IL-6 Amplifies TLR Mediated Cytokine and Chemokine Production: Implications for the Pathogenesis of Rheumatic Inflammatory Diseases

The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.     

Immunomodulatory Activity of a Novel,Synthetic Beta-glucan (β-glu6) in Murine Macrophages and Human Peripheral Blood Mononuclear Cells

Natural β-glucans extracted from plants and fungi have been used in clinical therapies since the late 20th century. However, the heterogeneity of natural β-glucans limits their clinical applicability. We have synthesized β-glu6, which is an analog of the lentinan basic unit, β-(1→6)-branched β-(1→3) glucohexaose, that contains an α-(1→3)-linked bond. We have demonstrated the stimulatory effect of this molecule on the immune response, but the mechanisms by which β-glu6 activates innate immunity have not been elucidated. In this study, murine macrophages and human PBMCs were used to evaluate the immunomodulatory effects of β-glu6. We showed that β-glu6 activated ERK and c-Raf phosphorylation but suppressed the AKT signaling pathway in murine macrophages. Additionally, β-glu6 enhanced the secretion of large levels of cytokines and chemokines, including CD54, IL-1α, IL-1β, IL-16, IL-17, IL-23, IFN-γ, CCL1, CCL3, CCL4, CCL12, CXCL10, tissue inhibitor of metalloproteinase-1 (TIMP-1) and G-CSF in murine macrophages as well as IL-6, CCL2, CCL3, CCL5, CXCL1 and macrophage migration inhibitory factor (MIF) in human PBMCs. In summary, it demonstrates the immunomodulatory activity of β-glu6 in innate immunity.     

Effect of Pregnancy on Anti-HEV Antibody Titres,Plasma Cytokines and the Corresponding Gene Expression Levels in the PBMCs of Patients Presenting with Self-Recovering Clinical and Subclinical Hepatitis E

High mortality in pregnant women (PR) is a characteristic of hepatitis E in developing countries. To understand the pathogenesis of HEV infection in self-limiting disease during pregnancy, we compared clinical (PR-patients) and subclinical-HEV-infections in pregnant women in the first (SC-PR-1) and later (2nd and 3rd, SC-PR-2+3) trimesters with the respective healthy controls and acute non-PR patients. The SC-PR-2+3 exhibited lower ALT, bilirubin levels, anti-HEV-IgM/IgG titres than the acute-PR/non-PR-patients (p<0.05–0.0001). IFNγ/IL4ratios indicated Th2/Th1 bias in non-PR and PR-patients respectively. Raised levels of 10/20 plasma cytokines in the non-PR-patients reflect predominant inflammatory response, unaltered- IFNγ/reduced-IFNα responses and a robust chemokine secretion. On contrary, the acute-PR-patients exhibited drastic reduction in majority of the cytokines relative to in the non-PR-patients. Importantly, diminished or unaltered response was noted in the acute-PR-group when compared to the corresponding controls. The only exception was sIL2RA, increasing in both patient categories. Of the 14 genes evaluated, the expression of IFNγ/IL10/IL1A/IL7/CCL2/CCL3/CXCL8/CXCL10 was higher in the non-PR patients. Of these, the expression of IFNγ/IL10/IL1A/CCL2/CCL3/CXCL8 and, additionally, IL2/IL6/TNF genes was higher in the clinical-PRs. Almost identical pattern was noted in the control-PR-2+3 category indicating no influence of HEV infection. Comparison of patient-categories identified significant elevation of IFNγ(P<0.001), CCL2(p<0.01), CXCL8(P<0.05), IL1B(p<0.05) and IL10(P<0.0001) and decrease in CXCL10(<0.05) in the PR-patients. The results suggest antibody-dependent disease severity and impaired immune response in the PR patients. Higher expression of cytokine-genes in the PBMCs did not correlate with the plasma-cytokine levels in the PR-patients.     

The Effect of Lung Cancer on Cytokine Expression in Peripheral Blood Mononuclear Cells

The purpose of this study is to evaluate cytokine expression by peripheral blood mononuclear cells (PBMC) from stage I lung cancer patients and to confirm these expression patterns by exposing PBMCs to lung cancer cells in vitro. Five altered cytokines in stage I lung cancer patients (CCL3, IL8, IL1β, CXCL10, sIL2Rα) were identified in plasma from subjects (n = 15) before and after resection using a 30-plex panel protein assay. Gene expression studies using quantitative RT-qPCR were performed on PBMCs from stage I lung cancer patients (n = 62) before and after resection, and compared to non-cancer patients (n = 32) before and after surgery for benign disease. Co-culture experiments that exposed healthy donor PBMCs to lung cancer cells in vitro were performed to evaluate the effect on PBMC cytokine expression. PBMC gene expression of CCL3, IL8 and IL1β was higher in lung cancer patients compared to the same patients at each of four sequential timepoints after removal of their tumors, while CXCL10 and IL2Rα were essentially unchanged. This pattern was also detected when lung cancer patients were compared to non-cancer patients. When non-cancer patients underwent surgery for benign diseases, these cytokine expression changes were not demonstrable. Lung cancer cell lines, but not benign bronchial epithelial cells, induced similar changes in cytokine gene and protein expression by healthy donor PBMCs in an in vitro co-culture system. We conclude that PBMCs from stage I lung cancer patients possess distinct cytokine expression patterns compared to both non-cancer patients, and lung cancer patients following tumor removal. These expression patterns are replicated by healthy donor PBMCs exposed to lung cancer cell lines, but not benign bronchial epithelial cells in vitro. These findings have implications for understanding the immune response to lung cancer.     

Effects of Reticuloendotheliosis Virus Infection on Cytokine Production in SPF Chickens

Infection with reticuloendotheliosis virus (REV), a gammaretrovirus in the Retroviridae family, can result in immunosuppression and subsequent increased susceptibility to secondary infections. The effects of REV infection on expression of mRNA for cytokine genes in chickens have not been completely elucidated. In this study, using multiplex branched DNA (bDNA) technology, we identified molecular mediators that participated in the regulation of the immune response during REV infection in chickens. Cytokine and chemokine mRNA expression levels were evaluated in the peripheral blood mononuclear cells (PBMCs). Expression levels of interleukin (IL)-4, IL-10, IL-13 and tumor necrosis factor (TNF)-α were significantly up-regulated while interferon (IFN)-α, IFN-β, IFN-γ, IL-1β，IL-2, IL-3, IL-15, IL-17F, IL-18 and colony-stimulating factor (CSF)-1 were markedly decreased in PBMCs at all stages of infection. Compared with controls, REV infected chickens showed greater expression levels of IL-8 in PBMCs 21 and 28 days post infection. In addition, REV regulates host immunity as a suppressor of T cell proliferative responses. The results in this study will help us to understand the host immune response to virus pathogens.     

HBsAg Inhibits IFN-α Production in Plasmacytoid Dendritic Cells through TNF-α and IL-10 Induction in Monocytes

Type I Interferon (IFN) is one of the first lines of defense against viral infection. Plasmacytoid dendritic cells (pDCs) are professional IFN-α-producing cells that play an important role in the antiviral immune response. Previous studies have reported that IFN-α production is impaired in chronic hepatitis B (CHB) patients. However, the mechanisms underlying the impairment in IFN-α production are not fully understood. Here, we report that plasma-derived hepatitis B surface antigen (HBsAg) and HBsAg expressed in CHO cells can significantly inhibit toll like receptor (TLR) 9-mediated Interferon-α (IFN-α) production in peripheral blood mononuclear cells (PBMCs) from healthy donors. Further analysis indicated that monocytes participate in the inhibitory effect of HBsAg on pDCs through the secretion of TNF-α and IL-10. Furthermore, TLR9 expression on pDCs was down-regulated by TNF-α, IL-10 and HBsAg treatment. This down-regulation may partially explain the inhibition of IFN-α production in pDCs. In conclusion, we determined that HBsAg inhibited the production of IFN-α by pDCs through the induction of monocytes that secreted TNF-α and IL-10 and through the down-regulation of TLR9 expression on pDCs. These data may aid in the development of effective antiviral treatments and lead to the immune control of the viral infections.     

Inflammatory Stress on Autophagy in Peripheral Blood Mononuclear Cells from Patients with Alzheimer's Disease during 24 Months of Follow-Up

Recent findings indicate that microglia in Alzheimer’s disease (AD) is senescent whereas peripheral blood mononuclear cells (PBMCs) could infiltrate the brain to phagocyte amyloid deposits. However, the molecular mechanisms involved in the amyloid peptide clearance remain unknown. Autophagy is a physiological degradation of proteins and organelles and can be controlled by pro-inflammatory cytokines. The purpose of this study was to evaluate the impact of inflammation on autophagy in PBMCs from AD patients at baseline, 12 and 24 months of follow-up. Furthermore, PBMCs from healthy patients were also included and treated with 20 μM amyloid peptide 1–42 to mimic AD environment. For each patient, PBMCs were stimulated with the mitogenic factor, phytohaemagglutin (PHA), and treated with either 1 μM C16 as an anti-inflammatory drug or its vehicle. Autophagic markers (Beclin-1, p62/sequestosome 1 and microtubule-associated protein-light chain 3: LC3) were quantified by western blot and cytokines (Interleukin (IL)-1β, Tumor necrosis Factor (TNF)-α and IL-6) by Luminex X-MAP^® technology. Beclin-1 and TNF-α levels were inversely correlated in AD PBMCs at 12 months post-inclusion. In addition, Beclin-1 and p62 increased in the low inflammatory environment induced by C16. Only LC3-I levels were inversely correlated with cognitive decline at baseline. For the first time, this study describes longitudinal changes in autophagic markers in PBMCs of AD patients under an inflammatory environment. Inflammation would induce autophagy in the PBMCs of AD patients while an anti-inflammatory environment could inhibit their autophagic response. However, this positive response could be altered in a highly aggressive environment.     

Interleukin-17A- or tumor necrosis factor α-mediated increase in proliferation of T cells cocultured with synovium-derived mesenchymal stem cells in rheumatoid arthritis

Introduction

Mesenchymal stem cells (MSCs) represent promising applications in rheumatoid arthritis (RA). However, the inflammatory niche in the RA synovium could adversely affect MSC function. This study was designed to investigate biologic and immunologic properties of synovium-derived MSCs (SMSCs) in RA, with particular focus on whether cytokines can mediate increase of proliferation of T cells cocultured with SMSCs in RA.

Methods

Compared with SMSCs from eight healthy donors (HDs), SMSCs from 22 patients with RA (RAp) were evaluated. The methyl thiazolyl tetrazolium (MTT) assay was used to assess cell-population doubling and viability. Multipotentiality of SMSCs was examined by using appropriate culture conditions. Flow cytometry was used to investigate the marker phenotype of SMSCs. Immunomodulation potential of SMSCs was examined by mixed peripheral blood mononuclear cells (PBMCs) reactions, and then by PBMCs or synovial T cells with or without the addition of inflammatory cytokines (interleukin-17A (IL-17A), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)) after stimulation with phytohemagglutinin (PHA), respectively.

Results

SMSCs from RA patients (RA-SMSCs) showed normal population doubling, cell viability, multiple differentiation characteristics, and surface markers. In either mixed PBMC reactions or PBMC proliferation stimulated with PHA, RA-SMSCs showed normal immunomodulation function compared with SMSCs from healthy donors (HD-SMSCs). However, the increase in proliferation of T cells was observed when IL-17A and TNF-α were added alone or in combination.

Conclusions

Our data suggest that the inflammatory niche, especially these cytokines, may increase the proliferation of T cells cocultured with SMSCs in RA.     

Effect of In Vitro and In Vivo Anakinra on Cytokines Production in Schnitzler Syndrome

IL-1 receptor antagonist anakinra is usually highly efficient in Schnitzler syndrome (SS), a rare inflammatory condition associating urticaria, fever, and IgM monoclonal gammopathy. In this study, we aimed to assess lipopolysaccharide (LPS)-induced production of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs) before and after 1 month of anakinra in patients with SS. LPS-induced production of IL-1β, IL-6 and TNFα was assessed by enzyme-linked immunosorbent assay with and without anakinra in vitro, and before and after 1 month (in vivo condition) of treatment in 2 patients with SS. Spontaneous production of IL-1β, IL-6 and TNF-α by PBMCs was similar in the patients and the healthy controls and was almost undetectable. Stimulation with LPS caused a higher release of cytokines from the patients than from the healthy controls. Before in vivo anakinra start, in vitro adjunction of anakinra reduced the high LPS-induced production of IL-1β and TNFα in both patients and of IL-6 in one patient. After 1 month of treatment with anakinra, while the patients had dramatically improved, there was also a marked reduction in LPS-induced cytokines production, which was almost normalized in one patient. This study shows an abnormal LPS-induced inflammatory cytokines production in SS, which can be decreased or even normalized by in vitro and in vivo anakinra.     

Role of the Sympathetic Nervous System in Carbon Tetrachloride-Induced Hepatotoxicity and Systemic Inflammation

Carbon tetrachloride (CCl₄) is widely used as an animal model of hepatotoxicity and the mechanisms have been arduously studied, however, the contribution of the sympathetic nervous system (SNS) in CCl₄-induced acute hepatotoxicity remains controversial. It is also known that either CCl₄ or SNS can affect systemic inflammatory responses. The aim of this study was to establish the effect of chemical sympathectomy with 6-hydroxydopamine (6-OHDA) in a mouse model of CCl₄-induced acute hepatotoxicity and systemic inflammatory response. Mice exposed to CCl₄ or vehicle were pretreated with 6-OHDA or saline. The serum levels of aminotransferases and alkaline phosphatase in the CCl₄-poisoning mice with sympathetic denervation were significantly lower than those without sympathetic denervation. With sympathetic denervation, hepatocellular necrosis and fat infiltration induced by CCl₄ were greatly decreased. Sympathetic denervation significantly attenuated CCl₄-induced lipid peroxidation in liver and serum. Acute CCl₄ intoxication showed increased expression of inflammatory cytokines/chemokines [eotaxin-2/CCL24, Fas ligand, interleukin (IL)-1α, IL-6, IL-12p40p70, monocyte chemoattractant protein-1 (MCP-1/CCL2), and tumor necrosis factor-α (TNF-α)], as well as decreased expression of granulocyte colony-stimulating factor and keratinocyte-derived chemokine. The overexpressed levels of IL-1α, IL-6, IL-12p40p70, MCP-1/CCL2, and TNF-α were attenuated by sympathetic denervation. Pretreatment with dexamethasone significantly reduced CCl₄-induced hepatic injury. Collectively, this study demonstrates that the SNS plays an important role in CCl₄-induced acute hepatotoxicity and systemic inflammation and the effect may be connected with chemical- or drug-induced hepatotoxicity and circulating immune response.     

Effect of erythropoietin on primed leucocyte expression profile

Resistance to erythropoietin (EPO) affects a significant number of anaemic patients with end-stage renal disease. Previous reports suggest that inflammation is one of the major independent predictors of EPO resistance, and the effects of EPO treatment on inflammatory mediators are not well established. The aim of this study was to investigate EPO-induced modification to gene expression in primary cultured leucocytes. Microarray experiments were performed on primed ex vivo peripheral blood mononuclear cells (PBMCs) and treated with human EPO-α. Data suggested that EPO-α modulated genes involved in cell movement and interaction in primed PBMCs. Of note, EPO-α exerts anti-inflammatory effects inhibiting the expression of pro-inflammatory cytokine IL-8 and its receptor CXCR2; by contrast, EPO-α increases expression of genes relating to promotion of inflammation encoding for IL-1β and CCL8, and induces de novo synthesis of IL-1α, CXCL1 and CXCL5 in primed cells. The reduction in MAPK p38-α activity is involved in modulating both IL-1β and IL-8 expression. Unlike the induction of MAPK, Erk1/2 activity leads to upregulation of IL-1β, but does not affect IL-8 expression and release. Furthermore, EPO-α treatment of primed cells induces the activation of caspase-1 upstream higher secretion of IL-1β, and this process is not dependent on caspase-8 activation. In conclusion, our findings highlight new potential molecules involved in EPO resistance and confirm the anti-inflammatory role for EPO, but also suggest a plausible in vivo scenario in which the positive correlation found between EPO resistance and elevated levels of some pro-inflammatory mediators is due to treatment with EPO itself.     

Angiotensin receptor type 1 and endothelin receptor type A on immune cells mediate migration and the expression of IL-8 and CCL18 when stimulated by autoantibodies from systemic sclerosis patients

Introduction

Agonistic autoantibodies (Aabs) against the angiotensin II receptor type 1 (AT1R) and the endothelin receptor type A (ETAR) have been identified in patients with systemic sclerosis (SSc). In our present study, we examined the expression of the AT1R and the ETAR in human immune cells and the pathological effects mediated through these receptors by their corresponding Aabs.

Methods

Protein expression of AT1R and ETAR on peripheral blood mononuclear cells (PBMCs) from healthy individuals and SSc patients was analyzed using flow cytometry, and mRNA expression of both receptors in PBMCs from healthy donors was examined by real-time PCR. In addition, PBMCs from healthy donors were stimulated in vitro with affinity-purified immunoglobulin G (IgG) fractions from SSc patients positive for AT1R and ETAR Aabs, as well as with IgG from healthy donors serving as controls. Alterations in cell surface marker expression, cytokine secretion and chemotactic motility were analyzed using flow cytometry, enzyme-linked immunosorbent assays and chemotaxis assays, respectively. The results were correlated with the characteristics and clinical findings of the IgG donors.

Results

Both AT1R and ETAR were expressed on PBMCs in humans. Protein expression of both receptors was decreased in SSc patients compared with that of healthy donors and declined during the course of disease. IgG fractions of SSc patients positive for AT1R and ETAR Aabs induced T-cell migration in an Aab level–dependent manner. Moreover, IgG of SSc patients stimulated PBMCs to produce more interleukin 8 (IL-8) and chemokine (C-C motif) ligand 18 (CCL18) than did the IgG of healthy donors. All effects were significantly reduced by selective AT1R and ETAR antagonists. Statistical analysis revealed an association of SSc-IgG induced high IL-8 concentrations with an early disease stage and of high CCL18 concentrations with lung fibrosis onset and vascular complications in the respective IgG donors.

Conclusion

In our present study, we could demonstrate the expression of both AT1R and ETAR on human peripheral T cells, B cells and monocytes. The decreased receptor expression in SSc patients, the inflammatory and profibrotic effects upon Aab stimulation of PBMCs in vitro and the associations with clinical findings suggest a role for Aab-induced activation of immune cells mediated by the AT1R and the ETAR in the pathogenesis or even the onset of the disease.     

Increased Numbers of IL-7 Receptor Molecules on CD4+CD25?CD107a+ T-Cells in Patients with Autoimmune Diseases Affecting the Central Nervous System

Background

High content immune profiling in peripheral blood may reflect immune aberrations associated with inflammation in multiple sclerosis (MS) and other autoimmune diseases affecting the central nervous system.

Methods and Findings

Peripheral blood mononuclear cells from 46 patients with multiple sclerosis (MS), 9 patients diagnosed with relapsing remitting MS (RRMS), 13 with secondary progressive multiple sclerosis (SPMS), 9 with other neurological diseases (OND) and well as 15 healthy donors (HD) were analyzed by 12 color flow cytometry (TCRαβ, TCRγδ, CD4, CD8α, CD8β, CD45RA, CCR7, CD27, CD28, CD107a, CD127, CD14) in a cross-sectional study to identify variables significantly different between controls (HD) and patients (OND, RRMS, SPMS). We analyzed 187 individual immune cell subsets (percentages) and the density of the IL-7 receptor alpha chain (CD127) on 59 individual immune phenotypes using a monoclonal anti-IL-7R antibody (clone R34.34) coupled to a single APC molecule in combination with an APC-bead array. A non-parametric analysis of variance (Kruskal-Wallis test) was conducted in order to test for differences among the groups in each of the variables. To correct for the multiplicity problem, the FDR correction was applied on the p-values. We identified 19 variables for immune cell subsets (percentages) which allowed to segregate healthy individuals and individuals with CNS disorders. We did not observe differences in the relative percentage of IL-7R-positive immune cells in PBMCs. In contrast, we identified significant differences in IL-7 density, measured on a single cell level, in 2/59 variables: increased numbers of CD127 molecules on TCRαβ+CD4+CD25 (intermed) T-cells and on TCRαβ+CD4+CD25−CD107a+ T-cells (mean: 28376 Il-7R binding sites on cells from HD, 48515 in patients with RRMS, 38195 in patients with SPMS and 33692 IL-7 receptor binding sites on cells from patients with OND).

Conclusion

These data show that immunophenotyping represents a powerful tool to differentiate healthy individuals from individuals suffering from neurological diseases and that the number of IL-7 receptor molecules on differentiated TCRαβ+CD4+CD25−CD107a+ T-cells, but not the percentage of IL-7R-positive cells, segregates healthy individuals from patients with neurological disorders.     

Spleen Tyrosine Kinase (Syk) Mediates IL-1β Induction by Primary Human Monocytes during Antibody-enhanced Dengue Virus Infection

Approximately 500,000 people are hospitalized with severe dengue illness annually. Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is believed to contribute to the pathogenic cytokine storm described in severe dengue patients, but the precise signaling pathways contributing to elevated cytokine production are not elucidated. IL-1β is a potent inflammatory cytokine that is frequently elevated during severe dengue, and the unique dual regulation of IL-1β provides an informative model to study ADE-induced cytokines. This work utilizes patient-derived anti-DENV mAbs and primary human monocytes to study ADE-induced IL-1β and other cytokines. ADE of DENV serotype 2 (DENV-2) elevates mature IL-1β secretion by monocytes independent of DENV replication by 4 h postinoculation (hpi). Prior to this, DENV immune complexes activate spleen tyrosine kinase (Syk) within 1 hpi. Syk induces elevated IL1B, TNF, and IL6 mRNA by 2 hpi. Syk mediates elevated IL-1β secretion by activating ERK1/2, and both Syk and ERK1/2 inhibitors ablated ADE-induced IL-1β secretion. Maturation of pro-IL-1β during ADE requires caspase-1 and NLRP3, but caspase-1 is suboptimally increased by ADE and can be significantly enhanced by a typical inflammasome agonist, ATP. Importantly, this inflammatory Syk-ERK signaling axis requires DENV immune complexes, because DENV-2 in the presence of serotype-matched anti-DENV-2 mAb, but not anti-DENV-1 mAb, activates Syk, ERK, and IL-1β secretion. This study provides evidence that DENV-2 immune complexes activate Syk to mediate elevated expression of inflammatory cytokines. Syk and ERK may serve as new therapeutic targets for interfering with ADE-induced cytokine expression during severe dengue.     

The Role of Cryptococcus in the Immune System of Pulmonary Cryptococcosis Patients

Objectives

To investigate the role of Cryptococcus in the immune system of immunocompetent patients with pulmonary cryptococcosis (PC) by analysing the dynamic changes of patients’ immune status before and after antifungal therapy.

Methods

The level of the serum interferon-γ (IFN-γ) and interleukin (IL)-2, -4, -10 and -12 was measured before and after 6-months of treatment. Peripheral blood samples were obtained from 30 immunocompetent PC patients and 30 age- and gender-matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated and incubated with recombinant human IL-12 (rhIL-12) for 48 h. Then the concentrations of IFN-γ and IL-4 in the supernatant were analysed.

Results

Baseline serum IFN-γ level was significantly lower in the PC patients as compared with the control group (P < 0.001). The serum IL-2 and IFN-γ of PC patients were significantly increased after appropriate treatments (P < 0.05 and P < 0.001 when compared to their baseline levels). The productions of IFN-γ in the culture supernatant of PBMCs showed no significant difference between the control and PC patients both before and after antifungal treatments. RhIL-12 is a potent stimulus for IFN-γ production. Culture PBMCs collected from PC patients before treatments had a smaller increase of IFN-γ production in the present of rhIL-12 than the control (P < 0.01); PBMCs from PC patients completing 6-months of treatment showed a comparable increase of IFN-γ production by rhIL-12 stimulation to the control group.

Conclusions

In apparently immunocompetent patients with PC, a normalization of serum IFN-γ was achieved after recovery from infection. This suggests that Cryptococcus infection per se can suppress the immune system and its elimination contributes to the reestablishment of an immune equilibrium.     

IL-4 Receptor-Alpha-Dependent Control of Cryptococcus neoformans in the Early Phase of Pulmonary Infection

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα–dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα^−/− mice unexpectedly show decreased fungal control early upon infection with C. neoformans, whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα^−/− mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα^−/− mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase.     

Effects of the Commercial Flame Retardant Mixture DE-71 on Cytokine Production by Human Immune Cells

IntroductionAlthough production of polybrominated diphenyl ethers (PBDEs) is now banned, release from existing products will continue for many years. The PBDEs are assumed to be neurotoxic and toxic to endocrine organs at low concentrations. Their effect on the immune system has not been investigated thoroughly. We aimed to investigate the influence of DE-71 on cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with Escherichia Coli lipopolysaccharide (LPS) or phytohaemagglutinin-L (PHA-L).ResultsAt non-cytotoxic concentrations (0.01–10 μg/mL), DE-71 significantly enhanced secretion of IL-1β, IL-6, CXCL8, IL-10, and TNF-α (p<0.001–0.019; n = 6) from LPS-stimulated PBMCs. IFN-γ, TNF-α, IL-17A, and IL-17F (p = <0.001–0.043; n = 6) secretion were enhanced from PHA-L-stimulated PBMCs as well. Secretion of IL-1β, IL-2, IL-10, IL-8 and IL-6 was not significantly affected by DE-71.ConclusionsWe demonstrate an enhancing effect of DE-71 on cytokine production by normal human PBMCs stimulated with LPS or PHA-L ex vivo.