Loss of CD4+ T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection.

Arginase 1 (Arg1), the enzyme catalyzing the conversion of arginine to ornithine, is a hallmark of IL-10-producing immunoregulatory M2 macrophages. However, its expression in T cells is disputed. Here, we demonstrate that induction of Arg1 expression is a key feature of lung CD4+ T cells during mouse in vivo influenza infection. Conditional ablation of Arg1 in CD4+ T cells accelerated both virus-specific T helper 1 (Th1) effector responses and its resolution, resulting in efficient viral clearance and reduced lung pathology. Using unbiased transcriptomics and metabolomics, we found that Arg1-deficiency was distinct from Arg2-deficiency and caused altered glutamine metabolism. Rebalancing this perturbed glutamine flux normalized the cellular Th1 response. CD4+ T cells from rare ARG1-deficient patients or CRISPR-Cas9-mediated ARG1-deletion in healthy donor cells phenocopied the murine cellular phenotype. Collectively, CD4+ T cell-intrinsic Arg1 functions as an unexpected rheostat regulating the kinetics of the mammalian Th1 lifecycle with implications for Th1-associated tissue pathologies.

Phosphatidylinositol 3-Kinase δ Deficiency Protects From Antimyeloperoxidase Vasculitis.

OBJECTIVE: Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is a systemic autoimmune disease in which glomerulonephritis is an important manifestation. Antibodies against myeloperoxidase (MPO) or proteinase 3 are thought to be important in pathogenesis. Phosphoinositide 3-kinase δ (PI3Kδ) mediates a number of effects in lymphocytes, but its role in myeloid cell responses is less clear. Therefore, this study was undertaken to assess this in a preclinical model of glomerulonephritis induced by the transfer of antibodies to MPO. METHODS: D910A mice with inactive PI3Kδ were compared with wild-type controls. Disease protocols allowed for a comparison of experimental groups in the setting of both mild and more severe disease. Adoptive transfer experiments were performed, with flow cytometric analysis of digested kidneys taken at the end of the experiment. RESULTS: With mild disease, D910A mice had fewer glomerular macrophages, fewer glomerular neutrophils, and reduced albuminuria compared with wild-type controls. With more severe disease, they also had fewer glomerular crescents and lower serum creatinine levels, indicating protection from acute kidney injury. Adoptive transfer experiments showed a defect in the recruitment of D910A monocytes to the diseased kidney. CONCLUSION: Mice with inactive PI3Kδ were protected from anti-MPO vasculitis. This is due to cell intrinsic defect in the recruitment of monocytes to the kidney. These findings suggest that PI3Kδ is a potential therapeutic target in AAV.

The lectin pathway does not contribute to glomerular injury in the nephrotoxic nephritis model.

AIMS: Rapidly progressive crescentic glomerulonephritis occurs in number systemic and primary glomerular diseases, including anti-glomerular basement membrane disease, anti-neutrophil cytoplasmic antibody vasculitis and lupus nephritis. Our understanding of pathogenic mechanisms comes from animal models of disease such as the nephrotoxic nephritis model. The lectin pathway of complement activation has been shown to play a key role in several models of inflammation including renal ischaemia reperfusion. However, the lectin pathway is not required for crescentic glomerulonephritis in the anti-myeloperoxidase model of anti-neutrophil cytoplasmic antibody vasculitis. The aim of the current study was to explore the role of the lectin pathway in the nephrotoxic nephritis model, which is another model of crescentic glomerulonephritis. METHODS: Nephrotoxic nephritis was induced in wild type and mannan-binding lectin-associated serine protease-2 deficient mice. Diseases were assessed by quantifying glomerular crescents and macrophages, in addition to albuminuria and serum creatinine. RESULTS: There was no difference between wild type and MASP-2 deficient mice in any of the histological or biochemical parameters of disease assessed. In addition, there was no difference in the humoral immune response to sheep IgG. CONCLUSION: These data show that the lectin pathway of complement activation is not required for the development of crescentic glomerulonephritis in the nephrotoxic nephritis model, reinforcing previous findings in the anti-myeloperoxidase model.

Experimentally Induced Anti-Myeloperoxidase Vasculitis Is Not Attenuated in Factor B or VISTA Deficient Mice.

Background: Anti-neutrophil cytoplasmic antibody vasculitis is characterized by antibodies to myeloperoxidase or proteinase 3. Previous work in murine anti-myeloperoxidase vasculitis has shown a role for the alternative pathway complement component factor B and the anaphylatoxin C5a. However, mice deficient in properdin, which stabilizes the alternative pathway convertase, were not protected. V-Type immunoglobulin domain-containing suppressor of T-cell activation (VISTA)-deficient mice were protected in the nephrotoxic nephritis model but the role of VISTA in anti-myeloperoxidase vasculitis is unknown. Objectives: This study had 2 aims. First, we attempted to reproduce previous findings on the role of factor B in anti-myeloperoxidase vasculitis. Second, we examined the role of VISTA in this model, in order to see if the protection in the nephrotoxic nephritis model extended to anti-myeloperoxidase vasculitis. Methods: Anti-myeloperoxidase vasculitis was induced in wild type, factor B, or VISTA deficient mice. Disease was assessed by quantifying glomerular crescents and macrophages, in addition to albuminuria and serum creatinine. Results: When wild type and factor B deficient mice were compared, there were no differences in any of the histological or biochemical parameters of disease assessed. Similarly, when wild type or VISTA deficient mice were compared, there were no differences. Conclusions: Factor B deficient mice were not protected which is in contrast to previous studies. Therefore alternative pathway activation is not essential in this model, under the conditions used in this study. VISTA deficient mice were not protected, suggesting that therapies targeting VISTA may not be effective in vasculitis.

VISTA deficiency protects from immune complex-mediated glomerulonephritis by inhibiting neutrophil activation.

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator of T cells. We assessed VISTA deficient mice in the murine nephrotoxic nephritis models of acute and chronic immune-complex mediated glomerulonephritis. We show that VISTA deficiency protects from crescentic glomerulonephritis, with no effect on the nephritogenic adaptive immune response. The early neutrophil influx was unaffected but proteinuria was reduced suggesting a reduction in neutrophil activation. In vivo, there was reduced neutrophil degranulation in VISTA deficienct mice and, in vitro, VISTA-deficient neutrophils had an impaired response to immune complexes but not to fMLP or PMA. Mice with a genetic deficiency of neutrophils due to myeloid-specific deletion of myeloid cell leukemia 1 (Mcl-1) were also protected from crescentic glomerulonephritis, indicating an essential role for neutrophils. Therefore, VISTA deficiency inhibits neutrophil activation by immune complexes and neutrophil-dependent crescentic glomerulonephritis. This suggests that VISTA is a therapeutic target for inflammatory disease. However, this would need to be balanced against a potential enhancing effect on autoimmunity.

Asparaginyl Endopeptidase (Legumain) Supports Human Th1 Induction via Cathepsin L-Mediated Intracellular C3 Activation.

Autocrine activation of the complement receptors C3aR and CD46 by complement activation components C3a and C3b produced through C3 cleavage by the protease cathepsin L (CTSL) during T cell stimulation is a requirement for IFN-γ production and Th1 induction in human CD4+ T cells. Thus, lack of autocrine CD46 activation, such as in CD46-deficient patients, is associated with defective Th1 responses and recurrent infections. We have identified LGMN [the gene coding for legumain, also known as asparaginyl endopeptidase (AEP)] as one of the key genes induced by CD46 co-stimulation during human CD4+ T cell activation. AEP processes and activates a range of proteins, among those α1-thymosin and CTSL, which both drive intrinsically Th1 activity-but has so far not been described to be functionally active in human T cells. Here we found that pharmacological inhibition of AEP during activation of human CD4+ T cells reduced CTSL activation and the CTSL-mediated generation of intracellular C3a. This translated into a specific reduction of IFN-γ production without affecting cell proliferation or survival. In line with these findings, CD4+ T cells isolated from Lgmn-/- mice also displayed a specific defect in IFN-γ secretion and Th1 induction. Furthermore, we did not observe a role for AEP-driven autocrine α1-thymosin activation in T cell-derived IFN-γ production. These data suggest that AEP is an "upstream" activator of the CTSL-C3-IFN-γ axis in human CD4+ T cells and hence an important supporter of human Th1 induction.

The "ins and outs" of complement-driven immune responses.

The complement system represents an evolutionary old and critical component of innate immunity where it forms the first line of defense against invading pathogens. Originally described as a heat-labile fraction of the serum responsible for the opsonization and subsequent lytic killing of bacteria, work over the last century firmly established complement as a key mediator of the general inflammatory response but also as an acknowledged vital bridge between innate and adaptive immunity. However, recent studies particularly spanning the last decade have provided new insights into the novel modes and locations of complement activation and highlighted unexpected additional biological functions for this ancient system, for example, in regulating basic processes of the cell. In this review, we will cover the current knowledge about complement's established and novel roles in innate and adaptive immunity with a focus on the functional differences between serum circulating and intracellularly active complement and will describe and discuss the newly discovered cross-talks of complement with other cell effector systems particularly during T-cell induction and contraction.

Experimentally-induced anti-myeloperoxidase vasculitis does not require properdin, MASP-2 or bone marrow-derived C5.

Anti-neutrophil cytoplasmic antibody vasculitis is a systemic autoimmune disease with glomerulonephritis and pulmonary haemorrhage as major clinical manifestations. The name reflects the presence of autoantibodies to myeloperoxidase and proteinase-3, which bind to both neutrophils and monocytes. Evidence of the pathogenicity of these autoantibodies is provided by the observation that injection of anti-myeloperoxidase antibodies into mice causes a pauci-immune focal segmental necrotizing glomerulonephritis which is histologically similar to the changes seen on renal biopsy in patients. Previous studies in this model have implicated the alternative pathway of complement activation and the anaphylatoxin C5a. Despite this progress, the factors that initiate complement activation have not been defined. In addition, the relative importance of bone marrow-derived and circulating C5 is not known. This is of interest given the recently identified roles for complement within leukocytes. We induced anti-myeloperoxidase vasculitis in mice and confirmed a role for complement activation by demonstrating protection in C3-deficient mice. We showed that neither MASP-2- nor properdin-deficient mice were protected, suggesting that alternative pathway activation does not require properdin or the lectin pathway. We induced disease in bone marrow chimaeric mice and found that circulating and not bone marrow-derived C5 was required for disease. We have therefore excluded properdin and the lectin pathway as initiators of complement activation and this means that future work should be directed at other potential factors within diseased tissue. In addition, in view of our finding that circulating and not bone marrow-derived C5 mediates disease, therapies that decrease hepatic C5 secretion may be considered as an alternative to those that target C5 and C5a. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Toll-like receptor 2 or toll-like receptor 4 deficiency does not modify lupus in MRLlpr mice.

Systemic lupus erythematosus is an autoimmune disease with a high morbidity and nephritis is a common manifestation. Previous studies in murine lupus models have suggest a role for Toll-like receptor 2 and 4. We examined the role of these molecules in MRL lpr mice which is one of the most established and robust murine models. We compared disease parameters in Toll-like receptor 2 or Toll-like receptor 4 deficient mice with their littermate controls. We found no difference in the severity of glomerulonephritis as assessed by histology, serum creatinine and albuminuria when Toll-like receptor 2 or Toll-like receptor 4 deficient MRLlpr mice were compared with Toll-like receptor sufficient controls. We also found similar levels of anti-dsDNA and anti-ssDNA antibodies. These results show that Toll-like receptor 2 and Toll-like receptor 4 do not play a significant role in MRLlpr mice, and therefore they may not be important in human lupus.

Granulocyte colony stimulating factor exacerbates antineutrophil cytoplasmic antibody vasculitis.

OBJECTIVES: Granulocyte colony stimulating factor (GCSF) is important in mobilising neutrophils from the bone marrow but also has a range of proinflammatory effects. We therefore decided to investigate the role of GCSF in antineutrophil cytoplasmic antibody (ANCA) vasculitis. METHODS: We measured GCSF levels in the serum of 38 patients with active ANCA vasculitis compared with 31 age-matched controls, and assessed the effect of GCSF priming on the response of human neutrophils to ANCA. We also examined the effect of exogenous GCSF administration in a murine model of antimyeloperoxidase (anti-MPO) vasculitis, and the effect of GCSF on murine neutrophil activation. RESULTS: The serum levels of GCSF in patients with active ANCA vasculitis were significantly higher than those of age matched healthy controls (mean 38.04 vs 18.35 pg/ml, p<0.001). Furthermore, we demonstrated that GCSF primed human neutrophils in vitro for a respiratory burst in response to anti-MPO ANCA. In an anti-MPO antibody transfer model, mice given GCSF had more crescents (mean 29.1% vs 5.8% per glomerular cross section, p<0.05), more macrophages (mean 3.2 vs 1.2 per glomerular cross-section, p<0.01), higher serum creatines (mean 13.6 vs 8.3 µmol/l, p<0.05) and more haematuria (p<0.05) compared with controls. In vivo administration of GCSF with lipopolysaccharide (LPS), but not LPS alone, led to upregulation of CD11c on murine neutrophils. CONCLUSIONS: These data suggest that GCSF, which is raised in patient serum, may play an important role in exacerbating disease in ANCA vasculitis. In addition, GCSF therapy for neutropenia should be used with caution in these patients.

Humanised mice have functional human neutrophils.

The differences between murine and human neutrophils mean that findings in mice may not translate to humans, and therefore an in vivo model with human neutrophils would be an important methodological advance. We generated humanised mice by injecting human cord blood derived CD34+ stem cells into irradiated NOD-scid-γc(-/-) mice. At least 3 months after engraftment, treatment of mice with GCSF mobilised circulating human neutrophils, which comprised 2.6% of human leukocytes, and led to L-selectin shedding and upregulation of CD66b, CD11b and CD63. Subsequent in vivo LPS treatment led to further downregulation of L-selectin with upregulation of CD66b and CD63, and also resulted in human neutrophil sequestration in the lungs. Furthermore, human neutrophils from these mice were capable of robust functional responses. They were shown to undergo a respiratory burst, and to degranulate with upregulation of CD63 and CD66b, in response to fMLP and Escherichia coli. These data show that functional human neutrophils develop from CD34+ cord blood stem cells in NOD-scid-γc(-/-) mice. They suggest that this approach may facilitate the in vivo study of human neutrophils in clinically relevant models of infection and autoimmunity.

Transferred antigen-specific T(H)17 but not T(H)1 cells induce crescentic glomerulonephritis in mice.

To explore the role of antigen-specific CD4(+) T cells in glomerulonephritis, we administered ovalbumin 323-339 peptide conjugated to glomerular-binding polyclonal antibody and induced disease in RAG1(-/-) mice with CD4(+) T cells from OT2 × RAG1(-/-) mice. These OT2 × RAG1(-/-) mice have a transgenic T-cell receptor specific for this peptide. When CD4(+) T cells were primed in vivo, crescentic glomerulonephritis developed after 21 days in mice given peptide-conjugated glomerular-binding antibody but not unconjugated antibody control. We then investigated the relative roles of T(H)1 and T(H)17 cells, using Fab(2) fragments of glomerular-binding antibody to exclude a role for antibody in this model. T cells from OT2 × RAG1(-/-) mice were polarized in vitro, and T(H)1 or T(H)17 cell lines were injected into mice that were also given peptide-conjugated Fab(2) or unconjugated Fab(2) control, giving four experimental groups. After 21 days crescentic glomerulonephritis was seen in mice receiving T(H)17 cells and peptide-conjugated Fab(2) but in none of the other three groups. These results suggest that T(H)17 but not T(H)1 cells can induce crescentic glomerulonephritis.