Anastasios E. Germenis

Liver FOXP3 and PD1/PDL1 expression is down-regulated in chronic HBV hepatitis on maintained remission related to the degree of inflammation

Background and Aim: T cell expression of PD1 and inhibition of T effector cells by Foxp3+-T regulatory cells are among the most powerful mechanisms for achieving a balanced immune response. Our aim was to investigate, how liver FOXP3 and PD1/PDL1 expression is regulated in chronic HBV hepatitis (CHB) on maintained long-term remission in comparison with active disease, and whether they are correlated to the expression of pro- and anti-inflammatory cytokines and apoptosis mediators, along with the degree of histological inflammation and markers of T cell effector restoration. Methods: Fifty-three HBeAg-negative CHB patients with both active (30) and completely remitted disease on long-term antiviral treatment (23) and four controls (submitted to liver biopsy due to a mild increase of aminotransferases but without liver necroinflammatory and architecture changes) were enrolled in the study. Liver mRNA levels of immunoregulatory genes (FOXP3, IL10, TGFB1, and those of PD1/PDL1/PDL2 pathway), major apoptosis mediators (FAS, FASL, TNFA, TRAIL), cytokines of effector T cell restoration (IL2, IFNG), and those of IL1B, CD4, and CD8, were evaluated by quantitative real-time reverse-transcriptase PCR and were correlated with each other, along with the intensity of liver inflammation and fibrosis staging. The expression and localization of FOXP3, PD1, PDL1, CD4, and CD8 were also assessed by immunohistochemistry. Results: The expression of FOXP3, IL10, TGFB1, PD1, PDL1, FASL, and CD8 was significantly down-regulated in the remission state. In contrast, liver expression of IL2 and IFNG, along with CD4, IL1B, TNFA, and FAS did not change significantly. Moreover, FOXP3, PD1, PDL1, and CD8 transcripts were positively correlated to the intensity of liver inflammation. Conclusion: Our data indicate that in the CHB disease model, the immunosuppressive liver environment is down-regulated in the maintained on-treatment long-term remission state and correlates with the intensity of liver inflammation, but not liver T cell restoration.

F12‐46C/T polymorphism as modifier of the clinical phenotype of hereditary angioedema

The factors influencing the heterogeneous clinical manifestation of hereditary angioedema due to C1‐INH deficiency (C1‐INH‐HAE) represent one of the oldest unsolved problems of the disease. Considering that factor XII (FXII) levels may affect bradykinin production, we investigated the contribution of the functional promoter polymorphism F12‐46C/T in disease phenotype. We studied 258 C1‐INH‐HAE patients from 113 European families, and we explored possible associations of F12‐46C/T with clinical features and the SERPING1 mutational status. Given that our cohort consisted of related subjects, we implemented generalized estimating equations (GEEs), an extension of the generalized linear model accounting for the within‐subject correlation. F12‐46C/T carriers exhibited a significantly delayed disease onset (P < 0.001) and did not need long‐term treatment (P = 0.02). In a GEE linear regression model, the presence of F12‐46C/T was significantly associated with a 7‐year delay in disease onset (P < 0.0001) regardless of SERPING1 mutational status. It is concluded that F12‐46C/T carriage acts as an independent modifier of C1‐INH‐HAE severity.

TGF-β signaling is activated in patients with chronic HBV infection and repressed by SMAD7 overexpression after successful antiviral treatment.

ObjectivesAlthough animal studies demonstrated that Smad7 induction ameliorates TGF-β/SMAD-mediated fibrogenesis, its role in human hepatic diseases is rather obscure. Our study explored the activation status of TGF-β/activin pathway in patients with chronic liver diseases, and how it is affected by successful antiviral treatment in chronic HBV hepatitis (CHB).MethodsThirty-seven CHB patients (19 with active disease, 14 completely remitted on long-term antiviral treatment and 4 with relapse after treatment withdrawal), 18 patients with chronic HCV hepatitis, 12 with non-alcoholic fatty liver disease (NAFLD), and 3 controls were enrolled in the study. Liver mRNA levels of CTGF, all TGF-β/activin isoforms, their receptors and intracellular mediators (SMADs) were evaluated using qRT-PCR and were correlated with the grade of liver inflammation and fibrosis staging. The expression and localization of pSMAD2 and pSMAD3 were assessed by immunohistochemistry.ResultsTGF-β signalling is activated in CHB patients with active disease, while SMAD7 is up-regulated during the resolution of inflammation after successful treatment. SMAD7 overexpression was also observed in NAFLD patients exhibiting no or minimal fibrosis, despite the activation of TGF-β/activin signaling.ConclusionsSMAD7 overexpression might represent a mechanism limiting TGF-β-mediated fibrogenesis in human hepatic diseases; therefore, SMAD7 induction likely represents a candidate for novel therapeutic approaches.

Genetic polymorphisms of innate and adaptive immunity as predictors of outcome in critically ill patients.

Sepsis and septic shock frequently cause the admission or complicate the clinical course of critically ill patients admitted in the intensive care units (ICU). Genetic variations disrupting the immune sensing of infectious organisms, could affect the ability of the immune system to respond to infection, and may influence both the genetic predisposition to infection and the diversity of the clinical presentation of sepsis. The aim of this study was to uncover possible associations between common functional immune gene polymorphisms (of both innate and adaptive immunity) and ICU-acquired sepsis and mortality. The TLR4-D299G (rs4986790), TLR4-T399I (rs4986791), C2-c.841_849+19del28 (rs9332736), TACI-C104R (rs34557412), BAFFR-P21R (rs77874543), and BAFFR-H159Y (rs61756766) polymorphisms were detected in a cohort of 215 critically ill patients, admitted in an 8-bed medical/surgical ICU. Interestingly, TLR4-D299G, TLR4-T399I and BAFFR-P21R carriage was associated with a lower risk of ICU-acquired sepsis. This association applied particularly in medical patients, while in trauma and surgical patients no significant associations were observed. Moreover, carriers of TACI-C104R displayed an undiagnosed mild to moderate hypogammaglobulinemia along with a significantly lower survival rate in the ICU, although lethal events were not attributed to sepsis. These findings further elucidate the role that host immune genetic variations may play in the susceptibility to ICU-acquired sepsis and ICU mortality.

Hereditary Angioedema with Normal C1 Inhibitor: Update on Evaluation and Treatment

A new form of hereditary angioedema (HAE) was identified in the year 2000. Its clinical appearance resembles HAE types I and II, which are caused by mutations that result in a deficiency of C1 inhibitor (C1-INH). In patients with the new form of HAE, C1-INH plasma levels and function values are normal, so its termed HAE with normal C1-INH (HAE-nC1). HAE-nC1, in a subgroup of patients, is thought to be caused by mutations that affect the F12 gene. The diagnosis of HAE-nC1 is based on history and clinical criteria. There are no licensed drugs with proven treatment effects for HAE-nC1.

The role of the NLRP3 inflammasome and the activation of IL-1β in the pathogenesis of chronic viral hepatic inflammation

Background and aims: Chronic viral hepatitis is a prevalent disease with major health implications. Its underlying pathophysiological mechanisms are not fully understood. IL‐1&bgr; and the NLRP3 inflammasome involvement has been suggested in recent years, from in vitro data and data from peripheral blood samples. Therefore, we investigated IL‐1&bgr; and the NLRP3 inflammasome in liver tissues in an effort to clarify their role in the pathophysiology of chronic viral hepatitis. Methods: We studied liver biopsies from patients with a new diagnosis of either chronic hepatitis B (CHB) and chronic hepatitis C (CHC) or patients with chronic hepatitis B in remission (CHB‐rem). The biopsies were separated in two parts. The first part was sent to histology to determine the grade of inflammation and fibrosis. From the second part, RNA was extracted and converted to cDNA used in semi‐quantitative Real‐Time PCR to measure the levels of IL1B, CASP1, NLRP3, ASC and IL1RA. The cell lines used in the in vitro experiments were Huh7.5, LX2 and THP‐1 in variety of combinations of monocultures, co‐cultures and triple cultures with one of the cell lines infected with the JFH‐1 HCV clone. From the cell cultures RNA was extracted and converted to cDNA. For cell lines, we focused in the expression of IL1B and NLRP3. Results: The expression of IL1B, CASP1 and NLRP3 were found significantly different between our groups (p = 0.001, p = 0.001 and p = 0.038, respectively). CHB patients displayed significantly higher IL1B and CASP1 mRNA levels compared to both CHB‐rem and CHC patients. IL1B expression significantly correlates with liver biochemical data in CHB patients (AST: p = 0.006, r = 0.457; ALT p = 0.002, r = 0.497). Finally, mRNA levels of IL1B in CHB patients significantly correlate with the degree of inflammation (p = 0.016) but not the stage of fibrosis (p = 0.362). Interestingly, the relative expression of IL1B in triple culture experiments in vitro was below of 1.5‐fold, suggesting no activation of IL1B. Moreover, no activation of NLRP3 was demonstrated in all investigated in vitro conditions. Conclusion: IL‐1&bgr; might play an important role in the pathogenesis of chronic hepatic inflammation from HBV, but not from HCV.

An Activating Janus Kinase-3 Mutation Is Associated with Cytotoxic T Lymphocyte Antigen-4-Dependent Immune Dysregulation Syndrome

Heterozygous mutations in the cytotoxic T lymphocyte antigen-4 (CTLA-4) are associated with lymphadenopathy, autoimmunity, immune dysregulation, and hypogammaglobulinemia in about 70% of the carriers. So far, the incomplete penetrance of CTLA-4 haploinsufficiency has been attributed to unknown genetic modifiers, epigenetic changes, or environmental effects. We sought to identify potential genetic modifiers in a family with differential clinical penetrance of CTLA-4 haploinsufficiency. Here, we report on a rare heterozygous gain-of-function mutation in Janus kinase-3 (JAK3) (p.R840C), which is associated with the clinical manifestation of CTLA-4 haploinsufficiency in a patient carrying a novel loss-of-function mutation in CTLA-4 (p.Y139C). While the asymptomatic parents carry either the CTLA-4 mutation or the JAK3 variant, their son has inherited both heterozygous mutations and suffers from hypogammaglobulinemia combined with autoimmunity and lymphoid hyperplasia. Although the patient’s lymph node and spleen contained many hyperplastic germinal centers with follicular helper T (TFH) cells and immunoglobulin (Ig) G-positive B cells, plasma cell, and memory B cell development was impaired. CXCR5+PD-1+TIGIT+ TFH cells contributed to a large part of circulating T cells, but they produced only very low amounts of interleukin (IL)-4, IL-10, and IL-21 required for the development of memory B cells and plasma cells. We, therefore, suggest that the combination of the loss-of-function mutation in CTLA-4 with the gain-of-function mutation in JAK3 directs the differentiation of CD4 T cells into dysfunctional TFH cells supporting the development of lymphadenopathy, hypogammaglobulinemia, and immunodeficiency. Thus, the combination of rare genetic heterozygous variants that remain clinically unnoticed individually may lead to T cell hyperactivity, impaired memory B cell, and plasma cell development resulting finally in combined immunodeficiency.