Michelle Spaan

Immunological Analysis During Interferon-Free Therapy for Chronic Hepatitis C Virus Infection Reveals Modulation of the Natural Killer Cell Compartment

BACKGROUND Chronic hepatitis C virus (HCV) infection is a global health problem, resulting in liver failure, hepatocellular carcinoma, and liver-related death. Natural killer (NK) cells are innate immune cells, and their activity is known to correlate to viral treatment response of HCV. In this study, we investigate the immune effects of viral load decline with direct-acting antivirals (DAAs) in blood. METHODS Twelve patients with chronic HCV were treated with asunaprevir and daclatasvir, and peripheral blood was analyzed at various time points during therapy. RESULTS In line with previous studies, we confirmed restoration of HCV-specific T-cell frequency upon viral load decline. In addition, we show that serum interferon (IFN)-γ inducible-protein 10, interleukin (IL)-12p40, and IL-18 levels decreased early after start of therapy. Surface expression of activation receptors NKp30, NKp46, and inhibitory receptor NKG2A on blood NK cells reduced during therapy. In addition, the expression of TRAIL on NK cells was reduced during IFN-free therapy, suggesting a decrease in TRAIL-mediated killing by NK cells. CONCLUSIONS We show that viral load decline as a consequence of treatment with novel DAAs in chronic HCV patients reduces serum levels of NK cell-stimulating cytokines and causes correction of the altered NK cell phenotype observed in chronic HCV patients. CLINICAL TRIALS REGISTRATION NCT02282709.

Healthy human salivary glands contain a DHEA-sulphate processing intracrine machinery, which is deranged in primary Sjögren’s syndrome

Sjögrens syndrome (SS) patients have low salivary dehydroepiandrosterone (DHEA) and androgen biomarker levels, but high salivary oestrogen levels. The hypothesis was that the healthy glands contain DHEA‐sulphate processing intracrine machinery; the local androgen/oestrogen imbalance suggests that this is disarranged in SS. Indirect immunofluorescence and quantitative real‐time PCR (qRT‐PCR) of steroid sulphatase, sulfotransferase, 3β‐ and 17β‐hydroxysteroid dehydrogenases (3β‐ and 17β‐HSD), 5α‐reductase and aromatase were performed for labial salivary glands of healthy controls and persons with SS. In control acini steroid sulphatase and sulfotransferase immunoreactivities were located in the basolateral cell parts. 3β‐ and 17β‐HSD formed strong, interrupted bands along the basal cell parts. 5α‐reductase was mainly located in acinar cell nuclei and aromatase in the apical cell membrane. All enzymes were more widespread in ducts. In SS, steroid sulphatase was weak and deranged, 3β‐ and 17β‐HSD had lost their strict basal acinar cell localization and 5α‐reductase was mainly found in the cytoplasm of the acinar cells, whereas aromatase showed similar staining in SS and controls. qRT‐PCR of labial salivary glands disclosed all corresponding enzyme mRNAs with the levels of 3β‐HSD in SS being the lowest. Healthy tubuloacinar epithelial cells contain complete intracrine machineries for DHEA(‐sulphate) pro‐hormone processing. These enzymes have in healthy acini an organized architecture, which corresponds with DHEA uptake from the circulation, nuclear site of production of the active dihydrotestosterone (DHT) end product and production of oestrogens into saliva for export to ductal and oral epithelial cells. SS is characterized by low 3β‐HSD levels, which together with impaired subcellular compartmentalization of HSDs and 5α‐reductase may explain the low local DHT and androgen biomarker levels in SS.

CD4+CXCR5+ T cells in chronic HCV infection produce less IL-21, yet are efficient at supporting B cell responses

BACKGROUND & AIMS During chronic HCV infection, T cell dependent virus-specific antibodies are produced. However, the role of B-T cell interaction in chronic HCV is largely unknown. CD4(+)CXCR5(+) T follicular helper (TFH)-cells activate B cells and are important for clearance of various chronic viral infections. We investigated the function of TFH cells and B cells in liver and in peripheral blood of chronic HCV patients. METHODS T cells from chronic HCV patients and healthy individuals were analysed for expression of CXCR5, PD-1, ICOS, and IL-21 and IFN-γ production by flow cytometry. CD19(+) B cell subpopulations were identified on the basis of CD27 and IgD expression. In order to assess the frequency and function of T cells and B cells in liver follicles, immunohistochemistry was performed for CD3, CXCR5, Bcl6, IL-21, CD20, IgD, IgM, and IgG. RESULTS The frequency of IL-21-producing CXCR5(+)CD4(+) T cells in blood was lower in HCV patients compared to healthy individuals (p=0.002), which was reflected by lower serum IL-21 levels (p<0.001). Nonetheless, CXCR5(+)CD4(+) T cells from HCV patients and healthy individuals were equally capable to stimulate CD19(+)CD27(+) memory B cells into IgG and IgM-producing plasmablasts. Importantly, human intrahepatic TFH cells and their related function were identified by immunohistochemistry on liver biopsies for CD3, Bcl6, and CD20 within portal areas and follicles. CONCLUSIONS The specific localization of TFH cells and IgG and IgD/IgM-producing B cells suggests a functional B-T cell environment in liver follicles during HCV infection. The decreased frequency of IL-21-producing CXCR5(+)CD4(+) T cells and lower serum IL-21 levels in chronic HCV patients did not lead to an altered TFH-B cell interaction.

Frequencies of circulating MAIT cells are diminished in chronic hCV, HIV and HCV/ HIV Co-Infection and do not recover during therapy

Objective Mucosal-associated invariant T (MAIT) cells comprise a subpopulation of T cells that can be activated by bacterial products and cytokines to produce IFN-γ. Since little is known on MAIT cells during HCV infection, we compared their phenotype and function in comparison to HIV and HCV/HIV co-infected patients, and determined the effect of IFN-α-based and direct-acting antiviral therapy on MAIT cells of HCV patients. Methods Blood samples from patients with chronic HCV (CHCV), virologically suppressed HIV, acute HCV/HIV co-infection (AHCV/HIV) and healthy individuals were examined by flowcytometry for phenotype and function of MAIT and NK cells. Results and Conclusions Compared to healthy individuals, the frequency of CD161+Vα7.2+ MAIT cells was significantly decreased in patients with CHCV, HIV and AHCV/HIV co-infection. CD38 expression on MAIT cells was increased in AHCV/HIV patients. MAIT cells were responsive to IFN-α in vitro as evidenced by enhanced frequencies of IFN-γ producing cells. IFN-α-based therapy for CHCV decreased the frequency of IFN-γ+ MAIT cells, which was still observed 24 weeks after successful therapy. Importantly, even after successful IFN-α-based as well as IFN-α-free therapy for CHCV, decreased frequencies of MAIT cells persisted. We show that the frequencies of MAIT cells are reduced in blood of patients with CHCV, HIV and in AHCV/HIV co-infection compared to healthy individuals. Successful therapy for CHCV did not normalize MAIT cell frequencies at 24 weeks follow up. The impact of HIV and HCV infection on the numbers and function of MAIT cells warrant further studies on the impact of viral infections and the antimicrobial function of MAIT cells.

The Intrahepatic T Cell Compartment Does Not Normalize Years After Therapy-Induced Hepatitis C Virus Eradication

Little is known about the immune status in liver and blood of patients with chronic hepatitis C virus (HCV) infection long after therapy-induced viral clearance. In this study, we demonstrate that, 4 years after clearance, regulation of HCV-specific immunity in blood by regulatory T cells (Tregs) and the immunosuppressive cytokines interleukin 10 and transforming growth factor β is still ongoing. Importantly, analysis of liver specimens collected 4 years after HCV clearance shows that intrahepatic Tregs are still present in all patients, suggesting that liver T cells remain regulated. Identifying mechanisms that regulate HCV-specific memory T-cell responses after clearance is highly relevant for the development of protective vaccines, especially in patients at high risk of reinfection.

Sustained virologic response after therapy with the HCV protease inhibitor narlaprevir in combination with peginterferon and ribavirin is durable through long‐term follow‐up

Achievement of a sustained virologic response (SVR) after peginterferon (PEG‐IFN) and ribavirin (RBV) treatment is considered to be a marker for the cure of chronic hepatitis C virus (HCV) infection. Long‐term follow‐up of patients with SVR after treatment with a direct acting antiviral has not yet been described. We used a randomized placebo‐controlled, double‐blind, two‐period phase 1b trial that was conducted in 40 HCV genotype 1 (treatment‐naïve and treatment‐experienced)‐infected patients. Nineteen patients achieved SVR after treatment with the HCV protease inhibitor narlaprevir followed by PEG‐IFN/RBV. In these patients, HCV‐RNA tests were scheduled at 3, 6, 12 and 24 months after end of treatment. Patients were followed for a median of 27 months (range 15–32) after end of treatment with a median number of follow‐up visits of 4 (range 3–8). All patients remained HCV‐RNA negative over time. SVR achieved following narlaprevir and PEG‐IFN/RBV‐therapy was durable up to 32 months after the end of treatment.

Erythropoietin administration suppresses human monocyte function in vitro and during therapy-induced anemia in HCV patients.

Erythropoietin (EPO) is a hormone that controls red blood cell production. Binding of EPO to the EPO-receptor results in increased numbers of red blood cells in the circulation, which makes EPO a potent molecule to treat anemia in various groups of patients. Although numerous studies have examined the clinical effects of EPO, its immunological effects have received less attention. In this study, we examined the immunological effects of EPO on human monocytes. We show that human monocytes express EPO receptor mRNA, and are responsive to EPO in cell culture. In vitro exposure of PBMC from individuals to EPO and the TLR4 ligand LPS showed a significant reduction of monocytes producing IL-6 and TNF, while the frequencies of IL-12p40, IL-10, MIP-1β and IL-8-producing cells did not change upon incubation with EPO. In addition, EPO did increase the phagocytic activity but did not affect the ability to produce ROS by monocytes. Moreover, we studied eight chronic HCV patients undergoing treatment with peg-IFN and ribavirin, who were administered EPO for treatment-induced anemia. Blood was collected before and 7 days after EPO injection. In 7 patients, we observed a significant decline at day 7 after EPO administration of the frequency of monocytes producing various pro-inflammatory cytokines following stimulation with the TLR4 ligand LPS and the TLR7/8 ligand R848, which is in line with our in vitro findings. Our findings demonstrate an inhibitory effect of EPO on the secretion of effector molecules by monocytes and a stimulatory effect on the phagocytic activity by monocytes.

Longitudinal analysis of peripheral and intrahepatic NK cells in chronic HCV patients during antiviral therapy

INTRODUCTION A strong immune response is integral to the clearance of HCV infection. NK cells are specialized cells that are able to inhibit replication of HCV in infected hepatocytes. Previous studies have correlated therapy outcome in HCV to the expression of various markers on NK cells. However, the effect of viral load reduction on NK cell function during therapy is still largely unknown, particularly in the liver. Therefore we investigated NK cell phenotype and effector function in both the peripheral and intrahepatic compartments during the course of antiviral therapy in chronic HCV patients. METHODS Chronic HCV patients were treated for 24 or 48weeks with triple therapy consisting of telaprevir, pegIFN-α and ribavirin. Blood and fine needle aspiration (FNA) biopsies of the liver were collected at start and 6h, 1week and 12weeks during therapy. Flowcytometry was performed for expression of different markers (NKG2A, NKG2D, NKp46, and CD69). RESULTS Our results demonstrate a highly activated phenotype of NK cells in liver compared to blood in chronic HCV patients. Six hours after start of triple therapy, no activation of intrahepatic NK cells was observed in the liver as compared to baseline. At 1week after start of triple therapy, the frequency of NK cells with the activating receptor NKp46 was increased in blood, whereas at week 12, the frequencies of the inhibitory receptor NKG2A was increased. No alterations were observed during therapy in liver NK cell phenotype. CONCLUSION IFN-based therapy for chronic HCV affects NK cell phenotype in peripheral blood more than in the liver.

Inhibitory receptor molecules in chronic hepatitis B and C infections: novel targets for immunotherapy?

Chronic HBV and HCV infections are the leading cause of liver‐related morbidity and mortality. For effective antiviral immunity, virus‐specific T cells are required, but these cells have been shown to be weak or absent in chronic HBV and HCV patients. One of the mechanisms that underlies the impaired T‐cell response is the result of the continuously high viral load that causes HBV‐specific and HCV‐specific T cells to become exhausted, which is characterized by impaired proliferation, cytokine production and cytotoxic activity of T cells as well as high susceptibility to apoptosis. In vitro studies from chronic HBV and HCV patients as well as in vivo studies in animal models demonstrated a reversible state of T‐cell exhaustion, which can be manipulated to reinvigorate the specific antiviral immune responses. In chronic HCV infection, this concept has been explored in clinical trials by administration of specific antibody to block the inhibitory pathways. The manipulation of inhibitory receptors is a promising and potential strategy for immunotherapeutic interventions in chronic HBV and HCV patients to facilitate complete elimination of the viruses or sustained viral control. Copyright