IFNγ and CTLA-4 Drive Hepatic CD4 T-Cell Tolerance and Protection From Autoimmunity in Mice.

BACKGROUND & AIMS: The liver has a distinct capacity to induce immune tolerance to hepatic antigens. Although liver tolerance can be advantageous for preventing autoimmune and inflammatory diseases, it also can be detrimental by preventing immune surveillance of infected or malignant cells. Here, we investigated the immune mechanisms that establish hepatic tolerance. METHODS: Tolerance was investigated in C-reactive protein (CRP)-myelin basic protein (MBP) mice expressing the neuroantigen MBP in hepatocytes, providing profound resistance to MBP-induced neuroinflammation. Tolerance induction was studied after transfer of MBP-specific CD4 T cells into CRP-MBP mice, and tolerance mechanisms were tested using depleting or blocking antibodies. RESULTS: Although tolerant CRP-MBP mice display increased numbers of forkhead box P3+ regulatory T cells, we here found them not essential for the maintenance of hepatic tolerance. Instead, upon MBP recognition in the liver, MBP-specific T cells became activated to produce interferon (IFN)γ, which, in turn, induced local up-regulation of recruitment molecules, including Chemokine (C-X-C motif) ligand9 and its receptor C-X-C motif chemokine receptor3, facilitating endothelial translocation and redirection of MBP-specific T cells into the hepatic parenchyma. There, the translocated MBP-specific CD4 T cells partly converted into interleukin 10-producing type 1 regulatory T cells, and significantly up-regulated the expression of immune checkpoint molecules, notably cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Intriguingly, although liver tolerance was not affected by impairment of interleukin 10 signaling, concomitant blockade of IFNγ and CTLA-4 abrogated hepatic tolerance induction to MBP, resulting in neuroinflammatory autoimmune disease in these mice. CONCLUSIONS: IFNγ-mediated redirection of autoreactive CD4 T cells into the liver and up-regulation of checkpoint molecules, including CTLA-4, were essential for tolerance induction in the liver, hence representing a potential treatment target for boosting or preventing liver tolerance.

Inflammatory type 2 conventional dendritic cells contribute to murine and human cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a progressive cholangiopathy characterised by fibrotic stricturing and inflammation of bile ducts, which seems to be driven by a maladaptive immune response to bile duct injury. The histological finding of dendritic cell expansion in portal fields of patients with PSC prompted us to investigate the role of dendritic cells in orchestrating the immune response to bile duct injury. METHODS: Dendritic cell numbers and subtypes were determined in different mouse models of cholangitis by flow cytometry based on lineage-imprinted markers. Findings were confirmed by immunofluorescence microscopy of murine livers, and liver samples from patients with PSC were compared to control samples from bariatric surgery patients. Using genetic tools, selected dendritic cell subsets were depleted in murine cholangitis. The dendritic cell response to bile duct injury was determined by single-cell transcriptomics. RESULTS: Cholangitis mouse models were characterised by selective intrahepatic expansion of type 2 conventional dendritic cells, whereas plasmacytoid and type 1 conventional dendritic cells were not expanded. Expansion of type 2 conventional dendritic cells in human PSC lesions was confirmed by histology. Depletion studies revealed a proinflammatory role of type 2 conventional dendritic cells. Single-cell transcriptomics confirmed inflammatory maturation of the intrahepatic type 2 conventional dendritic cells and identified dendritic cell-derived inflammatory mediators. CONCLUSIONS: Cholangitis is characterised by intrahepatic expansion and inflammatory maturation of type 2 conventional dendritic cells in response to biliary injury. Therefore, type 2 conventional dendritic cells and their inflammatory mediators might be potential therapeutic targets for the treatment of PSC. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease of the bile ducts for which there is no effective treatment. Herein, we show that the inflammatory immune response to bile duct injury is organised by a specific subtype of immune cell called conventional type 2 dendritic cells. Our findings suggest that this cell subtype and the inflammatory molecules it produces are potential therapeutic targets for PSC.

Failure of thymic deletion and instability of autoreactive Tregs drive autoimmunity in immune-privileged liver.

The liver is an immune-privileged organ that can deactivate autoreactive T cells. Yet in autoimmune hepatitis (AIH), autoreactive T cells can defy hepatic control and attack the liver. To elucidate how tolerance to self-antigens is lost during AIH pathogenesis, we generated a spontaneous mouse model of AIH, based on recognition of an MHC class II-restricted model peptide in hepatocytes by autoreactive CD4+ T cells. We found that the hepatic peptide was not expressed in the thymus, leading to deficient thymic deletion and resulting in peripheral abundance of autoreactive CD4+ T cells. In the liver, autoreactive CD4+ effector T cells accumulated within portal ectopic lymphoid structures and maturated toward pathogenic IFN-γ and TNF coproducing cells. Expansion and pathogenic maturation of autoreactive effector T cells was enabled by a selective increase of plasticity and instability of autoantigen-specific Tregs but not of nonspecific Tregs. Indeed, antigen-specific Tregs were reduced in frequency and manifested increased IL-17 production, reduced epigenetic demethylation, and reduced expression of Foxp3. As a consequence, autoantigen-specific Tregs had a reduced suppressive capacity, as compared with that of nonspecific Tregs. In conclusion, loss of tolerance and the pathogenesis of AIH were enabled by combined failure of thymic deletion and peripheral regulation.

Gene knockdown in malaria parasites via non-canonical RNAi.

The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called AgoshRNA. Using this strategy, we achieved robust and specific gene knockdown throughout the entire parasite life cycle. We also successfully silenced the endogenous gene perforin-like protein 2, phenocopying a full gene knockout. Transcriptionally restricting Ago2 expression to the liver stage further enabled us to perform a stage-specific gene knockout. The RNAi-competent Plasmodium lines reported here will be a valuable resource for loss-of-function phenotyping of the many uncharacterized genes of Plasmodium in low or high throughput, without the need to engineer the target gene locus. Thereby, our new strategy and transgenic Plasmodium lines will ultimately benefit the discovery of urgently needed antimalarial drug and vaccine candidates. Generally, the ability to render RNAi-negative organisms RNAi-competent by mere introduction of two components, Ago2 and AgoshRNA, is a unique paradigm that should find broad applicability in other species.