Search

Fermentation of dietary fibre by the gut microbiota leads to the production of metabolites called short-chain fatty acids (SCFAs), which have emerged as potent regulators of immune, metabolic, and tissue barrier functions. More recently, a high fibre diet and SCFA supplementation were shown to lower blood pressure and be cardio-protective. SCFAs activate host signalling responses via the receptors GPR41 and GPR43, which have redundancy in their signalling pathways. Whether these receptors play a role in hypertension or mediate the cardio-protective effects of fibre remains unknown. Using an experimental model that lacks both GPR41 and GPR43, we show that lack of signalling via these receptors increases risk to high blood pressure and leads to cardiorenal fibrosis and hypertrophy.Moreover, we demonstrate that GPR41/43 signalling is essential in maintaining gut epithelial barrier, which prevents the translocation of the bacterial toxins lipopolysaccharides (LPS) from entering the peripheral circulation. In the absence of GPR41/43, this is accompanied by macrophage infiltration to the kidneys, resulting in pro-inflammatory cytokine production.Using an antagonist against the LPS’ receptor, TLR4, a potent pro-inflammatory signalling pathway, we were able to rescue the cardiovascular phenotype in GPR41/43 knockout mice. We also demonstrate that GPR41/43 are, at least partially, responsible for the blood pressure- lowering and cardio-protective effects of a high fibre diet; however, improvements of gut barrier integrity and macrophages in the kidney were independent of GPR41/43 signalling.Finally, using the UK Biobank, we provide translational evidence that variants associated with lower expression of both GPR41/43 are more prevalent in hypertensive patients. Our findings highlight that lack of SCFA-receptor signalling via both GPR41/43 increases risk of high blood pressure, suggesting these receptors could be targeted as a new treatment.

Besides damaging the brain, stroke causes systemic changes, including to the gastrointestinal system. A growing body of evidence supports the role of the gut and its microbiota in stroke, stroke prognosis, and recovery. The gut microbiota can increase the risk of a cerebrovascular event, playing a role in the onset of stroke. Conversely, stroke can induce dysbiosis of the gut microbiota and epithelial barrier integrity. This has been proposed as a contributor to systemic infections. In this review, we describe the role of the gut microbiota, microbiome and microbiota-derived metabolites in experimental and clinical stroke, and their potential use as therapeutic targets. Fourteen clinical studies have identified 62 upregulated (eg, Streptococcus , Lactobacillus, Escherichia ) and 29 downregulated microbial taxa (eg, Eubacterium, Roseburia ) between stroke and healthy participants. The majority found that stroke patients have reduced gut microbiome diversity. However, other nonbacterial microorganisms are yet to be studied. In experimental stroke, severity is dependent on gut microbiome composition, whereas the latter can greatly change with antibiotics, age, and diet. Consumption of foods rich in choline and L-carnitine are positively associated with stroke onset via production of trimethylamine N-oxide in experimental and clinical stroke. Conversely, in mice, consumption of dietary fiber improves stroke outcome, likely via gut microbiota–derived metabolites called short-chain fatty acids, such as acetate, propionate, and butyrate. The majority of the evidence, however, comes from experimental studies. Clinical interventions targeted at gut microbiota–derived metabolites as new therapeutic opportunities for stroke prevention and treatment are warranted.

Elevated blood pressure (BP), or hypertension, is the main risk factor for cardiovascular disease. As a multifactorial and systemic disease that involves multiple organs and systems, hypertension remains a challenging disease to study. Models of hypertension are invaluable to support the discovery of the specific genetic, cellular and molecular mechanisms underlying essential hypertension, as well as to test new possible treatments to lower BP. Rodent models have proven to be an invaluable tool for advancing the field. In this review, we discuss the strengths and weaknesses of rodent models of hypertension through a systems approach. We highlight the ways how target organs and systems including the kidneys, vasculature, the sympathetic nervous system (SNS), immune system and the gut microbiota influence BP in each rodent model. We also discuss often overlooked hypertensive conditions such as pulmonary hypertension and hypertensive-pregnancy disorders, providing an important resource for researchers. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.

Dietary fibre regulates blood pressure (BP) through gut microbial production of acidic metabolites known as short-chain fatty acids (SCFAs). The specific mechanisms of how SCFAs regulate BP are still emerging. We hypothesised that acidic metabolites that are abundant in the large intestine may activate proton-sensing G-protein coupled receptors, such as GPR65, thus conferring BP regulating effects. Using mouse models, we found that dietary fibre levels determined the luminal pH in the large intestine, through production of SCFAs by the gut microbiota. We then investigated a new mouse model lacking GPR65, which spontaneously developed higher BP, cardiac and renal hypertrophy and fibrosis. We identified that low pH, acting via GPR65 signalling, increased cAMP production and phosphorylation of CREB, and regulated inflammatory cytokine production involved in hypertension. We showed that the benefits of diets high in fibre, which usually prevent hypertension and its associated phenotypes, were decreased in mice lacking GPR65. Finally, we provided proof-of-concept evidence that the luminal pH profile in the colon of hypertensive participants is higher than that of normotensive participants. Colonic pH was further associated with dietary fibre, particularly in the colonic regions where fibre is fermented by the gut microbiota. Together, we show that pH sensing by GPR65 underlies at least some of the cardiovascular benefits of dietary fibre.

Objective: Altered immune cell activation plays a key role in promoting hypertension. How the immune system becomes activated during hypertension is unknown. A possibility is necroptosis, a newly described form of cell death that alerts the immune system to dying cells, causing inflammation. Using Mlkl -/- mice that are genetically unable to undergo necroptosis, we explored the role of necroptosis in inflammation or blood pressure regulation during hypertension. We also examined whether Ang-II sensitizes human and murine cells to necroptosis. Methods: Monocytes were sorted from peripheral blood mononuclear cells of 3 normotensive and 3 untreated hypertensive participants diagnosed with 24h BP monitoring. Primary murine cardiomyocytes and fibroblasts were obtained from 3 litters of neonatal C57BL/6J mice. Necroptosis was induced using TNF-alpha, SMAC mimetic and zVAD in the presence or absence of Ang-II. Viability of human cells was measured using propidium iodide and in murine cells with CellTitre-Glo Viability Assay. Hypertension was induced in male Mlkl -/- mice and WT littermate controls by implanting minipumps containing Ang-II (0.5mg/kg/day, 28 days; n=6/group). BP was measured weekly by tail-cuff. Results: Monocytes from hypertensive participants were not more susceptible to necroptosis than normotensive participants (P=0.79). This was not altered by Ang-II treatment in normotensive (P=0.98) or hypertensive participants (P=0.92). Moreover, Ang-II did not alter the viability of primary murine fibroblasts in vitro (P=0.79). Mouse cardiac fibroblasts were unable to undergo necroptosis. In vivo, there was no difference in BP of Ang-II-treated Mlkl -/- and WT control mice at 4 weeks (P=0.87). Conclusions: We show that monocytes from hypertensive participants were not more susceptible to necroptosis, compared to normotensive participants. Furthermore, Ang-II did sensitize mouse cardiac fibroblasts or human monocytes to necroptosis. No changes in BP were observed between Ang-II treated WT and MLKL knockout mice, suggesting necroptosis does not impact BP regulation. Further analyses are being performed to determine if inflammation or fibrosis are altered when necroptosis is inhibited.

Background Risk factors for heart failure with preserved ejection fraction (HFpEF) include hypertension, age, sex, and obesity. Emerging evidence suggests that the gut microbiota independently contributes to each one of these risk factors, potentially mediated via gut microbial‐derived metabolites such as short‐chain fatty acids. In this study, we determined whether the gut microbiota were associated with HFpEF and its risk factors. Methods and Results We recruited 26 patients with HFpEF and 67 control participants from 2 independent communities. Patients with HFpEF were diagnosed by exercise right heart catheterization. We assessed the gut microbiome by bacterial 16S rRNA sequencing and food intake by the food frequency questionnaire. There was a significant difference in α‐diversity (eg, number of microbes) and β‐diversity (eg, type and abundance of microbes) between both cohorts of controls and patients with HFpEF ( P =0.001). We did not find an association between β‐diversity and specific demographic or hemodynamic parameters or risk factors for HFpEF. The Firmicutes to Bacteroidetes ratio, a commonly used marker of gut dysbiosis, was lower, but not significantly so ( P =0.093), in the patients with HFpEF. Compared with controls, the gut microbiome of patients with HFpEF was depleted of bacteria that are short‐chain fatty acid producers. Consistent with this, participants with HFpEF consumed less dietary fiber (17.6±7.7 versus 23.2±8.8 g/day; P =0.016). Conclusions We demonstrate key changes in the gut microbiota in patients with HFpEF, including the depletion of bacteria that generate metabolites known to be important for cardiovascular homeostasis. Further studies are required to validate the role of these gut microbiota and metabolites in the pathophysiology of HFpEF.

Necroptosis is a form of cell death associated with inflammation; however, the biological consequences of chronic necroptosis are unknown. Necroptosis is mediated by RIPK1, RIPK3, and MLKL kinases but in hematopoietic cells RIPK1 has anti-inflammatory roles and functions to prevent necroptosis. Here we interrogate the consequences of chronic necroptosis on immune homeostasis by deleting Ripk1 in mouse dendritic cells. We demonstrate that deregulated necroptosis results in systemic inflammation, tissue fibrosis, and autoimmunity. We show that inflammation and autoimmunity are prevented upon expression of kinase inactive RIPK1 or deletion of RIPK3 or MLKL. We provide evidence that the inflammation is not driven by microbial ligands, but depends on the release of danger-associated molecular patterns and MyD88-dependent signaling. Importantly, although the inflammation is independent of type I IFN and the nucleic acid sensing TLRs, blocking these pathways rescues the autoimmunity. These mouse genetic studies reveal that chronic necroptosis may underlie human fibrotic and autoimmune disorders.

Neutropenia represents one of the major dose-limiting toxicities of many current cancer therapies. To circumvent the off-target effects of cytotoxic chemotherapeutics, kinase inhibitors are increasingly being used as an adjunct therapy to target leukemia. In this study, we conducted a screen of leukemic cell lines in parallel with primary neutrophils to identify kinase inhibitors with the capacity to induce apoptosis of myeloid and lymphoid cell lines whilst sparing primary mouse and human neutrophils. We have utilized a high-throughput live cell imaging platform to demonstrate that cytotoxic drugs have limited effects on neutrophil viability but are toxic to hematopoietic progenitor cells, with the exception of the topoisomerase I inhibitor SN-38. The parallel screening of kinase inhibitors revealed that mouse and human neutrophil viability is dependent on cyclin-dependent kinase (CDK) activity but surprisingly only partially dependent on PI3 kinase and JAK/STAT signaling, revealing dominant pathways contributing to neutrophil viability. Mcl-1 haploinsufficiency sensitized neutrophils to CDK inhibition, demonstrating that Mcl-1 is a direct target for CDK inhibitors. This study reveals a therapeutic window for the kinase inhibitors BEZ235, BMS-3, AZD7762, and (R)-BI-2536 to induce apoptosis of leukemia cell lines whilst maintaining immunocompetence and hemostasis.

The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called “shuttling”. InTalaromyces marneffeiandAspergillus fumigatuszebrafishin vivoinfections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, involves alterations of phagocyte mobility, inter-cellular tethering, and phagosome transfer. Shuttling kinetics were fungal species-specific, implicating a fungal determinant. β-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttledin vitro. The impact of shuttling for microbiological outcomes ofin vivoinfections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favourable macrophage intracellular niche. Shuttling is a frequent host/pathogen interaction contributing to fungal infection establishment patterns.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/β release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/β expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/β release.

Receptor interacting protein kinase 1 (RIPK1) has important kinase-dependent and kinase-independent scaffolding functions that activate or prevent apoptosis or necroptosis in a cell context-dependent manner. The kinase activity of RIPK1 mediates hypothermia and lethality in a mouse model of TNF-induced shock, reflecting the hyperinflammatory state of systemic inflammatory response syndrome (SIRS), where the proinflammatory "cytokine storm" has long been viewed as detrimental. Here, we demonstrate that cytokine and chemokine levels did not predict survival and, importantly, that kinase-inactive Ripk1D138N/D138N hematopoietic cells afforded little protection from TNF- or TNF/zVAD-induced shock in reconstituted mice. Unexpectedly, RIPK1 kinase-inactive mice transplanted with WT hematopoietic cells remained resistant to TNF-induced shock, revealing that a nonhematopoietic lineage mediated protection. TNF-treated Ripk1D138N/D138N mice exhibited no significant increases in intestinal or vascular permeability, nor did they activate the clotting cascade. We show that TNF administration damaged the liver vascular endothelium and induced phosphorylated mixed lineage kinase domain-like (phospho-MLKL) reactivity in endothelial cells isolated from TNF/zVAD-treated WT, but not Ripk1D138N/D138N, mice. These data reveal that the tissue damage present in this SIRS model is reflected, in part, by breaks in the vasculature due to endothelial cell necroptosis and thereby predict that RIPK1 kinase inhibitors may provide clinical benefit to shock and/or sepsis patients.

Necroptosis is a form of cell death associated with inflammation, however the biological consequences of chronic necroptosis are unknown. Necroptosis is mediated by RIPK1, RIPK3 and MLKL kinases but in hematopoietic cells RIPK1 has anti-inflammatory roles and functions to prevent necroptosis. Here we interrogate the consequences of chronic necroptosis on immune homeostasis by deleting Ripk1 in mouse dendritic cells (DC). We demonstrate that deregulated necroptosis results in systemic inflammation, tissue fibrosis and autoimmunity. We show that inflammation and autoimmunity are prevented upon expression of kinase inactive RIPK1 or deletion of RIPK3 or MLKL. We provide evidence that the inflammation is not driven by microbial ligands, but depends on the release of danger-associated molecular patterns (DAMPs) and MyD88-dependent signaling. Importantly, whilst the inflammation is independent of type I interferon and the nucleic acid sensing TLRs, blocking these pathways rescues the autoimmunity. These mouse genetic studies reveal that chronic necroptosis may underlie human fibrotic and autoimmune disorders.

Neutrophils use Toll-like receptor and IL-18 signaling to reprogram Fas-induced death. The regulation of neutrophil lifespan is critical for a circumscribed immune response. Neutrophils are sensitive to Fas/CD95 death receptor signaling in vitro, but it is unknown if Fas regulates neutrophil lifespan in vivo. We hypothesized that FasL-expressing CD8+ T cells, which kill antigen-stimulated T cells during chronic viral infection, can also induce neutrophil death in tissues during infection. With the use of LysM-Cre Fasfl/fl mice, which lack Fas expression in macrophages and neutrophils, we show that Fas regulates neutrophil lifespan during lymphocytic choriomeningitis virus (LCMV) infection in the lung, peripheral blood, and spleen. Fas also contributed to the regulation of neutrophil numbers in the colon of Citrobacter rodentium-infected mice. To examine the effects of infection on Fas activation in neutrophils, we primed neutrophils with TLR ligands or IL-18, resulting in ablation of Fas death receptor signaling. These data provide the first in vivo genetic evidence that neutrophil lifespan is controlled by death receptor signaling and provide a mechanism to account for neutrophil resistance to Fas stimulation during infection.

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.

For over two decades, we have embraced the cytokine storm theory to explain sepsis, severe sepsis and septic shock. The failure of numerous large-scale clinical trials, which aimed to treat sepsis by neutralizing inflammatory cytokines and LPS, indicates that alternative pathophysiological mechanisms are likely to account for sepsis and the associated immune suppression in patients with severe infection. Recent insights that extricate pyroptotic death from inflammatory cytokine production in vivo have highlighted a need to investigate the consequences of apoptotic and non-apoptotic death in contributing to cytopenia and immune suppression. In this review, we will focus on the biochemical and cellular mechanisms controlling pyroptosis, a Caspase-1/11 dependent form of cell death during infection.

The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte–pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called “shuttling.” In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species–specific, implicating a fungal determinant. β-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host–pathogen interaction contributing to fungal infection establishment patterns., Imaging of the behaviour of white blood cells in living zebrafish embryos infected with fungi reveals “shuttling,” a specific and previously undescribed form of microorganism exchange between neutrophils and macrophages.

Neutrophilic dermatoses are a group of inflammatory skin disorders characterized by sterile infiltrates of neutrophils. These syndromes include pyoderma gangrenosum (PG) and Sweet’s syndrome (SS), and they are associated with an increased incidence of inflammatory bowel disease, rheumatoid arthritis, and acute myeloid leukemia. IL-1β is detectable in skin lesions, and IL-1 neutralizing therapies have met with success in some patients. Splicing variants and promoter region deletions of protein tyrosine phosphatase-6 (PTPN6) are a feature of SS and PG. Mice lacking Ptpn6 develop a cutaneous inflammatory disease that is dependent on the IL-1 receptor, G-CSF, and neutrophils, but the source of IL-1 and the mechanisms of IL-1 release remain unclear. Here, we investigated the mechanisms controlling IL-1α/β release specifically from neutrophils (Ptpn6ΔPMN) by inhibiting Caspase-8-dependent apoptosis and Ripk1/Ripk3/Mlkl-regulated necroptosis. Loss of Ripk1 from neutrophils accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6ΔPMN mice. Ptpn6ΔPMN neutrophils displayed increased p38-dependent Ripk1-independent IL-1 and TNF production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 MAP kinase activation to control TNF and IL-1α/β transcription, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3/Mlkl-dependent cell death and concomitant IL-1α/β release. These findings implicate neutrophils as the dominant producers of IL-1 in neutrophilic dermatoses, and identify novel therapeutic targets for the treatment of these skin disorders.

Glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly-functional T(H)1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR-domain-containing adapter inducing IFN-beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal T(H)1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA-SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild-type mice. We find that both MyD88 and TRIF are necessary for GLA-SE to induce a poly-functional T(H)1 immune response characterized by CD4(+) T cells producing IFN-γ, TNF, and IL-2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA-SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo T(H)1-skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.

During immune responses, neutrophils must integrate survival and death signals from multiple sources to regulate their lifespan. Signals that activate either the Bcl-2- or death receptor-regulated apoptosis pathways can provide powerful stimuli for neutrophils to undergo cell death, but whether they act cooperatively in parallel or directly cross-talk in neutrophils is not known. Previous studies suggested that Bcl-2 family proteins are not required for Fas-induced cell death in neutrophils, but did not examine whether they could modulate its rapid onset. By monitoring the rate of change in neutrophil viability associated with activation of the Fas-triggered death receptor pathway using real-time cell imaging, we show that the Bcl-2-related proteins Bid, Bax, and Bak accelerate neutrophil apoptosis but are not essential for cell death. Increased Bcl-2 or Mcl-1 expression prevents efficient induction of apoptosis by Fas stimulation indicating that the Bcl-2-regulated apoptosis pathway can directly interfere with Fas-triggered apoptosis. Fas has been shown to initiate NFκB activation and gene transcription in cell lines, however gene transcription is not altered in Fas-activated Bid −/− neutrophils, indicating that apoptosis occurs independently of gene transcription in neutrophils. The specification of kinetics of neutrophil apoptosis by Bid impacts on the magnitude of neutrophil IL-1β production, implicating a functional role for the Bcl-2-regulated pathway in controlling neutrophil responses to FasL. These data demonstrate that the intrinsic apoptosis pathway directly controls the kinetics of Fas-triggered apoptosis in neutrophils.

The lack of expression of the suppressor of cytokine signalling-3 (SOCS3) or inactivation of the negative regulatory capacity of SOCS3 has been well documented in rheumatoid arthritis, viral hepatitis and cancer. The specific qualitative and quantitative consequences of SOCS3 deficiency on interleukin-6 (IL-6)-mediated pro- and anti-inflammatory responses remain controversial in vitro and unknown in vivo. Mice with a conditional deletion of SOCS3 in hematopoietic cells develop lethal inflammatory disease during adult life and develop gross histopathological changes during experimental arthritis, typified by elevated IL-6 levels. To clarify the nature of the IL-6 responses in vivo, we generated mice deficient in SOCS3 (SOCS3(-/Δvav)) or both SOCS3 and IL-6 (IL-6(-/-)/SOCS3(-/Δvav)), and examined responses in models of acute and chronic inflammation. Acute responses to IL-1β were lethal to SOCS3(-/Δvav) mice but not IL-6(-/-)/SOCS3(-/Δvav) mice, indicating that IL-6 was required for the lethal inflammation induced by IL-1β. Administration of IL-1β to SOCS3(-/Δvav) mice induced systemic apoptosis of lymphocytes in the thymus, spleen and lymph nodes that was dependent on the presence of IL-6. IL-6 deficiency prolonged survival of SOCS3(-/Δvav) mice and ameliorated spontaneous inflammatory disease developing during adult life. Infection of SOCS3(-/Δvav) mice with LCMV induced a lethal inflammatory response that was dependent on IL-6, despite SOCS3(-/Δvav) mice controlling viral replication. We conclude that SOCS3 is required for survival during inflammatory responses and is a critical regulator of IL-6 in vivo.

The regulation of neutrophil recruitment, activation, and disposal is pivotal for circumscribed inflammation. SHP1Y208N/Y208N mutant mice develop severe cutaneous inflammatory disease that is IL-1R dependent. Genetic reduction in neutrophil numbers and neutrophilic responses to infection is sufficient to prevent the spontaneous initiation of this disease. Neutrophils from SHP1Y208N/Y208N mice display increased pro–IL-1β production due to altered responses to MyD88-dependent and MyD88-independent signals. The IL-1R–dependent inflammatory disease in SHP1Y208N/Y208N mice develops independently of caspase 1 and proteinase 3 and neutrophil elastase. In response to Fas ligand, a caspase 1-independent inducer of IL-1β production, neutrophils from SHP1Y208N/Y208N mice produce elevated levels of IL-1β but display reduced caspase 3 and caspase 7 activation. In neutrophils deficient in SHP1, IL-1β induces high levels of pro–IL-1β suggesting the presence of a paracrine IL-1β loop. These data indicate that the neutrophil- and IL-1–dependent disease in SHP1Y208N/Y208N mice is a consequence of loss of negative regulation of TLR and IL-1R signaling.

Neutrophilic dermatoses are a group of inflammatory skin disorders characterized by sterile infiltrates of neutrophils. These syndromes include pyoderma gangrenosum (PG) and Sweet's syndrome (SS), and they are associated with an increased risk of inflammatory bowel disease, rheumatoid arthritis, and hematologic malignancy, particularly monocytic or myelomonocytic leukemia (AML). IL-1 was first proposed in 1987 as a factor in SS, and the presence of "fragmented neutrophil nuclei" was hypothesized to contribute to disease. IL-1 has subsequently been reported at high levels in lesions of SS, and IL-1 neutralizing therapies have met with success in some SS patients. PG is also reported to be responsive to IL-1 neutralizing therapy, including PG patients with psoriatic arthritis. Splicing variants and promoter region deletions of protein tyrosine phosphatase-6 (PTPN6, or Src homology region 2 domain-containing phosphatase-1, SHP1) are a feature of SS and PG, consistent with the findings that mutations in Ptpn6 drive spontaneous IL-1R-dependent skin inflammation in mice. Mice lacking Ptpn6 develop a cutaneous inflammatory disease that is dependent on the IL-1 receptor (IL-1R), G-CSF, and neutrophils. We hypothesized that production of IL-1 by dying neutrophils drives skin inflammation in the setting of Ptpn6 deficiency. To investigate the mechanisms controlling pathogenic IL-1 release in mice lacking Ptpn6 specifically in neutrophils (Ptpn6∆PMN), we looked directly at cytokine production from neutrophils undergoing inflammatory forms of cell death. We found that stimuli engaging Ripk3/Mlkl-dependent necroptotic forms of cell death result in transcription, processing, and release of bioactive IL-1α and IL-1β from neutrophils. Production of IL-1α and IL-1β were increased in Ptpn6∆PMN neutrophils treated to undergo necroptosis. Ptpn6∆PMN neutrophils displayed increased rates of spontaneous cell death in the presence of G-CSF or IFN-γ alone, and were hypersensitive to necroptotic stimuli. These cell death abnormalities were absent in Ptpn6∆PMN Casp8∆PMN Mlkl-/- neutrophils, demonstrating that Ptpn6 regulates both apoptosis and necroptosis to prevent IL-1 release. We next examined the contribution of apoptosis and necroptosis in the development of spontaneous cutaneous inflammatory disease in Ptpn6∆PMN mice. Loss of either the Caspase-8-dependent apoptotic pathway or the Ripk3/Mlkl-dependent necroptotic pathway was not sufficient to prevent inflammation in mice. However, combined deletion of Caspase-8 and Ripk3/Mlkl protected Ptpn6∆PMN mice (Ptpn6∆PMN Casp8∆PMN Ripk3-/- and Ptpn6∆PMN Casp8∆PMN Mlkl-/- mice). Ripk1 is known to act as a physiological negative regulator of both Caspase-8-dependent apoptosis and Ripk3/Mlkl-dependent necroptosis. We found that the absence of Ripk1 in neutrophils in Ptpn6∆PMN Ripk1∆PMN mice resulted in heightened sensitivity of neutrophils to cell death and accelerated onset of cutaneous inflammatory disease. These results reveal Ripk1 as a critical physiological negative regulator of neutrophil inflammatory cell death and IL-1 production, and cutaneous inflammation. Together, these data emphasize dual functions for Ptpn6 in negative regulation of IL-1α/β transcription, and to prevent Caspase-8- and Ripk3/Mlkl-dependent cell death and concomitant IL-1α/β processing and release. These findings implicate neutrophils as the dominant producers of IL-1 in neutrophilic dermatoses, and identify novel therapeutic targets that could be exploited to control inflammatory forms of cell death in these skin disorders. Disclosures No relevant conflicts of interest to declare.

Leprosy elimination has been a goal of the WHO for the past 15 years. Widespread BCG vaccination and multidrug therapy have dramatically reduced worldwide leprosy prevalence, but new case detection rates have remained relatively constant. These data suggest that additional control strategies, such as a subunit vaccine, are required to block transmission and to improve leprosy control. We recently identified several M ycobacterium leprae antigens that stimulate gamma interferon (IFN-γ) secretion upon incubation with blood from paucibacillary leprosy patients, a group who limit M. leprae growth and dissemination. In this study, we demonstrate that M. leprae -specific mouse T-cell lines recognize several of these antigens, with the ML0276 protein stimulating the most IFN-γ secretion. We then examined if the ML0276 protein could be used in a subunit vaccine to provide protection against experimental M. leprae infection. Our data demonstrate that combining ML0276 with either a Toll-like receptor 4 (TLR4) (EM005), TLR7 (imiquimod), or TLR9 (CpG DNA) agonist during immunization induces Th1 responses that limit local inflammation upon experimental M. leprae infection. Our data indicate that only the ML0276/EM005 regimen is able to elicit a response that is transferable to recipient mice. Despite the potent Th1 response induced by this regimen, it could not provide protection in terms of limiting bacterial growth. We conclude that EM005 is the most potent adjuvant for stimulating a Th1 response and indicate that while a subunit vaccine containing the ML0276 protein may be useful for the prevention of immune pathology during leprosy, it will not control bacterial burden and is therefore unlikely to interrupt disease transmission.

RIPK1 has important kinase-dependent and kinase-independent scaffolding functions that prevent or activate necroptosis or apoptosis. Complete RIPK1 deficiency results in cell death and widespread inflammation yet tissue specific RIPK1 deletion can result in apoptosis, necroptosis and/or systemic inflammation, depending on the cell type. We have previously demonstrated that a hematopoietic RIPK1 deficiency results in constitutive activation of RIPK3 and MLKL and induction of necroptosis (Roderick et al, PNAS, 2014). These mice exhibit elevated serum TNFα and IFNγ levels, hematopoietic stem and progenitor cell (HSPC) loss, and ultimately succumb to bone marrow failure (BMF). When mice with a hematopoietic RIPK1 deficiency were placed on a RIPK3 deficient background, plasma pro-inflammatory cytokine and chemokine levels were reduced, HSPC numbers increased and BMF was significantly delayed. These mouse genetic data demonstrate that necroptotic death contributes to BMF in the mouse. To identify the receptor/ligands that trigger necroptosis, we generated mice with a hematopoietic RIPK1-deficiency on the Tnfr1-/-and Tnfr1-/-Tnfr2-/-genetic backgrounds. An absence of TNF signaling failed to prevent necroptosis and consequently, vav-iCre Ripk1f/f Tnfr1-/- Tnfr2-/-mice succumbed to BMF. Because type II interferons can also induce necroptosis, we generated vav-iCre Ripk1f/f Ifngr1-/- mice. These mice appear phenotypically normal demonstrating that an absence of IFNγ signaling prevents HSPC necroptosis and BMF.Collectively, these data may implicate IFNγ-mediated, RIPK3-dependent necroptosis in human BMF syndromes and raise the intriguing possibility that the progressive HSPC elimination observed in these patients reflects in part, IFNγ-induced necroptotic death. Disclosures No relevant conflicts of interest to declare.

Despite the advances toward the elimination of leprosy through widespread provision of multi-drug therapy to registered patients over the last 2 decades, new case detection rates have stabilized and leprosy remains endemic in a number of localized regions. A vaccine could overcome the inherent limitations of the drug treatment program by providing protection in individuals who are not already harboring the Mycobacterium leprae bacilli at the time of administration and effectively interrupt the transmission cycle over a wider timespan. In this report we present data validating the production of 73f, a chimeric fusion protein incorporating the M. leprae antigens ML2028, ML2346 and ML2044. The 73f protein was recognized by IgG in multibacillary (MB) leprosy patient sera and stimulated IFNγ production within whole blood assays of paucibacillary (PB) leprosy patient and healthy household contacts of MB patients (HHC). When formulated with a TLR4L-containing adjuvant (GLA-SE), 73f stimulated a strong and pluripotent Th1 response that inhibited M. leprae-induced inflammation in mice. We are using these data to develop new vaccine initiatives for the continued and long-term control of leprosy.

Leprosy is a debilitating chronic disease caused by infection with Mycobacterium leprae. A World Health Organization-directed control strategy based upon the identification and treatment of patients has resulted in a marked reduction in the number of registered worldwide leprosy cases over the last 20 years. Despite these efforts, the number of new leprosy cases detected each year now remains relatively stable, and M. leprae infection continues to pose a health problem. It is suggested that earlier diagnosis is required to strengthen control programs. In this study, we have examined the development of antigen-specific immunoglobulin responses within armadillos experimentally infected with M. leprae to identify those responses that develop most rapidly and robustly following infection. Antibody responses to the M. leprae-specific phenolic glycolipid I and several protein antigens previously demonstrated to have diagnostic potential were assessed. Our results identify several antigens that can provide early diagnosis of M. leprae infection but also indicate considerable variability in the development of antigen-specific antibodies. Our data suggest that a combination of antigens is likely required to provide accurate and early leprosy diagnosis. Leprosy is caused by infection with the bacterium Mycobacterium leprae, and its clinical symptoms, bacterial burdens, pathology, and underlying immunological responses vary widely. Diagnosis is complicated due to this wide range of signs but can be arranged histologically into the five distinct categories of the Ridley-Jopling scale (polar tuberculoid [TT], borderline tuberculoid [BT], mid-borderline [BB], borderline lepromatous [BL], and polar lepromatous [LL]) (22, 24). The World Health Organization (WHO) has suggested diagnosis of leprosy be made by the observation of one or more of the following: hypopigmented or reddish skin patches with definite loss of sensation; thickened peripheral nerves; and acid-fast bacilli on skin smears/biopsy specimens. In the classification based on skin smears, patients showing negative smears at all sites are grouped as paucibacillary (PB) leprosy, while those showing positive smears at any site are grouped as multibacillary (MB) leprosy. In practice, most programs use the clinical criteria for classifying and selecting the treatment regimen for individual patients. The clinical system includes counting the number of skin lesions and nerves involved as the basis for grouping leprosy patients into the PB (less than 5 lesions) and MB (typically 5 or more lesions) categories. It would be highly beneficial to identify M. leprae infection before such signs appear. It is believed that a significant number of individuals contain infection such that it remains subclinical or self-cures with

Therapy of intracellular pathogens can be complicated by drug toxicity, drug resistance, and the need for prolonged treatment regimens. One approach that has shown promise is immunotherapy. Leishmaniasis, a vector-borne disease ranked among the six most important tropical infectious diseases by the World Health Organization, has been treated clinically with crude or defined vaccine preparations or cytokines, such as IFN-γ and GM-CSF, in combination with chemotherapy. We have attempted to develop an improved and defined immunotherapeutic using a mouse model of cutaneous leishmaniasis. We hypothesized that immunotherapy may be improved by using TLR synergy to enhance the parasite-specific immune response. We formulated L110f, a well-established Leishmania poly-protein vaccine candidate, in conjunction with either monophosphoryl lipid A, a TLR4 agonist, or CpG, a TLR9 agonist, or a combination of these, and evaluated anti-Leishmania immune responses in absence or presence of active disease. Only mice treated with L110f plus monophosphoryl lipid A-CpG were able to induce a strong effective T cell response during disease and subsequently cured lesions and reduced parasite burden when compared with mice treated with L110f and either single adjuvant. Our data help to define a correlate of protection during active infection and indicate TLR synergy to be a potentially valuable tool in treating intracellular infections.

Reports describing the effect of interferon-gamma (IFNgamma) on interleukin-1beta (IL-1beta) production are conflicting. We resolve this controversy by showing that IFNgamma potentiates IL-1beta release from human cells, but transiently inhibits the production of IL-1beta from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL-1beta and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNgamma and IFNbeta are anti-inflammatory. We observed that these cytokines suppress IL-1beta production in response to MTB, resulting in a reduced number of IL-17-producing cells. In human cells, IFNgamma increased IL-1beta production, and this might explain why IFNgamma is detrimental for multiple sclerosis. In mice, IFNgamma decreased IL-1beta and subsequently IL-17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.

Rotaviruses have been implicated as a possible viral trigger for exacerbations in islet autoimmunity, suggesting they might modulate type 1 diabetes development. In this study, the ability of rotavirus strain RRV to infect the pancreas and affect insulitis and diabetes was examined in nonobese diabetic (NOD) mice, an experimental model of type 1 diabetes. Mice were inoculated either orally or intraperitoneally as infants or young adults. In infant mice inoculated orally, rotavirus antigen was detected in pancreatic macrophages outside islets and infectious virus was found in blood cells, pancreas, spleen, and liver. Extraintestinal RRV spread and pancreatic presence of infectious virus also occurred in intraperitoneally inoculated infant and adult mice. The initiation of insulitis was unaltered by infection. The onset of diabetes was delayed in infant mice inoculated orally and infant and adult mice inoculated intraperitoneally. In contrast, adult mice inoculated orally showed no evidence of pancreatic RRV, the lowest rate of detectable RRV replication, and no diabetes modulation. Thus, the ability of RRV infection to modulate diabetes development in infant and young adult NOD mice was related to the overall extent of detectable virus replication and the presence of infectious virus extraintestinally, including in the pancreas. These studies show that RRV infection of infant and young adult NOD mice provides significant protection against diabetes. As these findings do not support the hypothesis that rotavirus triggers autoimmunity related to type 1 diabetes, further research is needed to resolve this issue.

Introduction The regulation of neutrophil lifespan is critical for a circumscribed immune response. Neutrophils are sensitive to Fas/CD95 death receptor signaling in vitro but it is unknown if Fas regulates neutrophil lifespan in vivo. We hypothesized that FasL-expressing CD8+ T cells, which kill antigen-stimulated T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection, can also induce neutrophil death in tissues during infection. Infection of Fas- and FasL-deficient mice with the enteropathogenic-like mouse pathogen Citrobacter rodentium (C.rodentium) is associated with neutrophil infiltration and severe diarrhoea. We hypothesized that a deficiency of Fas in neutrophils prolongs neutrophil lifespan and contributes to the accumulation of neutrophils in C.rodentium-infected mice. Methods Fas signaling can drive IL-1β and IL-18 production and thereby affect neutrophil accumulation in tissues, so mixed bone marrow chimeras were generated to compare the accumulation of WT and Fas-deficient neutrophils in vivo. For mixed bone-marrow chimeras, irradiated Ly5.1 mice were reconstituted with 2.5 x 106 bone marrow cells from ubiquitin-GFP or Ly5.1/5.2 mice combined with LysM-Cre Fasfl/fl at a 1:1 ratio. For infection studies, mice were infected with LCMV docile or C.rodentium. For in vitro assays, neutrophils were primed for 1 h with GM-CSF prior to treatment with Toll-like receptor (TLR) ligands or IL-18 and FcFasL. Neutrophil viability was measured by live cell imaging and automated image analysis. Results Using LysM-Cre Fasfl/fl mice, which lack Fas expression in macrophages and neutrophils, we show that Fas regulates neutrophil lifespan in the colon during C.rodentium infection, and during LCMV infection in the lung, peripheral blood and spleen. To examine if pathogen-derived molecules can modulate Fas signaling in neutrophils, we primed neutrophils with TLR ligands, which ablated Caspase-8 processing and Fas signaling. Treatment of neutrophils with the TLR ligands Pam3CSK4, Pam2CSK4 or LPS or the MyD88-dependent cytokine IL-18 strongly protected neutrophils against Fas-induced death. Conclusion These data provide the first in vivo genetic evidence that neutrophil lifespan is controlled by death receptor signaling and provides a mechanism to account for neutrophil resistance to Fas stimulation during infection. Disclosures No relevant conflicts of interest to declare.

Abstract 1213 The inflammasome is an innate immune complex that recognizes a wide range of pathogen, cellular stress and damage signals. It triggers the Caspase-1-dependent production of inflammatory cytokines including IL-1β and IL-18. Sensors for the inflammasome include the nucleotide-binding oligomerisation domain, leucine-rich repeat (NLR) proteins. The best-characterised of these is NLRP3, which mediates antibacterial, viral, fungal and parasitic immune responses, and is mutated in a spectrum of autoinflammatory diseases. Activation of mammalian NLRs can also result in a Caspase-1-dependent form of cell death termed pyroptosis, the importance of which in disease states remains unclear. For example, it is known that NLRP3 activating mutations in humans can be effectively treated by neutralising IL-1β, suggesting that cell death induced by NLRP3 activation does not play a significant role in pathology. Similarly, the NLRP1b inflammasome is activated by anthrax lethal toxin to cause macrophage pyroptosis, but this does not play a role in anthrax sensitivity in vivo. Here we define a role for NLRP1 in autoinflammatory disease and the pyroptotic death of hematopoietic progenitor cells. We began by generating two mouse models, one carrying a point mutation in NLRP1a that results in constitutive activation of the protein, and another harbouring a deletion of the entire NLRP1 locus. Animals homozygous for the NRLP1a activating mutation developed a multi-organ neutrophilic inflammatory disease characterised by meningitis, hepatitis, pneumonitis, pancreatitis, pulmonary peri-arteritis, myocarditis and inflammatory bowel disease. Mean survival was approximately 3 months of age. Genetic crosses established that this inflammatory disease was driven by Caspase-1 and IL-1β, but was independent of ASC and Caspase-11, and ameliorated by IL-18. Surprisingly, in the absence of IL-1β-driven inflammation, constitutively active NLRP1a triggered the Caspase-1-dependent death of hematopoietic progenitor cells resulting in leukopenia at steady state. To evaluate the effect of activated NLRP1a during hematopoietic stress, IL-1 receptor-deficient mice homozygous for the NRLP1a activating mutation were challenged with 5-fluorouracil. Strikingly, these animals succumbed shortly after the nadir of leukopenia at 12 days post-injection. They exhibited hypoplastic bone marrow, lymphopenia, monocytopenia and a deficit of reticulocytes consistent with a functional deficiency in hematopoietic progenitor cells. Conversely, in mice lacking NLRP1, we observed improved recovery of the hematopoietic compartment following hemoablative chemotherapy, with increases in the numbers of platelets, lymphocytes and monocytes relative to littermate controls. Severe infections are commonly associated with a range of cytopenias including anemia, lymphopenia, neutropenia and thrombocytopenia, however, the etiological triggers for these conditions have not been elucidated. We therefore infected IL-1 receptor-deficient mice homozygous for the NRLP1a activating mutation with lymphocytic choriomeningitis virus (LCMV). Relative to controls, more severe cytopenia and bone marrow hypoplasia was observed. In contrast, NLRP1-deficient animals showed enhanced recovery from LCMV. Our results suggest that activation of the NLRP1 inflammasome in hematopoietic progenitors may contribute to cytopenias induced by hematopoietic stresses such as chemotherapy or infection. Disclosures: No relevant conflicts of interest to declare.

SummaryCytopenias are key prognostic indicators of life-threatening infection, contributing to immunosuppression and mortality. Here we define a role for Caspase-1-dependent death, known as pyroptosis, in infection-induced cytopenias by studying inflammasome activation in hematopoietic progenitor cells. The NLRP1a inflammasome is expressed in hematopoietic progenitor cells and its activation triggers their pyroptotic death. Active NLRP1a induced a lethal systemic inflammatory disease that was driven by Caspase-1 and IL-1β but was independent of apoptosis-associated speck-like protein containing a CARD (ASC) and ameliorated by IL-18. Surprisingly, in the absence of IL-1β-driven inflammation, active NLRP1a triggered pyroptosis of hematopoietic progenitor cells resulting in leukopenia at steady state. During periods of hematopoietic stress induced by chemotherapy or lymphocytic choriomeningitis virus (LCMV) infection, active NLRP1a caused prolonged cytopenia, bone marrow hypoplasia, and immunosuppression. Conversely, NLRP1-deficient mice showed enhanced recovery from chemotherapy and LCMV infection, demonstrating that NLRP1 acts as a cellular sentinel to alert Caspase-1 to hematopoietic and infectious stress.