Baseline Intrahepatic and Peripheral Innate Immunity are Associated with Hepatitis C Virus Clearance During Direct-Acting Antiviral Therapy.

Hepatitis C virus (HCV) infection induces interferon (IFN)-stimulated genes (ISGs) and downstream innate immune responses. This study investigated whether baseline and on-treatment differences in these responses predict response versus virological breakthrough during therapy with direct-acting antivirals (DAAs). Thirteen HCV genotype 1b-infected patients who had previously failed a course of pegylated IFN/ribavirin were retreated with asunaprevir/daclatasvir for 24 weeks. After pretreatment biopsy, patients were randomized to undergo a second biopsy at week 2 or 4 on therapy. Microarray and NanoString analyses were performed on paired liver biopsies and analyzed using linear mixed models. As biomarkers for peripheral IFN responses, peripheral blood natural killer cells were assessed for phosphorylated signal transducer and activator of transcription 1 (pSTAT1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and degranulation. Nine of 13 (69%) patients achieved sustained virological response at 12 weeks off therapy (SVR12), and 4 experienced virological breakthroughs between weeks 4 and 12. Patients who achieved SVR12 displayed higher ISG expression levels in baseline liver biopsies and a higher frequency of pSTAT1 and TRAIL-expressing, degranulating natural killer cells in baseline blood samples than those who experienced virological breakthrough. Comparing gene expression levels from baseline and on-therapy biopsies, 408 genes (±1.2-fold, P < 0.01) were differentially expressed. Genes down-regulated on treatment were predominantly ISGs. Down-regulation of ISGs was rapid and correlated with HCV RNA suppression. Conclusion: An enhanced IFN signature is observed at baseline in liver and blood of patients who achieve SVR12 compared to those who experience a virological breakthrough; the findings suggest that innate immunity may contribute to clearance of HCV during DAA therapy by preventing the emergence of resistance-associated substitutions that lead to viral breakthrough during DAA therapy.

Rapid decrease in hepatitis C viremia by direct acting antivirals improves the natural killer cell response to IFNα.

OBJECTIVE: Chronic HCV infection is characterised by innate immune activation with increased interferon-stimulated genes (ISG) expression and by an altered phenotype of interferon-responsive natural killer (NK) cells. Here, we asked whether a rapid reduction in viremia by daclatasvir (DCV) and asunaprevir (ASV) improves the response to pegylated interferon (PegIFN) in patients who had previously failed a standard course of PegIFN/ribavirin (RBV) therapy. DESIGN: Twenty-two HCV-infected non-responders to previous PegIFN/RBV therapy were studied for IFN-responsiveness of NK cells during quadruple (QUAD) therapy with DCV, ASV, PegIFN and RBV. A direct comparison of early NK cell responses in PegIFN/RBV therapy and QUAD therapy was performed for seven patients using paired cryopreserved peripheral blood mononuclear cells (PBMC) from both treatment courses. As a validation cohort, nine DCV/ASV-treated patients were studied for their NK cell response to in vitro stimulation with IFNα. RESULTS: The 24 h virological response to QUAD therapy correlated with an increase in signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (pSTAT1) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) expression in NK cells, and the STAT1/pSTAT1/TRAIL induction was greater during QUAD therapy than during previous PegIFN/RBV therapy. Successful QUAD therapy as well as successful IFN-free DCV/ASV regimen resulted in an improved functional NK cell response (degranulation and TRAIL expression) to in vitro stimulation with IFNα. CONCLUSIONS: IFN-responsiveness can be improved by inhibiting HCV replication and reducing the HCV-induced activation of the innate immune response. This may provide a rationale for clinical trials of a brief period of direct acting antiviral therapy followed by PegIFN/RBV therapy to reduce the overall treatment costs in low-income and middle-income countries. TRIAL REGISTRATION NUMBERS: NCT01888900 and NCT00718172.

IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection.

UNLABELLED: Early, vigorous intrahepatic induction of interferon (IFN)-stimulated gene (ISG) induction is a feature of hepatitis C virus (HCV) infection, even though HCV inhibits the induction of type I IFNs in vitro. To identify the cytokines and cells that drive ISG induction and mediate antiviral activity during acute HCV infection, type I and III IFN responses were studied in (1) serial liver biopsies and plasma samples obtained from 6 chimpanzees throughout acute HCV infection and (2) primary human hepatocyte (PHH) cultures upon HCV infection. Type I IFNs were minimally induced at the messenger RNA (mRNA) level in the liver and were undetectable at the protein level in plasma during acute HCV infection of chimpanzees. In contrast, type III IFNs, in particular, interleukin (IL)-29 mRNA and protein, were strongly induced and these levels correlated with ISG expression and viremia. However, there was no association between intrahepatic or peripheral type III IFN levels and the outcome of acute HCV infection. Infection of PHH with HCV recapitulated strong type III and weak type I IFN responses. Supernatants from HCV-infected PHH cultures mediated antiviral activity upon transfer to HCV-replicon-containing cells. This effect was significantly reduced by neutralization of type III IFNs and less by neutralization of type I IFNs. Furthermore, IL-29 production by HCV-infected PHH occurred independently from type I IFN signaling and was not enhanced by the presence of plasmacytoid dendritic cells. CONCLUSION: Hepatocyte-derived type III IFNs contribute to ISG induction and antiviral activity, but are not the principal determinant of the outcome of HCV infection.

Concerted action of wild-type and mutant TNF receptors enhances inflammation in TNF receptor 1-associated periodic fever syndrome.

TNF, acting through p55 tumor necrosis factor receptor 1 (TNFR1), contributes to the pathogenesis of many inflammatory diseases. TNFR-associated periodic syndrome (TRAPS, OMIM 142680) is an autosomal dominant autoinflammatory disorder characterized by prolonged attacks of fevers, peritonitis, and soft tissue inflammation. TRAPS is caused by missense mutations in the extracellular domain of TNFR1 that affect receptor folding and trafficking. These mutations lead to loss of normal function rather than gain of function, and thus the pathogenesis of TRAPS is an enigma. Here we show that mutant TNFR1 accumulates intracellularly in peripheral blood mononuclear cells of TRAPS patients and in multiple cell types from two independent lines of knockin mice harboring TRAPS-associated TNFR1 mutations. Mutant TNFR1 did not function as a surface receptor for TNF but rather enhanced activation of MAPKs and secretion of proinflammatory cytokines upon stimulation with LPS. Enhanced inflammation depended on autocrine TNF secretion and WT TNFR1 in mouse and human myeloid cells but not in fibroblasts. Heterozygous TNFR1-mutant mice were hypersensitive to LPS-induced septic shock, whereas homozygous TNFR1-mutant mice resembled TNFR1-deficient mice and were resistant to septic shock. Thus WT and mutant TNFR1 act in concert from distinct cellular locations to potentiate inflammation in TRAPS. These findings establish a mechanism of pathogenesis in autosomal dominant diseases where full expression of the disease phenotype depends on functional cooperation between WT and mutant proteins and also may explain partial responses of TRAPS patients to TNF blockade.

Spontaneous clearance of chronic hepatitis C virus infection is associated with appearance of neutralizing antibodies and reversal of T-cell exhaustion.

BACKGROUND: Hepatitis C virus (HCV) readily establishes chronic infection with exhaustion of HCV-specific T cells and escape from neutralizing antibodies. Spontaneous recovery from chronic infection is rare and has never to our knowledge been studied immunologically. METHODS: We prospectively studied, from prior to infection through >2 years of follow-up, cytokines, HCV-specific T cells, and antibodies, as well as viral sequence evolution in a white male who spontaneously cleared HCV genotype 1a after 65 weeks. RESULTS: Significant alanine aminotransferase and plasma cytokine elevation and broad HCV-specific T-cell responses did not result in HCV clearance in the acute phase. Frequency and effector function of HCV-specific T cells decreased thereafter, and HCV titers stabilized as is typical for the chronic phase. HCV clearance after 65 weeks followed the appearance of neutralizing antibodies at week 48 and was associated with reversal of HCV-specific T-cell exhaustion, as evidenced by reduced programmed death-1 (PD-1) expression and improved T-cell function. Clearance occurred without inflammation or superinfection with hepatitis B virus, human cytomegalovirus virus, influenza, and Epstein-Barr virus. CONCLUSIONS: T-cell exhaustion is reversible at least in the first 2 years of chronic HCV infection, and this reversion in conjunction with neutralizing antibodies may clear HCV. These findings are relevant for immunotherapy of chronic infections.

Serum neutralization activity declines but memory B cells persist after cure of chronic hepatitis C.

The increasing incidence of hepatitis C virus (HCV) infections underscores the need for an effective vaccine. Successful vaccines to other viruses generally depend on a long-lasting humoral response. However, data on the half-life of HCV-specific responses are lacking. Here we study archived sera and mononuclear cells that were prospectively collected up to 18 years after cure of chronic HCV infection to determine the role of HCV antigen in maintaining neutralizing antibody and B cell responses. We show that HCV-neutralizing activity decreases rapidly in potency and breadth after curative treatment. In contrast, HCV-specific memory B cells persist, and display a restored resting phenotype, normalized chemokine receptor expression and preserved ability to differentiate into antibody-secreting cells. The short half-life of HCV-neutralizing activity is consistent with a lack of long-lived plasma cells. The persistence of HCV-specific memory B cells and the reduced inflammation after cure provide an opportunity for vaccination to induce protective immunity against re-infection.

Clearance of pegylated interferon by Kupffer cells limits NK cell activation and therapy response of patients with HBV infection.

Pegylated interferon-α (PEG-IFN-α), where IFN-α is attached to polyethylene glycol (PEG), is an approved treatment for chronic hepatitis B virus (HBV) infection, a disease that causes liver-related morbidity and mortality in 257 million people worldwide. It is unknown why only a minority of patients respond to PEG-IFN-α. Using sequential blood samples and liver biopsies of patients with chronic HBV infection before, during, and after PEG-IFN-α treatment, we find that patients with early natural killer (NK) cell activation after PEG-IFN-α injection experienced greater liver inflammation, lysis of HBV-infected hepatocytes, and hepatitis B surface antigen (HBsAg) decline than those without. NK cell activation was associated with induction of interferon-stimulated genes and determined by PEG-IFN-α pharmacokinetics. Patients with delayed increases in PEG-IFN-α concentrations had greater amounts of PEG-specific immunoglobulin M (IgM) immune complexes in the blood and more PEG and IgM detected in the liver than patients with rapid increase in PEG-IFN-α concentration. This was associated with reduced NK cell activation. These results indicate that the immunomodulatory functions of PEG-IFN-α, particularly activation of NK cells, play a pivotal role in the response to treatment and further demonstrate that these functions are affected by PEG-IFN-α pharmacokinetics. Accelerated clearance of antibody-complexed pegylated drugs by Kupffer cells may be important beyond the field of HBV therapeutics. Thus, these findings may contribute to improving the efficacy of pegylated drugs that are now being developed for other chronic diseases and cancer.

Developing a core set of outcome measure domains to study Raynaud's phenomenon and digital ulcers in systemic sclerosis: Report from OMERACT 2020.

Raynaud's phenomenon (RP) and digital ulcers (DUs) are important disease manifestations of systemic sclerosis (SSc) that can lead to significant pain and disability. It is essential when studying these disease features to utilize outcome measures that fully evaluate the complexities of RP and DUs . The Outcome Measures in Rheumatology (OMERACT) Vascular Disease in SSc Working Group is applying the OMERACT filter 2.1 to identify a core set of disease domains that encompass the full burden of SSc-related RP and DUs. Progress to date and future research plans were presented during a Special Interest Group held in December 2020.

A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus.

Chronic infection with hepatitis C virus (HCV) is a common cause of liver cirrhosis and cancer. We performed RNA sequencing in primary human hepatocytes activated with synthetic double-stranded RNA to mimic HCV infection. Upstream of IFNL3 (IL28B) on chromosome 19q13.13, we discovered a new transiently induced region that harbors a dinucleotide variant ss469415590 (TT or ΔG), which is in high linkage disequilibrium with rs12979860, a genetic marker strongly associated with HCV clearance. ss469415590[ΔG] is a frameshift variant that creates a novel gene, designated IFNL4, encoding the interferon-λ4 protein (IFNL4), which is moderately similar to IFNL3. Compared to rs12979860, ss469415590 is more strongly associated with HCV clearance in individuals of African ancestry, although it provides comparable information in Europeans and Asians. Transient overexpression of IFNL4 in a hepatoma cell line induced STAT1 and STAT2 phosphorylation and the expression of interferon-stimulated genes. Our findings provide new insights into the genetic regulation of HCV clearance and its clinical management.

Lighting the fires within: the cell biology of autoinflammatory diseases.

Autoinflammatory diseases are characterized by seemingly unprovoked pathological activation of the innate immune system in the absence of autoantibodies or autoreactive T cells. Discovery of the causative mutations underlying several monogenic autoinflammatory diseases has identified key regulators of innate immune responses. Recent studies have highlighted the role of misfolding, oligomerization and abnormal trafficking of pathogenic mutant proteins in triggering autoinflammation, and suggest that more common rheumatic diseases may have an autoinflammatory component. This coincides with recent discoveries of new links between endoplasmic reticulum stress and inflammatory signalling pathways, which support the emerging view that autoinflammatory diseases may be due to pathological dysregulation of stress-sensing pathways that normally function in host defence.

Mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in TNFR1-associated periodic syndrome (TRAPS).

Reactive oxygen species (ROS) have an established role in inflammation and host defense, as they kill intracellular bacteria and have been shown to activate the NLRP3 inflammasome. Here, we find that ROS generated by mitochondrial respiration are important for normal lipopolysaccharide (LPS)-driven production of several proinflammatory cytokines and for the enhanced responsiveness to LPS seen in cells from patients with tumor necrosis factor receptor-associated periodic syndrome (TRAPS), an autoinflammatory disorder caused by missense mutations in the type 1 TNF receptor (TNFR1). We find elevated baseline ROS in both mouse embryonic fibroblasts and human immune cells harboring TRAPS-associated TNFR1 mutations. A variety of antioxidants dampen LPS-induced MAPK phosphorylation and inflammatory cytokine production. However, gp91(phox) and p22(phox) reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits are dispensable for inflammatory cytokine production, indicating that NADPH oxidases are not the source of proinflammatory ROS. TNFR1 mutant cells exhibit altered mitochondrial function with enhanced oxidative capacity and mitochondrial ROS generation, and pharmacological blockade of mitochondrial ROS efficiently reduces inflammatory cytokine production after LPS stimulation in cells from TRAPS patients and healthy controls. These findings suggest that mitochondrial ROS may be a novel therapeutic target for TRAPS and other inflammatory diseases.

The Yersinia effector protein YpkA induces apoptosis independently of actin depolymerization.

The pathogenicity of the plague agent Yersinia pestis is largely due to the injection of effector proteins that potently block immune responses into host cells through a type III secretion apparatus. One Yersinia effector protein, YpkA, a putative serine/threonine kinase, has been reported to act by depolymerizing actin and disrupting actin microfilament organization. Using YpkA-GFP fusion proteins to directly visualize cells expressing YpkA, we found instead that YpkA triggered rapid cell death that can be blocked by caspase inhibitors and Bcl-xL, but was not dependent on caspase-8. The actin depolymerization promoted by YpkA was only seen in cells with other features of apoptosis, and was blocked by inhibiting apoptosis, indicating that actin filament disruption is likely to be a result, rather than a cause of YpkA-induced apoptosis. A region including aa 133-262 in YpkA was sufficient for inducing apoptosis independent of localization to the plasma membrane. These data suggest that YpkA can act as a direct inducer of cell death.

The transcription factor ZBP-89 controls generation of the hematopoietic lineage in zebrafish and mouse embryonic stem cells.

Hematopoietic development is closely linked to that of blood vessels and the two processes are regulated in large part by transcription factors that control cell fate decisions and cellular differentiation. Both blood and blood vessels derive from a common progenitor, termed the hemangioblast, but the factor(s) specifying the development and differentiation of this stem cell population into the hematopoietic and vascular lineages remain ill defined. Here, we report that knockdown of the Krüppel-like transcription factor ZBP-89 in zebrafish embryos results in a bloodless phenotype, caused by disruption of both primitive and definitive hematopoiesis, while leaving primary blood vessel formation intact. Injection of ZBP-89 mRNA into cloche zebrafish embryos, which lack both the hematopoietic and endothelial lineages, rescues hematopoiesis but not vasculogenesis. Injection of mRNA for Stem Cell Leukemia (SCL), a transcription factor that directs hemangioblast development into blood cell precursors, rescues the bloodless phenotype in ZBP-89 zebrafish morphants. Forced expression of ZBP-89 induces the expansion of hematopoietic progenitors in wild-type zebrafish and in mouse embryonic stem cell cultures but inhibits angiogenesis in vivo and in vitro. These findings establish a unique regulatory role for ZBP-89, positioned at the interface between early blood and blood vessel development.

New strategies for immunosuppression: interfering with cytokines by targeting the Jak/Stat pathway.

PURPOSE OF REVIEW: Numerous immunosuppressants are available, but their adverse effects related to actions on nonlymphoid cells is problematic. Cytokines are key regulators of immune and inflammatory responses, and blocking their actions has become an important modality in treating autoimmune disorders. This review will discuss strategies to develop novel immunosuppressants that arise from advances in the understanding of cytokine signaling. RECENT FINDINGS: It is now recognized that large number of cytokines exert their effect by binding to receptors that activate the Janus kinase/signal transducer and activator of transcription pathway, so targeting intracellular signaling pathways is a logical strategy. A selective inhibitor of Janus kinase 3 has now been generated and is effective for transplant rejection in nonhuman primates and other models. Advances have also been made in understanding the functions of Stat family transcription factors, and approaches to interfering with the action of these DNA binding proteins are being devised. In addition, the identification of negative regulators of cytokine signaling offers additional therapeutic opportunities. SUMMARY: A selective inhibitor of Janus kinase 3 has now been generated and likely represents a new class of effective immunosuppressants. Strategies for targeting signal transducers and activators of transcription pathway are being intensively studied at present and hold potential promise. Multiple endogenous mechanisms exist for negatively regulating cytokine signaling; whether novel therapies can be devised that exploit these mechanisms remains to be determined.

The zinc finger transcription factor ZBP-89 is a repressor of the human beta 2-integrin CD11b gene.

Integrin CD11b is a differentiation marker of the myelomonocytic lineage and an important mediator of inflammation. Expression of the CD11b gene is transcriptionally induced as myeloid precursors differentiate into mature cells, then drops as monocytes further differentiate into macrophages. Previous studies have identified elements and factors involved in the transcriptional activation of the CD11b gene during myeloid differentiation, but no data exist regarding potential down-regulatory factors, especially in the later stages of differentiation. Using 2 copies of a GC-rich element (-141 to -110) in the CD11b promoter, we probed a cDNA expression library for interacting proteins. Three clones were identified among 9.1 million screened, all encoding the DNA-binding domain of the zinc finger factor ZBP-89. Overexpression of ZBP-89 in the monocyte precursor cell line U937 reduced CD11b promoter-driven luciferase activity when U937 cells were induced to differentiate into monocytelike cells using phorbol esters. To identify the differentiation stage at which ZBP-89 repression of the CD11b gene is exerted, the protein level of ZBP-89 was correlated with that of CD11b mRNA in differentiating U937 as well as in normal human monocytes undergoing in vitro differentiation into macrophages. A clear inverse relationship was observed in the latter but not the former state, suggesting that ZBP-89 represses CD11b gene expression during the further differentiation of monocytes into macrophages.

During differentiation of the monocytic cell line U937, Pur alpha mediates induction of the CD11c beta 2 integrin gene promoter.

CD11c is a member of the beta(2) integrin family of adhesion molecules that, together with CD18, forms a heterodimeric receptor on the surface of myeloid, NK, dendritic, and certain leukemic, lymphoma, and activated lymphoid cells. Monocytic differentiation is associated with an induction of both CD11c and CD18 gene expression. The resulting CD11c/CD18 receptor mediates firm adhesion to the vascular endothelium, transendothelial migration, chemotaxis, and phagocytosis. Monocytic differentiation can be mimicked in vitro by treatment of the promonocytic cell line U937 with PMA. Recently, we reported that in U937 cells, expression of the CD11c gene is controlled by an unidentified transcription factor that binds ssDNA. This finding suggested that DNA secondary structure plays an important role in controlling the CD11c gene and prompted us to search for additional ssDNA-binding activities with which this gene interacts. In this study, we report that in U937 cells, expression of the CD11c gene is mediated by the ssDNA-binding protein Puralpha. During PMA-induced differentiation, the ability of Puralpha to activate the CD11c promoter in U937 cells increases, as does that of Sp1. Together, these increases in the functional activity of both Puralpha and Sp1 combine to induce CD11c expression.

CD11c gene expression in hairy cell leukemia is dependent upon activation of the proto-oncogenes ras and junD.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative disease, the cause of which is unknown. Diagnostic of HCL is abnormal expression of the gene that encodes the beta2 integrin CD11c. In order to determine the cause of CD11c gene expression in HCL the CD11c gene promoter was characterized. Transfection of the CD11c promoter linked to a luciferase reporter gene indicated that it is sufficient to direct expression in hairy cells. Mutation analysis demonstrated that of predominant importance to the activity of the CD11c promoter is its interaction with the activator protein-1 (AP-1) family of transcription factors. Comparison of nuclear extracts prepared from hairy cells with those prepared from other cell types indicated that hairy cells exhibit abnormal constitutive expression of an AP-1 complex containing JunD. Functional inhibition of AP-1 expressed by hairy cells reduced CD11c promoter activity by 80%. Inhibition of Ras, which represents an upstream activator of AP-1, also significantly inhibited the CD11c promoter. Furthermore, in the hairy cell line EH, inhibition of Ras signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinases 1 and 2 (MEK1/2) reduced not only CD11c promoter activity but also reduced both CD11c surface expression and proliferation. Expression in nonhairy cells of a dominant-positive Ras mutant activated the CD11c promoter to levels equivalent to those in hairy cells. Together, these data indicate that the abnormal expression of the CD11c gene characteristic of HCL is dependent upon activation of the proto-oncogenes ras and junD.