Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by HCV are not defined. We provide a systems biology analysis with multiple lines of evidence to indicate that interleukin-1β (IL-1β) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1β compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells, resident hepatic macrophages, are the primary cellular source of hepatic IL-1β during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages, and Kupffer cells recovered from normal donor liver, produce IL-1β after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1β secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggers MyD88-mediated TLR7 signaling to induce IL-1β mRNA expression. HCV uptake concomitantly induces a potassium efflux that activates the NLRP3 inflammasome for IL-1β processing and secretion. RNA sequencing analysis comparing THP1 cells and chronic hepatitis C patient liver demonstrates that viral engagement of the NLRP3 inflammasome stimulates IL-1β production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identify intrahepatic IL-1β production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1β activity could offer therapeutic actions to reduce hepatic inflammation and mitigate disease. Hepatitis C virus (HCV) causes chronic infection of the liver and is a leading cause of liver inflammation, cirrhosis and liver cancer in nearly 200 million people worldwide. Importantly, hepatic inflammation during chronic HCV infection is considered to be the primary catalyst for progressive liver disease and development of liver cancer. However, the underlying molecular mechanism(s) of HCV-mediated hepatic inflammation are not well understood. The goal of this study was to determine the mechanisms of HCV-induced inflammation. We found that serum IL-1β levels are elevated in chronic hepatitis C patients. Furthermore, we found that hepatic macrophages or Kupffer cells are the major IL-1β-producing cell population within HCV infected livers. Our studies, using the THP1 cell culture model of HCV exposure, reveal that exposure of macrophages to HCV induces IL-1β through a process of infection-independent phagocytic virus uptake that triggers signaling through MyD88/TLR7 and NLRP3 inflammasome pathways to drive IL-1β expression and maturation/secretion, respectively. RNA sequencing (RNA-seq) analysis of patient liver biopsies shows that viral triggering of these signaling pathways drives an inflammatory response linked with liver disease in patients with chronic hepatitis C. Our results identify HCV-induced IL-1β production by hepatic macrophages as a critical and central process that promotes liver inflammation and disease.

Human mucosal-associated invariant T (MAIT) cells are a T cell population characterized by the expression of a semi-invariant TCR capable of recognizing bacterial products in the context of MR1. MAIT cells are enriched in the human liver, which is constantly exposed to bacterial products from the intestine. Whether this specific parenchymal localization influences their function remains unknown. We analyzed MAIT cells resident in the vascular bed of livers and showed that they represented the majority of T cells expressing NK markers and the dominant IL-17A⁺ T cell subset in the human liver sinusoids. In comparison with MAIT cells purified from peripheral blood, intrasinusoidal MAIT cells expressed markers of T cell activation; however, TCR-mediated cytokine production was equally suppressed in both circulating and intrasinusoidal MAIT cells. MAIT cells also expressed high levels of IL-7R, and we showed that IL-7, a cytokine produced by hepatocytes during inflammation, regulated TCR-mediated activation of MAIT cells, licensing them to dramatically increase Th1 cytokines and IL-17A production. Our quantitative and functional data indicate that MAIT cells are a specialized cell population highly adapted to exert their immune functions in the vascular network of the liver.

Background & Aims Chronic hepatitis B (CHB) infection acquired perinatally or in early childhood has been associated with a prolonged phase of immune tolerance from viral exposure into early adulthood. The immune-tolerant phase of the disease is characterized by high levels of hepatitis B virus (HBV) DNA and normal liver biochemistry, with minimal or no fibrosis. We investigated whether the age of patients with CHB affects their antiviral immunity and whether children and young adults have a veritable state of immunologic tolerance. Methods We isolated T cells from different age groups of patients with CHB and used flow cytometric methods to measure production of effector and inflammatory cytokines (interferon, tumor necrosis factor, interleukin [IL]-17A, IL-22, and IL-8), T-helper (Th)2 cytokines (IL-10, IL-4), Th1 cytokines (IL-2 and IL-21), and the CC chemokine CCL3 (MIP-1). We also measured markers of T-cell exhaustion or inhibition (PD-1, LAG-3, TIM3, LAIR-1, and CTLA-4) and HBV-specific T cells. Results Young patients with CHB have a Th1-cell cytokine profile and a partial profile of T-cell exhaustion. Direct quantification of the HBV-specific T-cell response showed that young patients with CHB have more HBV-specific T cells with the ability to proliferate and produce cytokines than adult patients with CHB. Conclusions HBV infection in younger patients is not associated with an immune profile of T-cell tolerance. On the contrary, children and young adults with chronic HBV infection have an HBV-specific immune profile that is less compromised than that observed in older patients.

The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161^Bright mucosal-associated invariant T (MAIT) and CD56^Bright NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies. The ability of human pathogens, like HBV, HCV or Plasmodium spp. to infect the liver might be influenced by its tolerogenic features. However, hepatic tolerance is not absolute since protective immunity can be triggered. Our goal was to define how to deliberately elicit an intrahepatic protective immune response. To achieve this, we purified immune cells residing in the vascular bed of human livers and we probed their reactivity against different pathogen-associated molecules, mimicking signature components of viruses or bacteria. We found that robust production of anti-viral cytokine IFN-γ was induced only by the TLR8 agonist ssRNA40. Mechanistically, ssRNA40 triggered hepatic monocytes to produce IL-12 and IL-18 cytokines, which stimulated IFN-γ production by liver-resident CD161^Bright MAIT and CD56^Bright NK cells. We also demonstrated that ssRNA40-mediated activation could occur in pathologic (HBV- or HCV-chronically infected) livers and that a similar cytokine-mediated activation of intrahepatic cells could also be triggered upon bacterial infection. Thus, we showed that the liver immune cells can respond vigorously to specific pathogen-associated molecules. The selective production of IFN-γ by liver-resident cells could have therapeutic implications for the treatment of chronic liver infections.