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Background Recent 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. Methods The 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. Results Renal 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. Conclusions AREG 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.

BACKGROUND: Recent 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. METHODS: The 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. RESULTS: Renal 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. CONCLUSIONS: AREG 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.

Background Amphiregulin (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]. Objectives Our 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. Methods PIL 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. Results Renal 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). Conclusion These 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 Interests None declared