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Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9 + TREM2 + macrophages that express SPP1 , GPNMB , FABP5 , and CD63 . In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-β1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-β1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-β1–induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-β1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in the progression and resolution of fibrosis. Identifying the unique fibrogenic macrophages that are found in human fibrotic tissues could lead to new and more effective treatments for fibrosis. Here we used human liver and lung single cell RNA sequencing datasets to identify a unique subset of CD9+ TREM2+ macrophages expressing SPP1, GPNMB, FABP5, and CD63 with strong pro-fibrotic activity. This population was validated across orthogonal techniques, species and tissues. These macrophages were enriched at the outside edges of scarring adjacent to activated mesenchymal cells, and in the fibrotic niche across species and organs. Neutrophils producing the type 3 cytokines GM-CSF and IL-17A, and expressing MMP9, which participates in the activation of TGF-β1, clustered with these scar-associated macrophages. Using in vitro primary human cell assays, we determined that GM-CSF, IL-17A and TGF-β1 drive the differentiation of these scar-associated macrophages, and that co-culture of monocyte-derived macrophages with hepatic stellate cells and TGF-β1 augmented type 1 collagen deposition. In vivo blockade of GM-CSF, IL-17A or TGF-β1 with small or large molecules reduced scar-associated macrophage expansion and fibrosis in multiple models of hepatic and pulmonary fibrosis. Our work demonstrates that a specific scar-associated macrophage population is linked with fibrosis across species and tissues. It further provides a strategy for unbiased discovery, triage and preclinical validation of therapeutic targets within this fibrogenic macrophage population.

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Inflammatory immune cells can modulate activation of hepatic stellate cells (HSCs) and progression of liver fibrosis. Type 3 inflammation characterized by production of interleukin-17A (IL-17) and IL-22 by innate and adaptive immune cells is implicated in many inflammatory conditions of the gut and can be counteracted by regulatory T cells (Tregs), but its contribution to liver fibrosis is still poorly understood. Here, we evaluated the contribution of type 3 inflammation in liver fibrosis using clinical liver biopsies, in vitro stimulation of primary HSCs, and in vivo mouse models. We report dysregulated type 3 responses in fibrotic lesions with increased IL-17+CD4+/FOXP3hiCD4+ ratio and increased IL-17 and IL-22 production in advanced liver fibrosis. Neutrophils and mast cells were the main sources of IL-17 in situ in humans. In addition, we demonstrate a new profibrotic function of IL-22 through enhancement of transforming growth factor-β signaling in HSCs in a p38 mitogen-activated protein kinase-dependent manner. In vivo, IL-22RA1 knockout mice exhibited reduced fibrosis in response to thioacetamide and carbon tetrachloride. Blocking either IL-22 or IL-17 production using aryl hydrocarbon receptor or RAR-related orphan receptor gamma-t antagonists resulted in reduced fibrosis. Together, these data have identified a pathogenic role for type 3 immune response mediated by IL-22 in driving liver fibrosis during chronic liver injury.

The type 3 cytokines IL-17 and IL-22 play a crucial, well synchronized physiological role in wound healing and repairing tissue damage due to infections or injury at barrier surfaces. These cytokines act on epithelial cells to induce secretion of early immune mediators, recruitment of inflammatory cells to the site of injury, and to trigger tissue repair mechanisms. However, if the damage persists or if these cytokines are dysregulated, then they contribute to a number of inflammatory pathologies, autoimmune conditions and cancer. The liver is a multifunctional organ that plays an essential role in metabolism, detoxification, and immune surveillance. It is also exposed to a variety of pathogens, toxins and injuries. Over the past decade, IL-17 and IL-22 have been implicated in various aspects of liver inflammation. IL-17 is upregulated in chronic liver injury and associated with liver disease progression. In contrast, IL-22 was shown to be hepatoprotective during acute liver injury but exhibited inflammatory effects in other models. Furthermore, IL-22 and IL-17 are both associated with poor prognosis in liver cancer. Finally, the regulatory mechanisms governing the physiological versus the pathological role of these two cytokines during acute and chronic liver injury remain poorly understood. In this review, we will summarize the current state of knowledge about IL-17 and IL-22 in wound healing during acute and chronic liver injury, their contribution to pathogenesis, their regulation, and their role in the transition from advanced liver disease to liver cancer.

Nonalcoholic fatty liver disease (NAFLD) is now the most common progressive liver disease in developed countries and is the second leading indication for liver transplantation due to the extensive fibrosis it causes. NAFLD progression is thought to be tied to chronic low-level type 1 inflammation originating in the adipose tissue during obesity; however, the specific immunological mechanisms regulating the progression of NAFLD-associated fibrosis in the liver are unclear. To investigate the immunopathogenesis of NAFLD more completely, we investigated adipose dysfunction, nonalcoholic steatohepatitis (NASH), and fibrosis in mice that develop polarized type 1 or type 2 immune responses. Unexpectedly, obese interleukin-10 (IL-10)/IL-4-deficient mice (type 1-polarized) were highly resistant to NASH. This protection was associated with an increased hepatic interferon-γ (IFN-γ) signature. Conversely, IFN-γ-deficient mice progressed rapidly to NASH with evidence of fibrosis dependent on transforming growth factor-β (TGF-β) and IL-13 signaling. Unlike increasing type 1 inflammation and the marked loss of eosinophils seen in expanding adipose tissue, progression of NASH was associated with increasing eosinophilic type 2 liver inflammation in mice and human patient biopsies. Finally, simultaneous inhibition of TGF-β and IL-13 signaling attenuated the fibrotic machinery more completely than TGF-β alone in NAFLD-associated fibrosis. Thus, although type 2 immunity maintains healthy metabolic signaling in adipose tissues, it exacerbates the progression of NAFLD collaboratively with TGF-β in the liver.

Single nucleotide polymorphisms (SNPs) in the proximity of the interleukin-28B (IL28B) gene can predict spontaneous resolution of hepatitis C virus (HCV) infection and response to interferon therapy. Screening for this polymorphism has become part of the standard criteria for the management of HCV-infected patients, hence the need for a rapid, cost-effective screening method. Here, we describe a rapid PCR-based test to screen for two IL28B SNPs (rs12979860 and rs8099917). We used this test to investigate IL28B polymorphism and prevalence in a cohort of French Canadian injection drug users who are part of a unique population known to have a strong genetic founder effect. This population had lower linkage disequilibrium between the two tested SNPs as compared to other cohorts (|d′| = 0.68, r = 0.59). The special genetic makeup should be considered in the management of HCV-infected patients within that population.

HIV-infected slow progressors (SP) represent a heterogeneous group of subjects who spontaneously control HIV infection without treatment for several years while showing moderate signs of disease progression. Under conditions that remain poorly understood, a subgroup of these subjects experience failure of spontaneous immunological and virological control. Here we determined the frequency of SP subjects who showed loss of HIV control within our Canadian Cohort of HIV+ Slow Progressors and identified the proinflammatory cytokine IL-32 as a robust biomarker for control failure. Plasmatic levels of the proinflammatory isoforms of IL-32 (mainly β and γ) at earlier clinic visits positively correlated with the decline of CD4 T-cell counts, increased viral load, lower CD4/CD8 ratio and levels of inflammatory markers (sCD14 and IL-6) at later clinic visits. We present here a proof-of-concept for the use of IL-32 as a predictive biomarker for disease progression in SP subjects and identify IL-32 as a potential therapeutic target.

The liver is an important metabolic organ with unique immunological properties. As a secondary lymphoid organ, the liver contains several populations of antigen-presenting cells including classical dendritic cells, but also hepatocytes, Kupffer cells, endothelial cells, and hepatic stellate cells that are able to uptake, process, and present antigens. Constantly exposed to self- and foreign antigen carried by gut-derived blood, the architecture and immune regulatory environment in the liver favor tolerance rather than activation of innate and adaptive immune responses. This tolerogenic environment is exploited by several hepatotropic pathogens to establish persistent infections with long-term liver damage. Here, we will discuss the functions of hepatic immune cells, the balance between tolerance versus activation, mechanisms facilitating the persistence of hepatotropic pathogens, and how immune responses influence the onset and progression of liver disease.

Activation of hepatic stellate cells (HSCs) is a key event in the initiation of liver fibrosis. We and others have demonstrated that IL-17A produced by Th17 cells promotes activation of HSCs. Th17 cells also produce IL-22, an enigmatic cytokine with pro-inflammatory and hepatoprotective properties. In addition, regulatory T cells (Treg) negatively modulate activation of HSCs. We hypothesized that liver fibrosis progression results from an alteration in the Th17/Treg ratio leading to an imbalance in pro-fibrotic Th17 cytokines in the liver. We examined ex vivo the frequency of Th17 and Treg populations and the cytokine profile of intrahepatic lymphocytes in liver biopsy samples (n=32). We observed increased Th17/Treg ratio in advanced as compared to moderate or no-fibrosis. Furthermore, we observed a bias towards Th17 cytokines in fibrotic livers with viral-hepatitis both in situ and ex vivo. All biopsies exhibited a 5-fold increase in IL-22 in fibrotic livers irrespective of aetiology. In vitro stimulation of HSCs with IL-22 sensitized them to suboptimal doses of TGF-β and activated p38 pathways. Chemical inhibition of p38 suppressed the pro-fibrogenic effect of IL-22. In vivo, lack of IL-22 signaling protected mice against liver fibrosis. IL-22RA1 Knockout mice exhibited reduced collagen deposition and expression of pro-fibrotic genes (ACTA2, LOXL2, TIMP-I, TGFb1, COL1A1) in comparison to wild-type littermates. Our results suggest a dysregulated Th17 response with increased IL-22 signaling in advanced fibrosis from all different aetiologies. Finally, we have identified IL-22 as a common factor in advanced liver fibrosis acting through sensitization of HSCs to TGF-β in a p38-dependent manner.

Activation of hepatic stellate cells (HSCs) is a key event in the initiation of liver fibrosis, characterized by enhanced extracellular matrix production and altered degradation. Activation of HSCs can be modulated by cytokines produced by immune cells. Recent reports have implicated the proinflammatory cytokine IL-17A in liver fibrosis progression. We hypothesized that IL-17A may enhance activation of HSCs and induction of the fibrogenic signals in these cells. The human HSC line LX2 and primary human HSCs were stimulated with increasing doses of IL-17A and compared with TGF-β– and PBS-treated cells as positive and negative controls, respectively. IL-17A alone did not induce activation of HSCs. However, IL-17A sensitized HSCs to the action of suboptimal doses of TGF-β as confirmed by strong induction of α–smooth muscle actin, collagen type I (COL1A1), and tissue inhibitor of matrix metalloproteinase I gene expression and protein production. IL-17A specifically upregulated the cell surface expression of TGF-βRII following stimulation. Pretreatment of HSCs with IL-17A enhanced signaling through TGF-βRII as observed by increased phosphorylation of SMAD2/3 in response to stimulation with suboptimal doses of TGF-β. This enhanced TGF-β response of HSCs induced by IL-17A was JNK-dependent. Our results suggest a novel profibrotic function for IL-17A by enhancing the response of HSCs to TGF-β through activation of the JNK pathway. IL-17A acts through upregulation and stabilization of TGF-βRII, leading to increased SMAD2/3 signaling. These findings represent a novel example of cooperative signaling between an immune cytokine and a fibrogenic receptor.

Single nucleotide polymorphisms (SNPs) in the proximity of the interleukin-28B (IL28B) gene can predict spontaneous resolution of hepatitis C virus (HCV) infection and response to interferon therapy. Screening for this polymorphism has become part of the standard criteria for the management of HCV-infected patients, hence the need for a rapid, cost-effective screening method. Here, we describe a rapid PCR-based test to screen for two IL28B SNPs (rs12979860 and rs8099917). We used this test to investigate IL28B polymorphism and prevalence in a cohort of French Canadian injection drug users who are part of a unique population known to have a strong genetic founder effect. This population had lower linkage disequilibrium between the two tested SNPs as compared to other cohorts (|d'| = 0.68, r = 0.59). The special genetic makeup should be considered in the management of HCV-infected patients within that population.

Loss of CD4 T cell help correlates with virus persistence during acute hepatitis C virus (HCV) infection, but the underlying mechanism(s) remain unknown. We developed a combined proliferation/intracellular cytokine staining assay to monitor expansion of HCV-specific CD4 T cells and helper cytokines expression patterns during acute infections with different outcomes. We demonstrate that acute resolving HCV is characterized by strong Th1/Th17 responses with specific expansion of IL-21-producing CD4 T cells and increased IL-21 levels in plasma. In contrast, viral persistence was associated with lower frequencies of IL-21-producing CD4 T cells, reduced proliferation and increased expression of the inhibitory receptors T cell immunoglobulin and mucin-domain-containing-molecule-3 (Tim-3), programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) on HCV-specific CD8 T cells. Progression to persistent infection was accompanied by increased plasma levels of the Tim-3 ligand Galectin-9 (Gal-9) and expansion of Gal-9 expressing regulatory T cells (Tregs). In vitro supplementation of Tim-3high HCV-specific CD8 T cells with IL-21 enhanced their proliferation and prevented Gal-9 induced apoptosis. siRNA-mediated knockdown of Gal-9 in Treg cells rescued IL-21 production by HCV-specific CD4 T cells. We propose that failure of CD4 T cell help during acute HCV is partially due to an imbalance between Th17 and Treg cells whereby exhaustion of both CD4 and CD8 T cells through the Tim-3/Gal-9 pathway may be limited by IL-21 producing Th17 cells or enhanced by Gal-9 producing Tregs., Author Summary In this study, we investigated the mechanisms underlying failure of the CD4 helper T cell response during acute hepatitis C infection. We demonstrate that this failure is primarily due to loss of IL-21-producing CD4 T cells in individuals who progress towards chronic infection. This is accompanied by exhaustion of virus-specific cytotoxic CD8 T cells through upregulation of the exhaustion markers Tim-3, PD-1 and CTLA-4, higher plasma levels of the Tim-3 ligand Galectin-9 (Gal-9) and increased frequency of Gal-9 producing regulatory T cells (Tregs). In vitro supplementation with IL-21 rescued HCV-specific CD8 T cells from Gal-9 induced apoptosis. Blocking Gal-9 expression in Tregs restored IL-21 production by virus-specific CD4 helper T cells. Altogether, our results suggest that failure of CD4 T cell help during acute HCV may be partially meditated by an imbalance between IL-21-producing CD4 T cells and Treg cells whereby exhaustion of both CD4 and CD8 T cells through the Tim-3/Gal-9 pathway is counteracted by IL-21.