Selective autophagy of the adaptor TRIF regulates innate inflammatory signaling.

Defective autophagy is linked to diseases such as rheumatoid arthritis, lupus and inflammatory bowel disease (IBD). However, the mechanisms by which autophagy limits inflammation remain poorly understood. Here we found that loss of the autophagy-related gene Atg16l1 promoted accumulation of the adaptor TRIF and downstream signaling in macrophages. Multiplex proteomic profiling identified SQSTM1 and Tax1BP1 as selective autophagy-related receptors that mediated the turnover of TRIF. Knockdown of Tax1bp1 increased production of the cytokines IFN-ß and IL-1ß. Mice lacking Atg16l1 in myeloid cells succumbed to lipopolysaccharide-mediated sepsis but enhanced their clearance of intestinal Salmonella typhimurium in an interferon receptor-dependent manner. Human macrophages with the Crohn's disease-associated Atg16l1 variant T300A exhibited more production of IFN-ß and IL-1ß. An elevated interferon-response gene signature was observed in patients with IBD who were resistant to treatment with an antibody to the cytokine TNF. These findings identify selective autophagy as a key regulator of signaling via the innate immune system.

NINJ1 mediates plasma membrane rupture during lytic cell death.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.

Integration of innate immune signalling by caspase-8 cleavage of N4BP1.

Mutations in the death receptor FAS1,2 or its ligand FASL3 cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD-which mediate cell death downstream of FAS and FASL-cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome4-6. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses7-12, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1-TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages13 was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.

Granulocyte Macrophage Colony-Stimulating Factor-Activated Eosinophils Promote Interleukin-23 Driven Chronic Colitis.

The role of intestinal eosinophils in immune homeostasis is enigmatic and the molecular signals that drive them from protective to tissue damaging are unknown. Most commonly associated with Th2 cell-mediated diseases, we describe a role for eosinophils as crucial effectors of the interleukin-23 (IL-23)-granulocyte macrophage colony-stimulating factor (GM-CSF) axis in colitis. Chronic intestinal inflammation was characterized by increased bone marrow eosinopoiesis and accumulation of activated intestinal eosinophils. IL-5 blockade or eosinophil depletion ameliorated colitis, implicating eosinophils in disease pathogenesis. GM-CSF was a potent activator of eosinophil effector functions and intestinal accumulation, and GM-CSF blockade inhibited chronic colitis. By contrast neutrophil accumulation was GM-CSF independent and dispensable for colitis. In addition to TNF secretion, release of eosinophil peroxidase promoted colitis identifying direct tissue-toxic mechanisms. Thus, eosinophils are key perpetrators of chronic inflammation and tissue damage in IL-23-mediated immune diseases and it suggests the GM-CSF-eosinophil axis as an attractive therapeutic target.

CD96 functions as a co-stimulatory receptor to enhance CD8+ T cell activation and effector responses.

CD96 is a member of the poliovirus receptor (PVR, CD155)-nectin family that includes T cell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate T cell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8+ T cells. Here, we demonstrate that CD96 has co-stimulatory function on CD8+ T cells. Crosslinking of CD96 on human or mouse CD8+ T cells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK-ERK pathway. CD96-mediated signaling led to increased frequencies of NUR77- and T-bet-expressing CD8+ T cells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector T cell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8+ T cells impaired expression of transcription factors and proinflammatory cytokines associated with CD8+ T cell activation in in vivo models. Taken together, CD96 has a co-stimulatory role in CD8+ T cell activation and effector function.

Dysregulated hematopoietic stem and progenitor cell activity promotes interleukin-23-driven chronic intestinal inflammation.

In interleukin-23 (IL-23)-dependent colitis, there is excessive accumulation of short-lived neutrophils and inflammatory monocytes in the intestine. It is unknown whether this reflects changes in mature cell populations or whether the IL-23-driven colitogenic T cell program regulates upstream hematopoietic stem and progenitor cells (HSPC). Here we have shown dysregulation of hematopoiesis in colitis mediated by inflammatory cytokines. First, there was an interferon-gamma-dependent accumulation of proliferating hematopoietic stem cells in the bone marrow and spleen. Second, there was a strong skew toward granulocyte-monocyte progenitor (GMP) production at the expense of erythroid and lymphoid progenitors. Extramedullary hematopoiesis was also evident, and granulocyte macrophage-colony stimulating factor (GM-CSF) blockade reduced the accumulation of splenic and colonic GMPs, resulting in amelioration of colitis. Importantly, transfer of GMPs exacerbated colitis. These data identify HSPCs as a major target of the IL-23-driven inflammatory axis suggesting therapeutic strategies for the treatment of inflammatory bowel disease.

Micro-CT imaging and structural analysis of glomeruli in a model of Adriamycin-induced nephropathy.

Glomeruli number and size are important for determining the pathogenesis of glomerular disease, chronic kidney disease, and hypertension. Moreover, renal injury can occur in specific cortical layers and alter glomerular spatial distribution. In this study, we present a comprehensive structural analysis of glomeruli in a model of Adriamycin (doxorubicin) nephropathy. Glomeruli are imaged (micro-CT at 10 × 10 × 10 µm3) in kidney specimens from C57Bl/6 mouse cohorts: control treated with saline ( n = 9) and Adriamycin treated with 20 mg/kg Adriamycin ( n = 7). Several indices were examined, including glomerular number, glomerular volume, glomerular volume heterogeneity, and spatial density at each glomerulus and in each cortical layer (superficial, midcortical, and juxtamedullary). In the Adriamycin-treated animals, glomerular number decreased significantly in the left kidney [control: 8,298 ± 221, Adriamycin: 6,781 ± 630 (mean ± SE)] and right kidney (control: 7,317 ± 367, Adriamycin: 5,522 ± 508), and glomerular volume heterogeneity increased significantly in the left kidney (control: 0.642 ± 0.015, Adriamycin: 0.786 ± 0.018) and right kidney (control: 0.739 ± 0.016, Adriamycin: 0.937 ± 0.023). Glomerular spatial density was not affected. Glomerular volume heterogeneity increased significantly in the superficial and midcortical layers of the Adriamycin cohort. Adriamycin did not affect glomerular volume or density metrics in the juxtamedullary region, suggesting a compensatory mechanism of juxtamedullary glomeruli to injury in the outer cortical layers. Left/right asymmetry was observed in kidney size and various glomeruli metrics. The methods presented here can be used to evaluate renal disease models with subtle changes in glomerular endowment locally or across the entire kidney, and they provide an imaging tool to investigate diverse interventions and therapeutic drugs.

TPL2 kinase action and control of inflammation.

Tumor progression locus 2 (TPL2, also known as COT or MAP3K8) is a mitogen-activated protein kinase kinase (MAP3K) activated downstream of TNFαR, IL1R, TLR, CD40, IL17R, and some GPCRs. TPL2 regulates the MEK1/2 and ERK1/2 pathways to regulate a cascade of inflammatory responses. In parallel to this, TPL2 also activates p38α and p38δ to drive the production of various inflammatory mediators in neutrophils. We discuss the implications of this finding in the context of various inflammatory diseases.

Therapeutic Targeting of the G-CSF Receptor Reduces Neutrophil Trafficking and Joint Inflammation in Antibody-Mediated Inflammatory Arthritis.

G-CSF is a hemopoietic growth factor that has a role in steady state granulopoiesis, as well as in mature neutrophil activation and function. G-CSF- and G-CSF receptor-deficient mice are profoundly protected in several models of rheumatoid arthritis, and Ab blockade of G-CSF also protects against disease. To further investigate the actions of blocking G-CSF/G-CSF receptor signaling in inflammatory disease, and as a prelude to human studies of the same approach, we developed a neutralizing mAb to the murine G-CSF receptor, which potently antagonizes binding of murine G-CSF and thereby inhibits STAT3 phosphorylation and G-CSF receptor signaling. Anti-G-CSF receptor rapidly halted the progression of established disease in collagen Ab-induced arthritis in mice. Neutrophil accumulation in joints was inhibited, without rendering animals neutropenic, suggesting an effect of G-CSF receptor blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti-G-CSF receptor-treated mice showed alterations in cell adhesion receptors, with reduced CXCR2 and increased CD62L expression. Furthermore, blocking neutrophil trafficking with anti-G-CSF receptor suppressed local production of proinflammatory cytokines (IL-1ß, IL-6) and chemokines (KC, MCP-1) known to drive tissue damage. Differential gene expression analysis of joint neutrophils showed a switch away from an inflammatory phenotype following anti-G-CSF receptor therapy in collagen Ab-induced arthritis. Importantly, G-CSF receptor blockade did not adversely affect viral clearance during influenza infection in mice. To our knowledge, we describe for the first time the effect of G-CSF receptor blockade in a therapeutic model of inflammatory joint disease and provide support for pursuing this therapeutic approach in treating neutrophil-associated inflammatory diseases.

G-CSF and Neutrophils Are Nonredundant Mediators of Murine Experimental Autoimmune Uveoretinitis.

Granulocyte colony-stimulating factor (G-CSF) is a regulator of neutrophil production, function, and survival. Herein, we investigated the role of G-CSF in a murine model of human uveitis-experimental autoimmune uveoretinitis. Experimental autoimmune uveoretinitis was dramatically reduced in G-CSF-deficient mice and in anti-G-CSF monoclonal antibody-treated, wild-type (WT) mice. Flow cytometric analysis of the ocular infiltrate in WT mice with experimental autoimmune uveoretinitis showed a mixed population, comprising neutrophils, macrophages, and T cells. The eyes of G-CSF-deficient and anti-G-CSF monoclonal antibody-treated WT mice had minimal neutrophil infiltrate, but no change in other myeloid-derived inflammatory cells. Antigen-specific T-cell responses were maintained, but the differentiation of pathogenic type 17 helper T cells in experimental autoimmune uveoretinitis was reduced with G-CSF deficiency. We show that G-CSF controls the ocular neutrophil infiltrate by modulating the expression of C-X-C chemokine receptors 2 and 4 on peripheral blood neutrophils, as well as actin polymerization and migration. These data reveal an integral role for G-CSF-driven neutrophil responses in ocular autoimmunity, operating within and outside of the bone marrow, and also identify G-CSF as a potential therapeutic target in the treatment of human uveoretinitis.

Therapeutic effect of IVIG on inflammatory arthritis in mice is dependent on the Fc portion and independent of sialylation or basophils.

High-dose i.v. Ig (IVIG) is used to treat various autoimmune and inflammatory diseases; however, the mechanism of action remains unclear. Based on the K/BxN serum transfer arthritis model in mice, IVIG suppression of inflammation has been attributed to a mechanism involving basophils and the binding of highly sialylated IgG Fc to DC-SIGN-expressing myeloid cells. The requirement for sialylation was examined in the collagen Ab-induced arthritis (CAbIA) and K/BxN serum transfer arthritis models in mice. High-dose IVIG (1-2 g/kg body weight) suppressed inflammatory arthritis when given prophylactically. The same doses were also effective in the CAbIA model when given subsequent to disease induction. In this therapeutic CAbIA model, the anti-inflammatory effect of IVIG was dependent on IgG Fc but not F(ab')2 fragments. Removal of sialic acid residues by neuraminidase had no impact on the anti-inflammatory activity of IVIG or Fc fragments. Treatment of mice with basophil-depleting mAbs did not abrogate the suppression of either CAbIA or K/BxN arthritis by IVIG. Our data confirm the therapeutic benefit of IVIG and IgG Fc in Ab-induced arthritis but fail to support the significance of sialylation and basophil involvement in the mechanism of action of IVIG therapy.

Opportunities for Treg cell therapy for the treatment of human disease.

Regulatory T (Treg) cells are essential for maintaining peripheral tolerance, preventing autoimmunity, and limiting chronic inflammatory diseases. This small CD4+ T cell population can develop in the thymus and in the peripheral tissues of the immune system through the expression of an epigenetically stabilized transcription factor, FOXP3. Treg cells mediate their tolerogenic effects using multiple modes of action, including the production of inhibitory cytokines, cytokine starvation of T effector (e.g., IL-2), Teff suppression by metabolic disruption, and modulation of antigen-presenting cell maturation or function. These activities together result in the broad control of various immune cell subsets, leading to the suppression of cell activation/expansion and effector functions. Moreover, these cells can facilitate tissue repair to complement their suppressive effects. In recent years, there has been an effort to harness Treg cells as a new therapeutic approach to treat autoimmune and other immunological diseases and, importantly, to re-establish tolerance. Recent synthetic biological advances have enabled the cells to be genetically engineered to achieve tolerance and antigen-specific immune suppression by increasing their specific activity, stability, and efficacy. These cells are now being tested in clinical trials. In this review, we highlight both the advances and the challenges in this arena, focusing on the efforts to develop this new pillar of medicine to treat and cure a variety of diseases.

Interleukin-23 drives innate and T cell-mediated intestinal inflammation.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract involving aberrant activation of innate and adaptive immune responses. We have used two complementary models of IBD to examine the roles of interleukin (IL)-12 family cytokines in bacterially induced intestinal inflammation. Our results clearly show that IL-23, but not IL-12, is essential for the induction of chronic intestinal inflammation mediated by innate or adaptive immune mechanisms. Depletion of IL-23 was associated with decreased proinflammatory responses in the intestine but had little impact on systemic T cell inflammatory responses. These results newly identify IL-23 as a driver of innate immune pathology in the intestine and suggest that selective targeting of IL-23 represents an attractive therapeutic approach in human IBD.

IL-23 plays a key role in Helicobacter hepaticus-induced T cell-dependent colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that is caused in part by a dysregulated immune response to the intestinal flora. The common interleukin (IL)-12/IL-23p40 subunit is thought to be critical for the pathogenesis of IBD. We have analyzed the role of IL-12 versus IL-23 in two models of Helicobacter hepaticus-triggered T cell-dependent colitis, one involving anti-IL-10R monoclonal antibody treatment of infected T cell-sufficient hosts, and the other involving CD4+ T cell transfer into infected Rag-/- recipients. Our data demonstrate that IL-23 and not IL-12 is essential for the development of maximal intestinal disease. Although IL-23 has been implicated in the differentiation of IL-17-producing CD4+ T cells that alone are sufficient to induce autoimmune tissue reactivity, our results instead support a model in which IL-23 drives both interferon gamma and IL-17 responses that together synergize to trigger severe intestinal inflammation.

Overcoming Preclinical Safety Obstacles to Discover (S)-N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl)-6-(methylamino)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonamide (GDC-2394): A Potent and Selective NLRP3 Inhibitor.

Inappropriate activation of the NLRP3 inflammasome has been implicated in multiple inflammatory and autoimmune diseases. Herein, we aimed to develop novel NLRP3 inhibitors that could minimize the risk of drug-induced liver injury. Lipophilic ligand efficiency was used as a guiding metric to identify a series of 6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazinesulfonylureas. A leading compound from this series was advanced into safety studies in cynomolgus monkeys, and renal toxicity, due to compound precipitation, was observed. To overcome this obstacle, we focused on improving the solubility of our compounds, specifically by introducing basic amine substituents into the scaffold. This led to the identification of GDC-2394, a potent and selective NLRP3 inhibitor, with an in vitro and in vivo safety profile suitable for advancement into human clinical trials.

Diverse cytokine production by NKT cell subsets and identification of an IL-17-producing CD4-NK1.1- NKT cell population.

NKT cell subsets can be divided based on CD4 and NK1.1 expression and tissue of origin, but the developmental and functional relationships between the different subsets still are poorly understood. A comprehensive study of 19 cytokines across different NKT cell subsets revealed that no two NKT subpopulations exhibited the same cytokine profile, and, remarkably, the amounts of each cytokine produced varied by up to 100-fold or more among subsets. This study also revealed the existence of a population of CD4(-)NK1.1(-) NKT cells that produce high levels of the proinflammatory cytokine IL-17 within 2-3 h of activation. On intrathymic transfer these cells develop into mature CD4(-)NK1.1(+) but not into CD4(+)NK1.1(+) NKT cells, indicating that CD4(-)NK1.1(-) NKT cells include an IL-17-producing subpopulation, and also mark the elusive branch point for CD4(+) and CD4(-) NKT cell sublineages.

Genetic inactivation of RIP1 kinase activity in rats protects against ischemic brain injury.

RIP1 kinase-mediated inflammatory and cell death pathways have been implicated in the pathology of acute and chronic disorders of the nervous system. Here, we describe a novel animal model of RIP1 kinase deficiency, generated by knock-in of the kinase-inactivating RIP1(D138N) mutation in rats. Homozygous RIP1 kinase-dead (KD) rats had normal development, reproduction and did not show any gross phenotypes at baseline. However, cells derived from RIP1 KD rats displayed resistance to necroptotic cell death. In addition, RIP1 KD rats were resistant to TNF-induced systemic shock. We studied the utility of RIP1 KD rats for neurological disorders by testing the efficacy of the genetic inactivation in the transient middle cerebral artery occlusion/reperfusion model of brain injury. RIP1 KD rats were protected in this model in a battery of behavioral, imaging, and histopathological endpoints. In addition, RIP1 KD rats had reduced inflammation and accumulation of neuronal injury biomarkers. Unbiased proteomics in the plasma identified additional changes that were ameliorated by RIP1 genetic inactivation. Together these data highlight the utility of the RIP1 KD rats for target validation and biomarker studies for neurological disorders.

Multiplexed proteomics of autophagy-deficient murine macrophages reveals enhanced antimicrobial immunity via the oxidative stress response.

Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn's disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri), a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo. Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens.

RIP1 kinase activity is critical for skin inflammation but not for viral propagation.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.

Discovery of Potent Benzolactam IRAK4 Inhibitors with Robust in Vivo Activity.

IRAK4 kinase activity transduces signaling from multiple IL-1Rs and TLRs to regulate cytokines and chemokines implicated in inflammatory diseases. As such, there is high interest in identifying selective IRAK4 inhibitors for the treatment of these disorders. We previously reported the discovery of potent and selective dihydrobenzofuran inhibitors of IRAK4. Subsequent studies, however, showed inconsistent inhibition in disease-relevant pharmacodynamic models. Herein, we describe application of a human whole blood assay to the discovery of a series of benzolactam IRAK4 inhibitors. We identified potent molecule 19 that achieves robust in vivo inhibition of cytokines relevant to human disease.