Targeted Induction of Interferon-λ in Humanized Chimeric Mouse Liver Abrogates Hepatotropic Virus Infection

Background & Aims

The interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV).

Methods

This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice.

Results

Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways.

Conclusions

These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.     

Interferon and Ribavirin Combination Treatment Synergistically Inhibit HCV Internal Ribosome Entry Site Mediated Translation at the Level of Polyribosome Formation

Purpose

Although chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture.

Methods

The antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software.

Results

RBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation.

Conclusions

We demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH.     

Upregulation of SOCS-3 and PIAS-3 Impairs IL-12-Mediated Interferon-Gamma Response in CD56+ T Cells in HCV-Infected Heroin Users

Background

CD56⁺ T cells are abundant in liver and play an important role in host innate immunity against viral infections, including hepatitis C virus (HCV) infection, a common infection among heroin abusers. We thus investigated the in vivo impact of heroin use or heroin use plus HCV infection on the CD56⁺ T cell frequency and function.

Methodology/Principal Findings

A total of 37 heroin users with (17) or without (20) HCV infection and 17 healthy subjects were included in the study. Although there was no significant difference in CD56⁺ T cell frequency in PBMCs among three study groups, CD56⁺ T cells isolated from the heroin users had significantly lower levels of constitutive interferon-gamma (IFN-γ) expression than those from the normal subjects. In addition, when stimulated by interleukin (IL)-12, CD56⁺ natural T cells from HCV-infected heroin users produced significantly lower levels of IFN-γ than those from the normal subjects. This diminished ability to produce IFN-γ by CD56⁺ T cells was associated with the increased plasma HCV viral loads in the HCV-infected heroin users. Investigation of the mechanisms showed that although heroin use or heroin use plus HCV infection had little impact on the expression of the key positive regulators (IL-12 receptors, STAT-1, 3, 4, 5, JAK-2, and TYK-2) in IL-12 pathway, heroin use or heroin use plus HCV infection induced the expression of suppressor of cytokine signaling protein-3 (SOCS-3) and protein inhibitors of activated STAT-3 (PIAS-3), two key inhibitors of IL-12 pathway.

Conclusion/Significance

These findings provide compelling in vivo evidence that heroin use or heroin use plus HCV infection impairs CD56⁺ T cell-mediated innate immune function, which may account for HCV infection and persistence in liver.     

IFN-β Inhibits the Increased Expression of IL-9 during Experimental Autoimmune Uveoretinitis

Purpose

It has been shown that IL-9 plays a proinflammatory role in the pathogenesis of certain autoimmune diseases. This study was designed to investigate the possible role of IL-9 in the development of experimental autoimmune uveoretinitis (EAU) and the effect of IFN-β on its expression.

Methods

EAU was induced in B10RIII mice by immunization with interphotoreceptor retinoid-binding protein peptide 161–180 (IRBP_161–180). IFN-β was administered subcutaneously to IRBP_161–180 immunized mice every other day from day one before immunization to the end of the study. Splenocytes and draining lymph node (DLN) cells from EAU mice or control mice or EAU mice treated with IFN-β or PBS were stimulated with anti-CD3/CD28 or IRBP_161–180 for 3 days. Naïve T cells cultured under Th1 or Th17 polarizing conditions were incubated in the presence or absence of IFN-β for 4 days. Effector/memory T cells were activated by anti-CD3/CD28 in the presence or absence of IFN-β for 3 days. IFN-β-treated monocytes were cocultured with naïve T cells or effector/memory T cells for 3 days. Culture supernatants were collected and IL-9 was detected by ELISA.

Results

IL-9 expression in splenocytes and DLN cells was increased in EAU mice during the inflammatory phase and returned back to lower levels during the recovery phase. IFN-β in vivo treatment significantly inhibited EAU activity in association with a down-regulated expression of IL-9. In vitro polarized Th1 and Th17 cells both secreted IL-9 and the addition of IFN-β suppressed production of IL-9 by both Th subsets. Beside its effect on polarized Th cells, IFN-β also suppressed the secretion of IL-9 by effector/memory T cells. However, IFN-β-treated monocytes had no effect on the production of IL-9 when cocultured with naïve or effector/memory T cells.

Conclusion

IL-9 expression is increased during EAU which could be suppressed by IFN-β.     

Impaired Expression of Type I and Type II Interferon Receptors in HCV-Associated Chronic Liver Disease and Liver Cirrhosis

Purpose

Chronic Hepatitis C Virus (HCV)-infected patients with liver cirrhosis (LC) respond poorly to interferon-alpha (IFN-α) and ribavirin (RBV) combination therapy, but the reason for this is unclear. We previously reported that HCV-infection induces endoplasmic reticulum (ER) stress and autophagy response that selectively down regulates the type I IFN-α receptor-1 (IFNAR1) and RBV transporters (CNT1 and ENT1), leading to IFN-α/RBV resistance. The goal of this study is to verify whether an increase in ER stress and autophagy response is also associated with the reduced expression of IFNAR1 and RBV transporters in chronic HCV-infected patients.

Methods

Primary human hepatocytes (PHH) were infected with cell culture grown HCV particles (JFH-ΔV3-Rluc). HCV replication was confirmed by the detection of viral RNA by RT-qPCR and HCV-core protein by Western blotting. The ER stress and autophagy response and expression of IFN receptors and RBV transporters in HCV infected PHH and liver tissues derived from patients were measured by Western blotting.

Result

HCV infection of PHH showed impaired expression of IFNAR1, IFNγR1 (Type II IFN receptor) and RBV transporters but not IL10Rβ (Type III IFN-λ receptor). ER stress markers (BiP, IRE1α and peIF2α) and autophagy response (LC3II, Beclin 1 and ATG5) were induced in HCV infected chronic liver disease (CLD) and LC patients. Liver biopsies (CLD) show a 50% reduced expression of IFNAR1 and RBV transporters. Furthermore, the expression of IFNAR1 and RBV transporters was impaired in almost all LC patients.

Conclusion

HCV infection induces ER stress and autophagy response in infected PHH and chronically infected liver tissues. The expression of IFNAR1, IFNγR1 and RBV transporters were significantly impaired in CLD and cirrhotic livers. Our study provides a potential explanation for the reduced response rate of IFN-α and RBV combination therapy in HCV infected patients with liver cirrhosis.     

Protective immunity to Mycobacterium tuberculosis infection by chemokine and cytokine conditioned CFP-10 differentiated dendritic cells

Background

Dendritic cells (DCs) play major roles in mediating immune responses to mycobacteria. A crucial aspect of this is the priming of T cells via chemokines and cytokines. In this study we investigated the roles of chemokines RANTES and IP-10 in regulating protective responses from Mycobacterium tuberculosis (M. tb) 10 kDa Culture Filtrate Protein-10 (CFP-10) differentiated DCs (CFP10-DCs).

Methods and Findings

Infection of CFP10-DCs with mycobacteria down-modulated RANTES and IP-10 levels. Pathway specific microarray analyses showed that in addition to RANTES and IP-10, mycobacteria infected CFP10-DCs showed reduced expression of many Th1 promoting chemokines and chemokine receptors. Importantly, T cells co-cultured with RANTES and IP-10 conditioned CFP10-DCs mediated killing of mycobacteria from infected macrophages. Similarly, T cells recruited by RANTES and IP-10 conditioned CFP10-DCs mediated significant killing of mycobacteria from infected macrophages. IFN-gamma treatment of CFP10-DCs restored RANTES and IP-10 levels and T cells activated by these DCs mediated significant killing of virulent M. tb inside macrophages. Adoptive transfer of either RANTES and IP-10 or IL-12 and IFN-gamma conditioned CFP10-DCs cleared an established M. tb infection in mice. The extent of clearance was similar to that obtained with drug treatment.

Conclusions

These results indicate that chemokine and cytokine secretion by DCs differentiated by M. tb antigens such as CFP-10 play major roles in regulating protective immune responses at sites of infection.     

TNF-α and Tumor Lysate Promote the Maturation of Dendritic Cells for Immunotherapy for Advanced Malignant Bone and Soft Tissue Tumors

Background

Dendritic cells (DCs) play a pivotal role in the immune system. There are many reports concerning DC-based immunotherapy. The differentiation and maturation of DCs is a critical part of DC-based immunotherapy. We investigated the differentiation and maturation of DCs in response to various stimuli.

Methods

Thirty-one patients with malignant bone and soft tissue tumors were enrolled in this study. All the patients had metastatic tumors and/or recurrent tumors. Peripheral blood mononuclear cells (PBMCs) were suspended in media containing interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF). These cells were then treated with or without 1) tumor lysate (TL), 2) TL + TNF-α, 3) OK-432. The generated DCs were mixed and injected in the inguinal or axillary region. Treatment courses were performed every week and repeated 6 times. A portion of the cells were analyzed by flow cytometry to determine the degree of differentiation and maturation of the DCs. Serum IFN-γ and serum IL-12 were measured in order to determine the immune response following the DC-based immunotherapy.

Results

Approximately 50% of PBMCs differentiated into DCs. Maturation of the lysate-pulsed DCs was slightly increased. Maturation of the TL/TNF-α-pulsed DCs was increased, commensurate with OK-432-pulsed DCs. Serum IFN-γ and serum IL-12 showed significant elevation at one and three months after DC-based immunotherapy.

Conclusions

Although TL-pulsed DCs exhibit tumor specific immunity, TL-pulsed cells showed low levels of maturation. Conversely, the TL/TNF-α-pulsed DCs showed remarkable maturation. The combination of IL-4/GM-CSF/TL/TNF-α resulted in the greatest differentiation and maturation for DC-based immunotherapy for patients with bone and soft tissue tumors.     

CD100 Up-Regulation Induced by Interferon-α on B Cells Is Related to Hepatitis C Virus Infection

Objectives

CD100, also known as Sema4D, is a member of the semaphorin family and has important regulatory functions that promote immune cell activation and responses. The role of CD100 expression on B cells in immune regulation during chronic hepatitis C virus (HCV) infection remains unclear.

Materials and Methods

We longitudinally investigated the altered expression of CD100, its receptor CD72, and other activation markers CD69 and CD86 on B cells in 20 chronic HCV-infected patients before and after treatment with pegylated interferon-alpha (Peg-IFN-α) and ribavirin (RBV) by flow cytometry.

Results

The frequency of CD5⁺ B cells as well as the expression levels of CD100, CD69 and CD86 was significantly increased in chronic HCV patients and returned to normal in patients with sustained virological response after discontinuation of IFN-α/RBV therapy. Upon IFN-α treatment, CD100 expression on B cells and the two subsets was further up-regulated in patients who achieved early virological response, and this was confirmed by in vitro experiments. Moreover, the increased CD100 expression via IFN-α was inversely correlated with the decline of the HCV-RNA titer during early-phase treatment.

Conclusions

Peripheral B cells show an activated phenotype during chronic HCV infection. Moreover, IFN-α therapy facilitates the reversion of disrupted B cell homeostasis, and up-regulated expression of CD100 may be indirectly related to HCV clearance.     

Increased Baseline Proinflammatory Cytokine Production in Chronic Hepatitis C Patients with Rapid Virological Response to Peginterferon Plus Ribavirin

Background

Chronic hepatitis C (CHC) patients achieving rapid virological response (RVR) on PEG-IFN/ribavirin (P/R) therapy have high chance of sustained virological response (SVR). To analyze host immunological factors associated with RVR, viral kinetics, phenotype distribution and Th1/Th2 cytokine production by peripheral blood mononuclear cells (PBMC) were studied prior to and during P/R therapy.

Methods

TNF-α, IFN-γ, IL-2, IL-6, IL-4 and IL-10 production by PBMC were measured after Toll-like receptor 4 (TLR-4) or phorbol myristate acetate/Ionomycin stimulation in 20 healthy controls and in 50 CHC patients before receiving and during P/R therapy. RVR was achieved by 14, complete early virological response (cEVR) by 19 patients and 17 patients were null-responders (NR).

Results

Patients with RVR showed an increased baseline TNF-α and IL-6 production by TLR-4 activated monocytes and increased IFN-γ, decreased IL-4 and IL-10 production by lymphocytes compared to non-RVR patients. SVR was also associated with increased baseline TNF-α production and decreased IL-10 levels compared to patients who did not achieve SVR. Baseline IL-2 production was higher in cEVR compared to NR patients. Antiviral treatment increased TNF-α, IL-6 production by monocytes and IFN-γ secretion by lymphocytes and decreased IL-4 and IL-10 production by lymphocytes in cEVR compared to NR patients.

Conclusion

RVR was associated with increased baseline proinflammatory cytokine production by TLR-4 stimulated monocytes and by activated lymphocytes. In null-responders and in patients who did not achieve SVR both TLR-4 sensing function and proinflammatory cytokine production were impaired, suggesting that modulation of TLR activity and controlled induction of inflammatory cytokine production may provide further therapeutic strategy for CHC patients non-responding to P/R treatment.     

CD86 and IL-12p70 Are Key Players for T Helper 1 Polarization and Natural Killer Cell Activation by Toll-Like Receptor-Induced Dendritic Cells

Background

Dendritic cells (DCs) determine the activation and polarization of T cells via expression of costimulatory molecules and secretion of cytokines. The function of DCs derived from monocytes ex vivo strongly depends on the composition of the maturation cocktail used.

Methodology/Principal Findings

We analyzed the effect of costimulatory molecule expression and cytokine secretion by DCs on T and natural killer (NK) cell activation by conducting a head-to-head comparison of a Toll-like receptor (TLR) agonist-based cocktail with the standard combination of proinflammatory cytokines or IL-10 alone. We could show that TLR-induced DCs are characterized by a predominance of costimulatory over coinhibitory molecules and by high secretion of IL-12p70, but not IL-10. Functionally, these signals translated into an increase in IFN-γ secreting Th1 cells and a decrease in regulatory T cells. T cell activation and polarization were dependent on IL-12p70 and CD86, but remarkably not on CD80 signaling. By means of IL-12p70 secretion, only TLR-induced DCs activated NK cells.

Conclusions/Significance

TLR-matured DCs are highly suitable for application in immunotherapeutic strategies that rely on strong type 1 polarization and NK cell activation. Their effects particularly depend on high CD86 expression and IL-12p70 secretion.     

Molecular targeting of hepatocyte growth factor by an antagonist,NK4, in the treatment of rheumatoid arthritis

Introduction

Hepatocyte growth factor (HGF) is a potent proangiogenic molecule that induces neovascularization. The HGF antagonist, NK4, competitively antagonizes HGF binding to its receptor. In the present study, we determined the inhibitory effect of NK4 in a rheumatoid arthritis (RA) model using SKG mice.

Methods

Arthritis was induced in SKG mice by a single intraperitoneal injection of β-glucan. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was also injected intravenously at the time of or 1 month after β-glucan injection. Ankle bone destruction was examined radiographically. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Enzyme-linked immunosorbent assays were used to determine the serum levels of HGF, interferon γ (IFN-γ, interleukin 4 (IL-4) and IL-17 production by CD4⁺ T cells stimulated with allogeneic spleen cells.

Results

The intravenous injection of AdCMV.NK4 into SKG mice suppressed the progression of β-glucan-induced arthritis. Bone destruction was also inhibited by NK4 treatment. The histopathologic findings of the ankles revealed that angiogenesis, inflammatory cytokines and RANKL expression in synovial tissues were significantly inhibited by NK4 treatment. Recombinant NK4 (rNK4) proteins inhibited IFN-γ, IL-4 and IL-17 production by CD4⁺ T cells stimulated with allogeneic spleen cells.

Conclusions

These results indicate that NK4 inhibits arthritis by inhibition of angiogenesis and inflammatory cytokine production by CD4⁺ T cells. Therefore, molecular targeting of angiogenic inducers by NK4 can potentially be used as a novel therapeutic approach for the treatment of RA.     

Increased Cytokines Response in Patients with Tuberculosis Complicated with Chronic Obstructive Pulmonary Disease

Objectives

To explore the change and its significance of cytokines in patients with pulmonary tuberculosis complicated with COPD.

Methods

The immune function of 152 cases of pulmonary tuberculosis with COPD was detected to compare with 150 cases of patients with pulmonary tuberculosis, 157 cases of patients with COPD and 50 cases of healthy volunteers who were in the hospital during the same period. T lymphocyte cell population in peripheral blood was detected by flow cytometry. The serum levels of sIL-2R, IL-6, IFN-γ, TNF-α were measured using ELISA.

Results

The percentage of CD4+ T cells in TB patients with or without COPD and COPD patients without TB was significantly lower than that in control group. The percentage of CD4+ T cells in patients with TB and COPD was significantly lower than that in the non-COPD TB patients. The percentage of CD8+ T cells was higher in the TB patients group than that in control group. The CD4+/CD8+ ratio in the TB patients group was significantly lower than that in control group. The concentrations of sIL-2R, IL-6, TNF-α, IFN-γ in TB patients with or without COPD and COPD patients without TB were significantly higher than those in control group. In addition, sIL-2R, IL-6, TNF-α concentrations in the patients with TB and COPD were higher than those in the non-COPD TB patients. The concentrations of sIL-2R, IL-6, TNF-α, IFN-γ in COPD patients with TB were significantly higher than those in COPD patients without TB. There was a significant negative correlation between serum levels of TNF-α, IL-6 and FEV1 (%, predicted) in COPD without TB group.

Conclusions

The patients with pulmonary tuberculosis complicated with COPD were impaired in cellular immunity, and its extent of immune impairment is more serious than those of the patients with pulmonary tuberculosis and the patients with COPD.     

The Placental Protein Syncytin-1 Impairs Antiviral Responses and Exaggerates Inflammatory Responses to Influenza

Background

Pregnancy increases susceptibility to influenza. The placenta releases an immunosuppressive endogenous retroviral protein syncytin-1. We hypothesised that exposure of peripheral monocytes (PBMCs) to syncytin-1 would impair responses to H1N1pdm09 influenza.

Methods and Findings

Recombinant syncytin-1 was produced. PBMCs from non-pregnant women (n=10) were exposed to H1N1pdm09 in the presence and absence of syncytin-1 and compared to responses of PBMCs from pregnant women (n=12). PBMCs were characterised using flow cytometry, release of interferon (IFN)-α, IFN-λ, IFN-γ, IL-10, IL-2, IL-6 and IL-1β were measured by cytometric bead array or ELISA. Exposure of PBMCs to H1N1pdm09 resulted in the release of IFN-α, (14,787 pg/mL, 95% CI 7311-22,264 pg/mL) IFN-λ (1486 pg/mL, 95% CI 756-2216 pg/mL) and IFN-γ (852 pg/mL, 95% CI 193-1511 pg/mL) after 48 hours. This was significantly impaired in pregnant women (IFN-α; p<0.0001 and IFN-λ; p<0.001). Furthermore, in the presence of syncytin-1, PBMCs demonstrated marked reductions in IFN-α and IFN-λ, while enhanced release of IL-10 as well as IL-6 and IL-1β.

Conclusions

Our data indicates that a placental derived protein, syncytin-1 may be responsible for the heightened vulnerability of pregnant women to influenza.     

MDSCs Mediate Angiogenesis and Predispose Canine Mammary Tumor Cells for Metastasis via IL-28/IL-28RA (IFN-λ) Signaling

Background

Myeloid-derived suppressor cells (MDSCs) function in immunosuppression and tumor development by induction of angiogenesis in a STAT3-dependent manner. Knowledge of MDSC biology is mainly limited to mice studies, and more clinical investigations using spontaneous tumor models are required. Here we performed in vitro experiments and clinical data analysis obtained from canine patients.

Methods

Using microarrays we examined changes in gene expression in canine mammary cancer cells due to their co-culture with MDSCs. Further, using Real-time rt-PCR, Western blot, IHC, siRNA, angiogenesis assay and migration/invasion tests we examined a role of the most important signaling pathway.

Results

In dogs with mammary cancer, the number of circulating MDSCs increases with tumor clinical stage. Microarray analysis revealed that MDSCs had significantly altered molecular pathways in tumor cells in vitro. Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling. The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs. IL-28 secreted by MDSCs stimulates STAT3 in tumor cells, which results in increased expression of angiogenic factors and subsequent induction of angiogenesis by endothelial cells, epithelial-mesenchymal transition (EMT) and increased migration of tumor cells in vitro. Knockdown of IL-28RA decreased angiogenesis, tumor cell invasion and migration.

Conclusions

We showed for the first time that MDSCs secrete IL-28 (IFN-λ), which promotes angiogenesis, EMT, invasion and migration of tumor cells. Thus, IL-28 may constitute an interesting target for further therapies. Moreover, the similarity in circulating MDSC levels at various tumor clinical stages between canine and human patients indicates canines as a good model for clinical trials of drugs targeting MDSCs.     

Serum Inflammatory Cytokine Levels Correlate with Hand-Foot-Mouth Disease Severity: A Nested Serial Case-Control Study

Background

Hand-food-mouth disease (HFMD) cases can be fatal. These cases develop rapidly, and it is important to predict the severity of HFMD from mild to fatal and to identify risk factors for mild HFMD. The objective of this study was to correlate the levels of serum inflammatory cytokines with HFMD severity.

Methods

This study was designed as a nested serial case-control study. The data collected included general information, clinical symptoms and signs, laboratory findings and serum cytokine levels.

Results

The levels of IL-4, IL-6, IL-10, TNF-α and IFN-γ in patients with severe HFMD were significantly higher than in mild patients during the 2nd to 5th day after disease onset. The levels of IL-4, IL-6, IL-10 and IFN-γ increased from the 2nd day to the 4th day and later decreased. The levels of TNF-α were high on the first two days and subsequently decreased. The changes of IL-10, TNF-α and IFN-γ in the controls were similar for all cases. The levels of IL-4, IL-6 and IL-17 in the controls were not significantly different with the progression of HFMD.

Conclusions

Our findings indicate that the IL-4, IL-6, IL-10, TNF-α and IFN-γ levels correlate with HFMD severity.     

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction

Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines.

Methods

To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays.

Results

Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays.

Conclusion

Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.