Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides are a heterogeneous group of diseases corresponding to necrotising inflammation of small vessels with a wide range of clinical presentations. At least two of the diseases are believed to exhibit a common ground of pathophysiological mechanisms. These are granulomatosis with polyangiitis (GPA, formerly known as Wegener's granulomatosis) and microscopic polyangiitis (MPA). ANCA directed against proteinase 3 (PR3) are preferentially associated with GPA, and anti-myeloperoxidase (MPO) ANCA are associated mainly with MPA and eosinophilic GPA (formerly known as Churg-Strauss syndrome). Anti-MPO and anti-PR3 antibodies can activate neutrophils in vitro. In vivo data are available for humans and mice on the pathogenicity of anti-MPO but it is more controversial for PR3-ANCA. A recent genome-wide association study of patients with ANCA-associated vasculitides confirmed the genetic contribution to the pathogenesis of these conditions, with significant association of PR3-ANCA and human leukocyte antigen-DP and the genes encoding α1-antitrypsin and PR3. MPO-ANCA were significantly associated with human leukocyte antigen-DQ. Thus, recent results from epidemiological studies, genome-wide association study and therapeutic trials have suggested that these entities are, in fact, distinct. We have summarised these results and discuss the idea that these two entities should be studied separately as the nature of the two auto-antigens suggests at a molecular level despite shared ANCA involvement.

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4⁺ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. The majority of HCC cases are associated with liver fibrosis or cirrhosis developing from chronic liver injuries. The immune system of the liver contributes to the severity of tissue damage, the establishment of fibrosis and the disease’s progression towards HCC. Herein, we provide a detailed characterization of the DEN-induced HCC rat model during fibrosis progression and HCC development with a special focus on the liver’s inflammatory microenvironment. Fischer 344 male rats were treated weekly for 14 weeks with intra-peritoneal injections of 50 mg/kg DEN. The rats were sacrificed before starting DEN-injections at 0 weeks, after 8 weeks, 14 weeks and 20 weeks after the start of DEN-injections. We performed histopathological, immunohistochemical, RT-qPCR, RNA-seq and flow cytometry analysis. Data were compared between tumor and non-tumor samples from the DEN-treated versus untreated rats, as well as versus human HCCs. Chronic DEN injections lead to liver damage, hepatocytes proliferation, liver fibrosis and cirrhosis, disorganized vasculature, and a modulated immune microenvironment that mimics the usual events observed during human HCC development. The RNA-seq results showed that DEN-induced liver tumors in the rat model shared remarkable molecular characteristics with human HCC, especially with HCC associated with high proliferation. In conclusion, our study provides detailed insight into hepatocarcinogenesis in a commonly used model of HCC, facilitating the future use of this model for preclinical testing.

Calreticulin (CRT) is a well-known “eat-me” signal harbored by dying cells participating in their recognition by phagocytes. CRT is also recognized to deeply impact the immune response to altered self-cells. In this study we focus on the role of the newly exposure of CRT following cell death induction. We show that if CRT increases at the outer face of the plasma membrane and is well recognized by C1q even when phosphatidylserine is not yet detected, CRT is also released in the surrounding milieu and is able to interact with phagocytes. We observed that exogenous CRT is endocytosed by THP1 macrophages through macropinocytosis and that internalization is associated with a particular phenotype characterized by an increase of cell spreading and migration, an up regulation of CD14, an increase of IL8 release and a decrease of early apoptotic cell uptake. Importantly, CRT-induced pro-inflammatory phenotype was confirmed on human monocytes-derived macrophages by the over expression of CD40 and CD274 and we found that monocytes-derived macrophages exposed to CRT display a peculiar polarization notably associated with a down regulation of the histocompatibility complex of class II molecules hampering its description through the classical M1/M2 dichotomy. Altogether our results highlight the role of soluble CRT with strong possible consequences on the macrophage-mediated immune response to dying cell.

Proteinase 3 (PR3) is the target of anti-neutrophil cytoplasm Abs in granulomatosis with polyangiitis, a form of systemic vasculitis. Upon neutrophil apoptosis, PR3 is coexternalized with phosphatidylserine and impaired macrophage phagocytosis. Calreticulin (CRT), a protein involved in apoptotic cell recognition, was found to be a new PR3 partner coexpressed with PR3 on the neutrophil plasma membrane during apoptosis, but not after degranulation. The association between PR3 and CRT was demonstrated in neutrophils by confocal microscopy and coimmunoprecipitation. Evidence for a direct interaction between PR3 and the globular domain of CRT, but not with its P domain, was provided by surface plasmon resonance spectroscopy. Phagocytosis of apoptotic neutrophils from healthy donors was decreased after blocking lipoprotein receptor-related protein (LRP), a CRT receptor on macrophages. In contrast, neutrophils from patients with granulomatosis with polyangiitis expressing high membrane PR3 levels showed a lower rate of phagocytosis than those from healthy controls not affected by anti-LRP, suggesting that the LRP-CRT pathway was disturbed by PR3-CRT association. Moreover, phagocytosis of apoptotic PR3-expressing cells potentiated proinflammatory cytokine in vitro by human monocyte-derived macrophages and in vivo by resident murine peritoneal macrophages, and diverted the anti-inflammatory response triggered by the phagocytosis of apoptotic cells after LPS challenge in thioglycolate-elicited murine macrophages. Therefore, membrane PR3 expressed on apoptotic neutrophils might amplify inflammation and promote autoimmunity by affecting the anti-inflammatory “reprogramming” of macrophages.

Colorectal adenocarcinoma is a leading cause of death worldwide, and immune infiltration in colorectal tumors has been recognized recently as an important pathophysiologic event. In this context, tumor-associated macrophages (TAM) have been related to chemoresistance to 5-fluorouracil (5-FU), the first-line chemotherapeutic agent used in treating colorectal cancers. Nevertheless, the details of this chemoresistance mechanism are still poorly elucidated. In the current study, we report that macrophages specifically overexpress dihydropyrimidine dehydrogenase (DPD) in hypoxia, leading to macrophage-induced chemoresistance to 5-FU via inactivation of the drug. Hypoxia-induced macrophage DPD expression was controlled by HIF2α. TAMs constituted the main contributors to DPD activity in human colorectal primary or secondary tumors, while cancer cells did not express significant levels of DPD. In addition, contrary to humans, macrophages in mice do not express DPD. Together, these findings shed light on the role of TAMs in promoting chemoresistance in colorectal cancers and identify potential new therapeutic targets.Significance: Hypoxia induces HIF2α-mediated overexpression of dihydropyrimidine dehydrogenase in TAMs, leading to chemoresistance to 5-FU in colon cancers.