Can Ergen

UNLABELLED: The liver is essential for inducing immunological tolerance toward harmless antigens to maintain immune system homeostasis. However, the precise cellular mechanisms of tolerance induction against particle-bound antigens, the role of the local hepatic microenvironment, and implications for therapeutic targets in immune-mediated diseases are currently unclear. In order to elucidate cellular mechanisms of tolerance induction in healthy and injured liver, we developed a novel in vivo system combining the systemic delivery of low-dose peptide antigens coupled to inert particles, immunological readouts, and sophisticated intravital multiphoton microscopy-based imaging of liver in mice. We show that liver resident macrophages, Kupffer cells (KCs), but not hepatic monocyte-derived macrophages or dendritic cells (DCs), are the central cellular scavenger for circulating particle-associated antigens in homeostasis. KC-associated antigen presentation induces CD4 T-cell arrest, expansion of naturally occurring Foxp3(+) CD25(+) interleukin-10-producing antigen-specific regulatory T cells (Tregs) and tolerogenic immunity. Particle-associated tolerance induction in the liver protected mice from kidney inflammation in T-cell-mediated glomerulonephritis, indicating therapeutic potential of targeting KC for immune-mediated extrahepatic disorders. Liver inflammation in two independent experimental models of chronic liver injury and fibrosis abrogated tolerance induction and led to an immunogenic reprogramming of antigen-specific CD4 T cells. In injured liver, infiltrating monocyte-derived macrophages largely augment the hepatic phagocyte compartment, resulting in antigen redistribution between myeloid cell populations and, simultaneously, KCs lose signature markers of their tolerogenic phenotype. CONCLUSIONS: Hepatic induction of tissue-protective immunological tolerance against particulate antigens is dependent on KCs as well as on a noninflamed liver microenvironment, thereby providing mechanistic explanations for the clinical observation of immune dysfunction and tolerance break in patients with advanced liver diseases.

Acetaminophen (APAP, paracetamol) poisoning is a leading cause of acute liver failure (ALF) in humans and induces hepatocyte necrosis, followed by activation of the innate immune system, further aggravating liver injury. The role of infiltrating monocytes during the early phase of ALF is still ambiguous. Upon experimental APAP overdose in mice, monocyte‐derived macrophages (MoMFs) massively accumulated in injured liver within 12‐24 hours, whereas the number of tissue‐resident macrophages (Kupffer cells) decreased. Influx of MoMFs is dependent on the chemokine receptor, chemokine (C‐C motif) receptor 2 (CCR2), given that Ccr2−/− mice display reduced infiltration of monocytes and attenuated liver injury post‐APAP overdose at early time points. As evidenced by intravital multiphoton microscopy of Ccr2 reporter mice, CCR2 + monocytes infiltrate liver as early as 8‐12 hours post‐APAP overdose and form dense cellular clusters around necrotic areas. CCR2 + MoMFs express a distinct pattern of inflammatory, but also repair‐associated, genes in injured livers. Adoptive transfer experiments revealed that MoMFs primarily exert proinflammatory functions early post‐APAP, thereby aggravating liver injury. Consequently, early pharmacological inhibition of either chemokine (C‐C motif) ligand (CCL2; by the inhibitor, mNOX‐E36) or CCR2 (by the orally available dual CCR2/CCR5 inhibitor, cenicriviroc) reduces monocyte infiltration and APAP‐induced liver injury (AILI) in mice. Importantly, neither the early nor continuous inhibition of CCR2 hinder repair processes during resolution from injury. In line with this, human livers of ALF patients requiring liver transplantation reveal increased CD68 + hepatic macrophage numbers with massive infiltrates of periportal CCR2 + macrophages that display a proinflammatory polarization. Conclusion : Infiltrating monocyte‐derived macrophages aggravate APAP hepatotoxicity, and the pharmacological inhibition of either CCL2 or CCR2 might bear therapeutic potential by reducing the inflammatory reaction during the early phase of AILI. (H epatology 2016;64:1667‐1682)

Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II(hi) and MHC-II(lo) tumor-associated macrophage (TAM) subpopulations that originate from Ly6C(hi) monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C(hi) monocytic precursors. M-CSFR signaling blockade shifted the MHC-II(lo)/MHC-II(hi) TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C(hi) monocytes into MHC-II(hi) TAMs. In addition, the genetic and functional signatures of MHC-II(lo) TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II(lo) TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II(hi) phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade.

Abstract IL-10 is a prototypical anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4 + T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3 neg CD4 + T cells that displays regulatory activity unlike other IL-10-producing CD4 + T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4 + T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3 neg CD4 + T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation.