Tick saliva increases production of three chemokines including monocyte chemoattractant protein‐1, a histamine‐releasing cytokine

The effect of Ixodes ricinus tick saliva on the production of various cytokines and chemokines by mouse splenocytes was tested by a cytokine array. We demonstrated a strong upregulation of three chemokines, monocyte chemoattractant protein‐1 (MCP‐1), thymus‐derived chemotactic agent 3 (TCA‐3) and macrophage inflammatory protein 2 (MIP‐2). MCP‐1 could be induced by tick saliva itself. While TCA‐3 and MIP‐2 are engaged in Th2 polarization of the host immune response associated with tick feeding, MCP‐1 may act as a histamine release factor, increasing blood flow into the feeding lesion thus facilitating tick engorgement in the late, rapid feeding phase.     

Effect of tick saliva on signalling pathways activated by TLR-2 ligand and Borrelia afzelii in dendritic cells

Dendritic cells are a sentinel in defending against pathogens and tick saliva facilitates transmission of tick-borne pathogens by modulating the host immune response. The maturation of dendritic cells is inhibited by tick saliva. To elucidate the mechanism of this inhibition, we tested the impact of Ixodes ricinus tick saliva on signalling pathways activated by Toll-like receptor (TLR-2) ligand and Borrelia afzelii in spleen dendritic cells. The activation of nuclear factor-κB (NF-κB) p65 and phosphatidylinositol-3 kinase (PI3K)/Akt pathways was decreased by tick saliva upon both TLR-2 and Borrelia stimulation. Among the mitogen-activated protein kinases (MAPK), the activation of extracellular matrix-regulated kinase (Erk1/2) was suppressed by tick saliva, but not p38. In response to spirochaetes, the amount of TNF-α decreased in the presence of tick saliva which was mediated by selective suppression of Erk1/2, NF-κB and Akt as tick saliva mimicked the effect of their specific inhibitors, UO126, IKK-IV and LY294002, respectively. Saliva-induced enhancement of IL-10 was not observed in the presence of specific inhibitor of Protein Kinase A (PKA), H-89, suggesting the involvement of PKA pathway in IL-10 production. Our cumulative data show that tick saliva interferes with several signalling pathways, thus modulating the immune functions of dendritic cells.     

Increase in platelet count based on inosine triphosphatase genotype during interferon beta plus ribavirin combination therapy

Background and Aim: The inosine triphosphatase (ITPA) genotype is associated with ribavirin‐induced anemia and pegylated interferon α (PEG IFN‐α)‐induced platelet reduction during PEG IFN‐α plus ribavirin combination therapy. Natural IFN‐β plus ribavirin therapy is associated with increases in platelet counts during treatment. We investigated decreases in platelet counts according to ITPA genotype during natural IFN‐β/ribavirin therapy to determine if patients with low platelet counts were eligible for this combination therapy. Methods: A total of 187 patients with chronic hepatitis C received PEG IFN‐α/ribavirin or natural IFN‐β/ribavirin therapy. Decreases in platelet counts based on ITPA genotype were investigated during treatment through 24 weeks. Results: Platelet counts decreased during week 1 of PEG IFN‐α/ribavirin therapy, but increased during week 2, after which platelet counts decreased gradually. Platelet counts decreased until week 4 of natural IFN‐β/ribavirin therapy, after which platelet counts increased. Platelet counts after week 8 were higher relative to pretreatment platelet counts. Patients with the ITPA‐CC genotype showed a smaller decrease in platelet counts during natural IFN‐β/ribavirin therapy than those with the ITPA‐CA/AA genotype; platelet counts after week 8 of this therapy were higher than pretreatment platelet counts, regardless of pretreatment platelet counts. Multivariate logistic regression analyses showed that natural INF‐β/ribavirin therapy was the only significant independent predictor for an increase in platelets through week 8. Conclusion: Natural IFN‐β/ribavirin therapy is safe for patients with the ITPA‐CC genotype, even if their pretreatment platelet counts are low.     

Efficacy of the regimen using twice-daily β-interferon followed by the standard of care for chronic hepatitis C genotype 1b with high viral load

Aim: In patients with refractory genotype 1b chronic hepatitis C with high viral loads, we retrospectively compared the efficacy of standard of care treatment (SOC: combined PEG‐IFN‐α‐2b/ribavirin for 48 weeks) and a regimen in which 2 weeks of SOC induction was replaced by twice‐daily β‐interferon alone (IFN‐β induction therapy). Methods: Seventeen patients received the IFN‐β induction therapy plus SOC (IFN‐β induction group) and 13 patients received SOC alone (SOC group). Results: In the IFN‐β induction group and SOC group, early virological response (EVR) rates were 88.2% and 53.8%, respectively. The end of treatment rates were 100.0% and 92.3%, and sustained virological response (SVR) rates were 70.6% and 53.8%, respectively. By induction with IFN‐β, even in refractory cases, the high virus negative conversion rate in the early treatment phase and actions of pegylated IFN‐α‐2b and ribavirin in the maintenance treatment phase led to an additive effect. In the analysis of contributing factors, only the achievement of EVR was associated with a significant difference in SVR (P = 0.0011). The univariate logistic regression analysis showed that only IFN‐β treatment was associated with a significant difference in EVR (P = 0.0492, odds ratio = 6.248, 95% confidence interval = 1.026–40.252), whereas no significant factors were found in the multivariate analysis due to small samples. Conclusion: IFN‐β induction therapy with higher EVR might be beneficial for protease inhibitor‐refractory chronic hepatitis C patients.     

Baseline sensitivity of T cells to alpha‐IFN correlates with sustained virological response to IFN‐based triple therapy in HCV infection

Chronic infection with HCV is a public health problem with approximately 170 million people infected worldwide. Interferon alpha (IFNα) sensitivity in liver and IL28B genotype has been identified as important determinants of HCV clearance in the setting of pegylated interferon/ribavirin treatment. Herein, we explored IFNα sensitivity in PBMC from 21 healthy donors and 21 HCV‐infected patients treated with pegylated interferon/ribavirin and HCV nonstructural protein‐3 inhibitors (i.e. telaprevir/boceprevir). We explored phospho‐STAT1 level as read‐out for IFN signalling pathway activation in PBMC, T cells and monocytes and correlated results with virological response. We found that PBMC from healthy donors are desensitized to IFNα after priming and challenged with IFNα, with a subsequent decrease of phospho‐STAT1 and interferon‐stimulated genes. Furthermore, we show that CD3+ T cells, but not monocytes, become desensitized after 4 weeks of treatment, with a significant decrease of phospho‐STAT1 after ex vivo IFNα stimulation. Finally, we identified baseline phospho‐STAT1 level in CD3+ T cells as a potential biomarker of sustained virological response, regardless of the IL28B genotype. In the upcoming costly era of IFN‐sparing regimen, baseline IFNα sensitivity could act as biomarker to define cost‐effectiveness strategies of treatment by identifying patients who will or will not respond to IFN‐based treatments.     

The effect of Echinococcus granulosus on spleen cells and TGF‐β expression in the peripheral blood of BALB/c mice

Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus (E. granulosus) is a zoonotic parasitic disease. The effective immune evasion mechanisms of E. granulosus allow it to parasitize its hosts. However, the status of the innate and adaptive immune cells and their contributions to E. granulosus progression remain poorly understood. In this study, we aimed to determine the impact of E. granulosus infection on T cells, NK cell responses and TGF‐β expression during the early infection phase in BALB/c mice. In E. granulosus infections, there was an increasing tendency in the percentage of CD4⁺CD25⁺ T cells and CD4⁺Foxp3⁺ T cells and peripheral blood TGF‐β levels and relative expression of the Foxp3 gene. Moreover, there were a decreasing tendency in the percentage of NK cells and NK cell cytotoxicity and the expression of NKG2D on NK cells. The TGF‐β1/Smad pathway was activated by E. granulosus in mice. Above results can be reversed by the inhibitor SB‐525334 (potent activin receptor‐like kinase 5 inhibitor). These results suggest that the TGF‐β/Smad pathway plays an important role in changes of T‐cell or NK cell responses. These results may contribute to revealing the preliminary molecular mechanisms in establishing hydatid infection.     

Inhibitory effect of human interferon-beta-1a on activated rat and human hepatic stellate cells

Background and Aims: Hepatic stellate cells (HSC) are the primary cell type mediating hepatic fibrosis. Although known for its antiviral effects, the inhibitory effects of interferon‐beta (IFN‐β) on HSC treatment have not yet been established. Methods: Both human and rat activated HSC cell lines were incubated with increasing concentrations of recombinant human IFN‐β1a (rhIFN‐β1a) for 24, 48 or 72 h. The effects of rhIFN‐β1a on α‐smooth muscle actin (α‐SMA), collagen types I and III, transforming growth factor‐β1 (TGF‐β1), platelet‐derived growth factor‐BB (PDGF‐BB), and mothers against decapentaplegic homolog (Smad4, Smad7) expression in HSC were examined using Western blotting and immunocytochemistry. Proliferation of HSC was evaluated via bromodeoxyuridine assay. Results: rhIFN‐β1a treatment had a dose‐dependent, inhibitory effect on α‐SMA and collagen type I protein expression. In addition, rhIFN‐β1a decreased the expression of collagen type III, TGF‐β1, PDGF‐BB and Smad4 protein expression in HSC compared with untreated cells. We also observed increased Smad7 protein expression and decreased proliferation in rhIFN‐β1a‐treated HSC. Conclusions: Our data suggest that rhIFN‐β1a treatment decreased α‐SMA and collagen expression and inhibited the activation of HSC through the inhibition of the TGF‐β and PDGF pathways.     

Tamoxifen alleviates hepatitis C virus‐induced inhibition of both toll‐like receptor 7 and JAK‐STAT signalling pathways in PBMCs of infected Egyptian females

Summary. Hepatitis C virus (HCV) is a major health concern in Egypt being highly prevalent among Egyptians. The two genders experience different responses to HCV infection and show variations in response to interferon (IFN)‐based therapy that may be attributed to sex hormones. We previously demonstrated the suppressive effect of 17β‐estradiol (E2) on the expression of the IFN‐stimulated gene MxA in HCV‐infected peripheral blood mononuclear cells (PBMCs). The selective oestrogen receptor (ER) modulator Tamoxifen has been shown to have an antiviral effect against HCV, but its effect on the host immune response is unknown. We investigated the effect of Tamoxifen on the IFN signalling pathways in PBMCs of HCV‐infected Egyptian females. We pooled PBMCs and treated then with exogenous interferon alpha (IFNα) or the TLR7 ligand, Imiquimod, and quantified the relative expressions of MxA using RTqPCR. Studies were performed with and without Tamoxifen pretreatment. Pretreatment with Tamoxifen reversed the suppressive effect of E2 on the JAK‐STAT pathway in IFNα‐treated PBMCs as indicated by a significant increase in MxA expression (P = 0.05*). Tamoxifen pretreatment also significantly upregulated MxA expression in Imiquimod‐treated PBMCs (P = 0.0011**), an effect not ascribed to ER blocking nor to an upregulation in TLR7 expression because Tamoxifen showed no potentiating effect on the expression of the receptor. In conclusion, our findings reveal that Tamoxifen has immunomodulatory effects whereby it enhances the host IFN signalling pathways during HCV infection.     

Leishmania braziliensis amastigotes stimulate production of IL‐1β, IL‐6, IL‐10 and TGF‐β by peripheral blood mononuclear cells from nonendemic area healthy residents

Leishmania (Viannia) braziliensis causes cutaneous and mucosal leishmaniasis in several countries in Latin America. In mammals, the parasites live as amastigotes, interacting with host immune cells and stimulating cytokine production that will drive the type of the specific immune responses. Generation of Th17 lymphocytes is associated with tissue destruction and depends on IL‐1β, IL‐6, TGF‐β and IL‐23 production, whereas IL‐10 and TGF‐β are associated with tissue protection. Here, we evaluate whether amastigotes stimulate peripheral blood mononuclear cells (PBMCs) from healthy donors to produce the major cytokines responsible for the generation of Th17. Seven L. (V.) braziliensis isolates from patients with different clinical forms of leishmaniasis were expanded in interferon‐γ knockout mice to obtain amastigotes and in culture to get promastigotes. The parasites were used to stimulate PBMCs from healthy donors, and cytokine production was evaluated by ELISA or qPCR. Amastigotes and promastigotes induced IL‐10 production in PBMCs; however, only amastigotes induced IL‐1β, IL‐6 and TGF‐β. These data demonstrate for the first time that L. (V.) braziliensis amastigotes directly stimulate production of a unique pattern of cytokines that could contribute to the generation of Th17.     

Interferon signals and monocytic sensitization of the interferon‐γ signaling pathway in the peripheral blood of patients with rheumatoid arthritis

Objective

Both type I interferons (IFNα and IFNβ) and type II IFN (IFNγ) signal via pSTAT‐1. Immunohistochemistry and the gene expression signatures of rheumatoid arthritis (RA) synovial tissue suggest an activated IFN/STAT‐1 signaling pathway. The aim of this study was to determine the systemic activity of the IFN/STAT‐1 signaling pathway in the peripheral blood cells of patients with RA.

Methods

Fluorocytometry or quantitative polymerase chain reaction was used to measure the expression of STAT‐1, pSTAT‐1, and IFN‐inducible genes (monokine induced by interferon‐γ [MIG], interferon‐γ–inducible protein 10 [IP‐10], and 2′,5′‐oligoadenylate synthetase [OAS]) in the peripheral blood mononuclear cells (PBMCs) and purified CD14+ peripheral blood monocytes of patients with RA and healthy control subjects. PBMCs were also incubated for 48 hours with IFNs and several other cytokines to investigate influences on STAT‐1 levels. To examine the significance of STAT‐1 activation in RA monocytes after stimulation with IFNγ, the expression of pSTAT‐1 and of the IFNγ‐inducible chemokine MIG was measured using fluorocytometry.

Results

Levels of STAT‐1 were significantly increased in peripheral lymphocytes and monocytes from patients with RA compared with those from healthy control subjects. STAT‐1 levels correlated well with RA disease activity, as measured by the Disease Activity Score in 28 joints and the Clinical Disease Activity Index. Furthermore, STAT‐1 messenger RNA expression in RA CD14+ monocytes correlated with the expression of other IFN‐target genes, such as IP‐10, OAS, or MIG. In RA PBMCs, STAT‐1 expression was increased not only by IFNs but also by tumor necrosis factor. RA monocytes demonstrated a considerably higher increase in pSTAT‐1 and MIG levels upon IFNγ stimulation when compared with monocytes from control subjects, indicating that RA monocytes are more sensitive to IFNγ stimulation.

Conclusion

In addition to supporting the role of IFNs in systemic proinflammatory activity, the results of this study further suggest preactivation of the IFNγ/STAT‐1 signaling pathway, especially in RA monocytes.

     

Type I IFNs promote the early IFN‐γ response and the IL‐10 response in Leishmania mexicana infection

The protozoan parasite Leishmania mexicana causes chronic cutaneous disease in humans and most mouse strains. We previously showed that STAT4‐deficient mice, but not IL‐12p40‐deficient mice, have more parasites and progressively growing lesions unlike those of wild‐type mice, the lesions and parasite burdens of which plateau by 10–12 weeks post‐infection. This demonstrates a STAT4‐dependent, IL‐12/IL‐23‐independent pathway of parasite control. Type I IFNs are important in viral and other infections and can activate STAT4. We found that IFN‐α/βR‐deficient mice have a nonpersistent, early IFN‐γ defect, and a persistent, early IL‐10 defect, without changes in serum IL‐12 or LN‐derived nitric oxide. We found less IL‐10 per cell in CD25⁺CD4⁺ T cells and possibly fewer IL‐10‐producing cells in the draining LN of IFN‐α/βR‐deficient vs. wild‐type mice. IFN‐α/βR‐deficient mice have chronic, nonprogressive disease, like wild‐type mice, suggesting that IL‐10 and IFN‐γ defects may balance each other. Our data indicate that although type I IFNs help promote early Th1 responses, they are not the missing activators of STAT4 responsible for partial control of L. mexicana. Also, the lack of lesion resolution in IFN‐α/βR‐deficient mice despite lower IL‐10 levels indicates that other pathways independent of T cell IL‐10 help prevent an IL‐12‐driven clearance of parasites.     

MicroRNA‐155 controls Toll‐like receptor 3‐ and hepatitis C virus‐induced immune responses in the liver

The hepatitis C virus (HCV) establishes persistent infections despite strong activation of the innate immune system through TLR3 and other sensors. Therefore, we analysed regulatory mechanisms of TLR3‐induced immune responses in nonparenchymal liver cells (NPCs). Effects of Interleukin‐10 (IL‐10), transforming growth factor beta (TGF‐β) and immunoregulatory miR‐155 on poly I:C‐activated murine (C57BL/6) Kupffer cells (KC) and sinusoidal endothelial cells (LSEC) were assessed in vitro. NPCs were assayed for inflammatory and antiviral cytokines and T‐cell (Balb/c)‐activating factors. Gene expression of miR‐155, IL‐10, TGF‐β and interferon sensitive genes (ISGs) in biopsies of patients with HCV was determined by qrt‐PCR. TLR3‐induced antiviral activity in murine NPCs was potently suppressed by IL‐10 and TGF‐β which correlated with decreased TLR3 expression and inhibition of NF‐κB and IRF‐3 activation. T‐cell activation, induced by TLR3‐activated NPCs, was also suppressed by IL‐10 and TGF‐β, which was associated with a down‐regulation of CD80 and CD86. Pretreatment with IL‐10 or TGF‐β suppressed TLR3‐induced miR‐155 expression, which itself positively regulated poly I:C‐mediated immune responses, thus counteracting IL‐10 or TGF‐β‐induced immunosuppression. In addition, hepatic expression of miR‐155 was elevated in chronically infected patients with HCV, was associated with an IL‐28B SNP (rs12979860) and was inversely correlated with HCV serum load and ISG expression levels. As miR‐155 is a key regulator of anti‐inflammatory mechanisms that control innate and adaptive hepatic immune responses during HCV infection, miR‐155 based therapies may represent a novel mechanism to control HCV in the future.     

Immunological interaction between Giardia cyst extract and experimental toxoplasmosis

Toxoplasmosis is mostly associated with other intestinal parasitic infections especially Giardia due to shared mode of peroral infection. Toxoplasma and Giardia induce a strong T‐helper 1‐ immune response. Our aim was to induce a protective immune response that results in significant impact on intestinal and extra‐intestinal phases of Toxoplasma infection. This study was conducted in experimental animals and assessment of Giardia cyst extract effect on Toxoplasma infection was investigated by histopathological examination of small intestine and brain, Toxoplasma cyst count and iNOS staining of the brain, measurement of IFN‐γ and TGF‐β in intestinal tissues. Results showed that the brain Toxoplasma cyst number was decreased in mice infected with Toxoplasma then received Giardia cyst extract as compared to mice infected with Toxoplasma only. This effect was produced because Giardia cyst extract augmented the immune response to Toxoplasma infection as evidenced by severe inflammatory reaction in the intestinal and brain tissues, increased levels of IFN‐γ and TGF‐β in intestinal tissues and strong iNOS staining of the brain. In conclusion, Giardia cyst extract generated a protective response against T. gondii infection. Therefore, Giardia antigen will be a suitable candidate for further researches as an immunomodulatory agent against Toxoplasma infection.     

Innate immunity evasion by Dengue virus

For viruses to productively infect their hosts, they must evade or inhibit important elements of the innate immune system, namely the type I interferon (IFN) response, which negatively influences the subsequent development of antigen-specific adaptive immunity against those viruses. Dengue virus (DENV) can inhibit both type I IFN production and signaling in susceptible human cells, including dendritic cells (DCs). The NS2B3 protease complex of DENV functions as an antagonist of type I IFN production, and its proteolytic activity is necessary for this function. DENV also encodes proteins that antagonize type I IFN signaling, including NS2A, NS4A, NS4B and NS5 by targeting different components of this signaling pathway, such as STATs. Importantly, the ability of the NS5 protein to bind and degrade STAT2 contributes to the limited host tropism of DENV to humans and non-human primates. In this review, we will evaluate the contribution of innate immunity evasion by DENV to the pathogenesis and host tropism of this virus.