BackgroundNew therapies blocking the IL-6 receptor (IL-6R) have recently become available and are successfully being used to treat inflammatory diseases like arthritis. Whether IL-6 blockers may help patients with kidney inflammation currently remains unknown.MethodsTo learn more about the complex role of CD4+ T cell-intrinsic IL-6R signaling, we induced nephrotoxic nephritis, a mouse model for crescentic GN, in mice lacking T cell–specific IL-6Ra. We used adoptive transfer experiments and studies in reporter mice to analyze immune responses and Treg subpopulations.ResultsLack of IL-6Ra signaling in mouse CD4+ T cells impaired the generation of proinflammatory Th17 cells, but surprisingly did not ameliorate the course of GN. In contrast, renal damage was significantly reduced by restricting IL-6Ra deficiency to T effector cells and excluding Tregs. Detailed studies of Tregs revealed unaltered IL-10 production despite IL-6Ra deficiency. However, in vivo and in vitro, IL-6Ra classic signaling induced RORγt+Foxp3+ double-positive Tregs (biTregs), which carry the trafficking receptor CCR6 and have potent immunoregulatory properties. Indeed, lack of IL-6Ra significantly reduced Treg in vitro suppressive capacity. Finally, adoptive transfer of T cells containing IL-6Ra−/− Tregs resulted in severe aggravation of GN in mice.ConclusionsOur data refine the old paradigm, that IL-6 enhances Th17 responses and suppresses Tregs. We here provide evidence that T cell–intrinsic IL-6Ra classic signaling indeed induces the generation of Th17 cells but at the same time highly immunosuppressive RORγt+ biTregs. These results advocate caution and indicate that IL-6–directed therapies for GN need to be cell-type specific.

BackgroundRecent studies have identified the EGF receptor (EGFR) ligand amphiregulin (AREG) as an important mediator of inflammatory diseases. Both pro- and anti-inflammatory functions have been described, but the role of AREG in GN remains unknown.MethodsThe nephrotoxic nephritis model of GN was studied in AREG−/− mice after bone marrow transplantation, and in mice with myeloid cell–specific EGFR deficiency. Therapeutic utility of AREG neutralization was assessed. Furthermore, AREG's effects on renal cells and monocytes/macrophages (M/M) were analyzed. Finally, we evaluated AREG expression in human renal biopsies.ResultsRenal AREG mRNA was strongly upregulated in murine GN. Renal resident cells were the most functionally relevant source of AREG. Importantly, the observation that knockout mice showed significant amelioration of disease indicates that AREG is pathogenic in GN. AREG enhanced myeloid cell responses via inducing chemokine and colony stimulating factor 2 (CSF2) expression in kidney resident cells. Furthermore, AREG directly skewed M/M to a proinflammatory M1 phenotype and protected them from apoptosis. Consequently, anti-AREG antibody treatment dose-dependently ameliorated GN. Notably, selective abrogation of EGFR signaling in myeloid cells was sufficient to protect against nephritis. Finally, strong upregulation of AREG expression was also detected in kidneys of patients with two forms of crescentic GN.ConclusionsAREG is a proinflammatory mediator of GN via (1) enhancing renal pathogenic myeloid cell infiltration and (2) direct effects on M/M polarization, proliferation, and cytokine secretion. The AREG/EGFR axis is a potential therapeutic target for acute GN.

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Interleukin-2 is central to the induction and maintenance of both natural (nTreg) and induced Foxp3-expressing regulatory T cells (iTreg). Thus, signals that modulate IL-2 availability may, in turn, also influence Treg homeostasis. Using global knockout and cell-specific knockout mouse models, we evaluated the role of the small GTPase ADP-ribosylation factor 4d (Arl4d) in regulatory T-cell biology. We show that the expression of Arl4d in T cells restricts both IL-2 production and responsiveness to IL-2, as measured by the phosphorylation of STAT5. Arl4d-deficient CD4 T cells converted more efficiently into Foxp3+ iTreg in vitro in the presence of αCD3ε and TGFβ, which was associated with their enhanced IL-2 secretion. As such, Arl4d−/− CD4 T cells induced significantly less colonic inflammation and lymphocytic infiltration in a model of transfer colitis. Thus, our data reveal a negative regulatory role for Arl4d in CD4 T-cell biology, limiting iTreg conversion via the restriction of IL-2 production, leading to reduced induction of Treg from conventional CD4 T cells.

Myeloid cells play an essential role in the maintenance of liver homeostasis, as well as the initiation and termination of innate and adaptive immune responses. In chronic hepatic inflammation, the production of transforming growth factor beta (TGF-β) is pivotal for scarring and fibrosis induction and progression. TGF-β signalling is tightly regulated via the Smad protein family. Smad7 acts as an inhibitor of the TGF-β-signalling pathway, rendering cells that express high levels of it resistant to TGF-β-dependent signal transduction. In hepatocytes, the absence of Smad7 promotes liver fibrosis. Here, we examine whether Smad7 expression in myeloid cells affects the extent of liver inflammation, injury and fibrosis induction during chronic liver inflammation. Using the well-established model of chronic carbon tetrachloride (CCl4)-mediated liver injury, we investigated the role of Smad7 in myeloid cells in LysM-Cre Smadfl/fl mice that harbour a myeloid-specific knock-down of Smad7. We found that the chronic application of CCl4 induces severe liver injury, with elevated serum alanine transaminase (ALT)/aspartate transaminase (AST) levels, centrilobular and periportal necrosis and immune-cell infiltration. However, the myeloid-specific knock-down of Smad7 did not influence these and other parameters in the CCl4-treated animals. In summary, our results suggest that, during long-term application of CCl4, Smad7 expression in myeloid cells and its potential effects on the TGF-β-signalling pathway are dispensable for regulating the extent of chronic liver injury and inflammation.

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BackgroundAmphiregulin (AREG) is a member of the epidermal growth factor (EGF) family and plays a role in development, tissue homeostasis and tumorigenesis. Recently, however, AREG has also emerged as novel player in immunity. Interestingly, AREG expression was shown to be one of the most highly upregulated transcripts in peripheral blood leukocytes of patient with systemic lupus erythematosus (SLE) [1]. The functional role of AREG in SLE, and inflammation in general, remains unclear to date and both, pro- and anti-inflammatory functions have been postulated [2, 3].ObjectivesOur aim was to investigate the role of AREG in the mouse model of pristane induced lupus (PIL) with particular focus on lupus nephritis (LN). We further wanted to identify target cells and mechanisms by which AREG exerts its immunomodulatory effects.MethodsPIL was induced in AREG knock-out (KO) mice and wild type controls. Animals were sacrificed at pre-specified time points. Renal histology, immune complex deposition, leukocyte influx and mRNA expression levels were analyzed. Furthermore, broad in vivo and in vitro analyses of renal and systemic immune responses were carried out.ResultsRenal AREG mRNA expression significantly increased during development of LN, indicating functional relevance. Indeed, lupus nephritis was significantly aggravated in AREG-KO mice both at early (9 months) and later (12 months) stages after PIL induction. In line with this, we noted an increased deposition of immune complexes and renal influx of pro-inflammatory leukocytes (CD3+ T-cells, macrophages and neutrophils). In addition, we found the CD4+ T-cells of AREG-KO mice to have a more pro-inflammatory phenotype with significantly increased production of pro-inflammatory cytokines (IFNγ and IL-17A) both in ex-vivo culture, as well as FACS-analyses of nephritic kidneys. Mechanistically, we found that AREG treatment of spleen cell cultures potently suppressed cytokine production. More detailed evaluation by further in-vitro studies indicated, that AREG can directly suppress cytokine production by effector CD4+ T-cells as well as enhance the suppressive capacity of FoxP3+ regulatory T-cells (Tregs).ConclusionThese data show that AREG has a protective role on development of LN induced by pristane, which might be therapeutically exploited. Our results further suggest, that direct effects on CD4+ T-effector cells, as well as indirect effects via Tregs, are two mechanisms by which AREG exerts its protective role.References[1] Ishii, T., et al., Isolation and expression profiling of genes upregulated in the peripheral blood cells of systemic lupus erythematosus patients. DNA Res, 2005. 12(6): p.429-39.[2] Kefaloyianni, E., et al.,ADAM17 substrate release in proximal tubule drives kidney fibrosis. JCI Insight, 2016. 1(13).[3] Zaiss, D.M., et al., Amphiregulin enhances regulatory T cell-suppressive function via the epidermal growth factor receptor. Immunity, 2013. 38(2): p.275-84.Disclosure of InterestsNone declared