Sorted B cell transcriptomes point towards actively regulated B cell responses during ongoing chronic hepatitis B infections

The natural course of chronic hepatitis B virus (HBV) infections follows distinct clinical disease phases, char-acterized by fluctuating levels of serum HBV DNA and ALT. The immune cells and their features that govern these clinical disease transitions remain unknown. In the current study, we performed RNA sequencing on pu-rified B cells from blood (n = 42) and liver (n = 10) of healthy controls and chronic HBV patients. We found distinct gene expression profiles between healthy controls and chronic HBV patients, as evidenced by 190 differentially expressed genes (DEG), but also between the clinical phenotypes of a chronic HBV infection (17?110 DEG between each phase). Numerous immune pathways, including the B cell receptor pathway were upregulated in liver B cells when compared to peripheral B cells. Further investigation of the detected DEG suggested an activation of B cells during HBeAg seroconversion and an active regulation of B cell signalling in the liver

Circulating Cytokines Reflect the Etiology-Specific Immune Environment in Cirrhosis and HCC

Background and Aims: Chronic liver disease—from any etiology—can progress to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The progression of liver cirrhosis to the end stages of disease is influenced by a variety of factors, including inflammatory cytokines. We pursued a study of cytokine-mediated inflammatory responses in hepatitis B (HBV), hepatitis C (HCV), alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) patients with liver cirrhosis. Methods: Immune profiles were determined through the serum multiplex profiling of >100 cytokines in a 188 cirrhotic patients, 35 healthy controls and 196 early-stage HCC patients. Results: Patients with liver cirrhosis exhibited a vast upregulation of proinflammatory cytokines (p < 0.0001), including those with pro-oncogenic features, when compared to healthy individuals. In contrast to prevailing assumptions, each etiological cause of cirrhosis exhibited a unique cytokine profile in blood. Regardless of antiviral therapy, HBV cirrhosis patients had the largest number of upregulated proinflammatory mediators, compared to HCV, ALD and NAFLD (p < 0.0001). To further evaluate the etiology-dependent modulation of cytokine response in relation to liver cancer, we studied cytokine profiles in early-stage HCC patients strictly stratified by underlying liver disease. We observed unique sets of differentially expressed cytokines in each cohort of early-stage HCC patients of different cirrhosis etiologies. Conclusions: Our findings, therefore, underscore the importance of stratification by the etiological cause of liver cirrhosis in immune-based studies

Monocytes from Chronic HBV Patients React <i>In Vitro</i> to HBsAg and TLR by Producing Cytokines Irrespective of Stage of Disease

<div><p>Individuals who are chronically infected with the hepatitis B virus (HBV) are highly heterogenous with respect to serum levels of HBV DNA, HBV particles and viral proteins. Since circulating leukocytes, such as monocytes, are constantly exposed to these viral components, it is likely that the functionality of these cells is affected. However, at present, little information is available on the consequences of the interaction between monocytes and viral components. Therefore, we examined the <i>in vitro</i> effects of HBV surface antigen (HBsAg) on monocytes and evaluated whether these effects were reflected <i>in vivo</i>. We observed that <i>in vitro</i> HBsAg exposure of monocytes induced robust production of IL-6 and TNF. However, between chronic HBV patients with distinct levels of serum HBsAg, HBV early antigen (HBeAg), and HBV DNA, TLR-induced monocyte cytokine production did not differ. Importantly, HBsAg-induced cytokine production by monocytes was similar between patients and healthy controls showing that earlier <i>in vivo</i> exposure to HBsAg does not affect the <i>in vitro</i> response. Additionally, we show that IL-10 is able to inhibit cytokine production by HBsAg-induced monocytes. In conclusion, we demonstrate that monocytes can recognize and respond to HBsAg, resulting in vigorous pro-inflammatory cytokine production <i>in vitro</i>. However, phenotype and function of the monocyte compartment in chronic HBV patients are not influenced by differences in levels of serum viral components, suggesting that regulatory mechanisms are active to avoid excessive <i>in vivo</i> monocyte activation.</p></div

Negative Regulation of Hepatitis C Virus Specific Immunity Is Highly Heterogeneous and Modulated by Pegylated Interferon-Alpha/Ribavirin Therapy

<div><p>Specific inhibitory mechanisms suppress the T-cell response against the hepatitis C virus (HCV) in chronically infected patients. However, the relative importance of suppression by IL-10, TGF-β and regulatory T-cells and the impact of pegylated interferon-alpha and ribavirin (PegIFN-α/ribavirin) therapy on these inhibitory mechanisms are still unclear. We revealed that coregulation of the HCV-specific T-cell responses in blood of 43 chronic HCV patients showed a highly heterogeneous pattern before, during and after PegIFN-α/ribavirin. Prior to treatment, IL-10 mediated suppression of HCV-specific IFN-γ production in therapy-naive chronic HCV patients was associated with higher HCV-RNA loads, which suggests that protective antiviral immunity is controlled by IL-10. In addition, as a consequence of PegIFN-α/ribavirin therapy, negative regulation of especially HCV-specific IFN-γ production by TGF-β and IL-10 changed dramatically. Our findings emphasize the importance of negative regulation for the dysfunctional HCV-specific immunity, which should be considered in the design of future immunomodulatory therapies.</p> </div

Different HBV DNA, HBeAg and HBsAg levels do not alter the frequency of monocyte subpopulations.

<p>Whole blood samples from chronic HBV patients were stained for CD14 and CD16 to analyze frequencies of the monocyte CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ subpopulations. (<b>A</b>) Gating strategy: monocytes were identified on the basis of their forward-sideward scatter, and divided into CD14⁺⁺CD16^- and CD14⁺CD16⁺ populations. (<b>B</b>) The frequencies of monocyte subpopulations were compared between groups 1–3 (n = 45).</p

IL-10 inhibits the frequency of monocytes producing cytokines upon HBsAg exposure.

<p>PBMC from healthy individuals were stimulated with HBsAg, with or without IL-10. After overnight incubation, cells were stained for CD14 and intracellularly for cytokines. The frequencies of IL-6-positive (<b>A</b>) and TNF-positive (<b>B</b>) monocytes were compared between HBsAg and HBsAg and IL10 (n = 3).</p

Chronic HCV infected patients show dysfunctional HCV-specific T-cell responses.

<p>Forty-three chronic HCV-infected patients were investigated (21 therapy-naive and 22 PegIFN-α/ribavirin experienced). T-cell proliferation (cpm) to HCV (<b>A</b>) or CMV (<b>B</b>) were determined at day 5 upon stimulation of PBMC, and IFN-γ production (<b>C</b>) at day 3. The sensitivity of the IFN-γ ELISA was 20 pg/mL, and therefore data of 17 patients are shown as one single horizontal line in the graph. For all graphs, patients showing significant responses are depicted to the left (YES) and those without are shown to the right (NO).</p