial mediator of septic shock

Host innate responses to bacterial infections are primarily mediated by neutrophils and monocytes/macrophages. These cells express pattern recognition receptors (PRRs) that bind conserved molecular structures shared by groups of microorganisms. Stimulation of PRR signalling pathways initiates secretion of proinflammatory mediators, which promote the elimination of infectious agents and the induction of tissue repair. Excessive inflammation owing to bacterial infections can lead to tissue damage and septic shock. Here we show that inflammatory responses to microbial products are amplified by a pathway mediated by triggering receptor expressed on myeloid cells (TREM)-1. TREM-1 is an activating receptor expressed at high levels on neutrophils and monocytes that infiltrate human tissues infected with bacteria. Furthermore, it is upregulated on peritoneal neutrophils of patients with microbial sepsis and mice with experimental lipopolysaccaride (LPS)-induced shock. Notably, blockade of TREM-1 protects mice against LPS-induced shock, as well as microbial sepsis caused by live Escherichia coli or caecal ligation and puncture. These results demonstrate a critical function of TREM-1 in acute inflammatory responses to bacteria and implicate TREM-1 as a potential therapeutic target for septic shock.     

RIP3 mediates the embryonic lethality of caspase-8-deficient mice

Apoptosis and necroptosis are complementary pathways controlled by common signalling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor-induced apoptosis. This caspase has also been implicated in non-apoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signalling and other less defined biological processes as diverse as innate immune signalling and myeloid or lymphoid differentiation patterns. Casp8 suppresses RIP3-RIP1 (also known as RIPK3-RIPK1) kinase complex-dependent necroptosis that follows death receptor activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defence mechanism against murine cytomegalovirus. Disruption of Casp8 expression leads to embryonic lethality in mice between embryonic days 10.5 and 11.5 (ref. 7). Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to promoting apoptosis. We find that RIP3 is responsible for the mid-gestational death of Casp8-deficient embryos. Remarkably, Casp8(-/-)Rip3(-/-) double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice seem immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr; also known as Fas). Thus, Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.     

Calcineurin sets the bandwidth for discrimination of signals during thymocyte development

At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the alphabeta T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.     

Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation

Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.     

Enhanced bacterial clearance and sepsis resistance in caspase-12-deficient mice

Caspases function in both apoptosis and inflammatory cytokine processing and thereby have a role in resistance to sepsis. Here we describe a novel role for a caspase in dampening responses to bacterial infection. We show that in mice, gene-targeted deletion of caspase-12 renders animals resistant to peritonitis and septic shock. The resulting survival advantage was conferred by the ability of the caspase-12-deficient mice to clear bacterial infection more efficiently than wild-type littermates. Caspase-12 dampened the production of the pro-inflammatory cytokines interleukin (IL)-1beta, IL-18 (interferon (IFN)-gamma inducing factor) and IFN-gamma, but not tumour-necrosis factor-alpha and IL-6, in response to various bacterial components that stimulate Toll-like receptor and NOD pathways. The IFN-gamma pathway was crucial in mediating survival of septic caspase-12-deficient mice, because administration of neutralizing antibodies to IFN-gamma receptors ablated the survival advantage that otherwise occurred in these animals. Mechanistically, caspase-12 associated with caspase-1 and inhibited its activity. Notably, the protease function of caspase-12 was not necessary for this effect, as the catalytically inactive caspase-12 mutant Cys299Ala also inhibited caspase-1 and IL-1beta production to the same extent as wild-type caspase-12. In this regard, caspase-12 seems to be the cFLIP counterpart for regulating the inflammatory branch of the caspase cascade. In mice, caspase-12 deficiency confers resistance to sepsis and its presence exerts a dominant-negative suppressive effect on caspase-1, resulting in enhanced vulnerability to bacterial infection and septic mortality.     

Progression of autoimmune diabetes driven by avidity maturation of a T-cell population

For unknown reasons, autoimmune diseases such as type 1 diabetes develop after prolonged periods of inflammation of mononuclear cells in target tissues. Here we show that progression of pancreatic islet inflammation to overt diabetes in nonobese diabetic (NOD) mice is driven by the 'avidity maturation' of a prevailing, pancreatic beta-cell-specific T-lymphocyte population carrying the CD8 antigen. This T-lymphocyte population recognizes two related peptides (NRP and NRP-A7) in the context of H-2Kd class I molecules of the major histocompatibility complex (MHC). As pre-diabetic NOD mice age, their islet-associated CD8+ T lymphocytes contain increasing numbers of NRP-A7-reactive cells, and these cells bind NRP-A7/H-2Kd tetramers with increased specificity, increased avidity and longer half-lives. Repeated treatment of pre-diabetic NOD mice with soluble NRP-A7 peptide blunts the avidity maturation of the NRP-A7-reactive CD8+ T-cell population by selectively deleting those clonotypes expressing T-cell receptors with the highest affinity and lowest dissociation rates for peptide-MHC binding. This inhibits the local production of T cells that are cytotoxic to beta cells, and halts the progression from severe insulitis to diabetes. We conclude that avidity maturation of pathogenic T-cell populations may be the key event in the progression of benign inflammation to overt disease in autoimmunity.