Induction and regulation of matrix metalloproteinase-12in human airway smooth muscle cells

Background

The elastolytic enzyme matrix metalloproteinase (MMP)-12 has been implicated in the development of airway inflammation and remodeling. We investigated whether human airway smooth muscle cells could express and secrete MMP-12, thereby participating in the pathogenesis of airway inflammatory diseases.

Methods

Laser capture microdissection was used to collect smooth muscle cells from human bronchial biopsy sections. MMP-12 mRNA expression was analysed by quantitative real-time RT-PCR. MMP-12 protein expression and secretion from cultured primary airway smooth muscle cells was further analysed by Western blot. MMP-12 protein localization in bronchial tissue sections was detected by immunohistochemistry. MMP-12 activity was determined by zymography. The TransAM AP-1 family kit was used to measure c-Jun activation and nuclear binding. Analysis of variance was used to determine statistical significance.

Results

We provide evidence that MMP-12 mRNA and protein are expressed by in-situ human airway smooth muscle cells obtained from bronchial biopsies of normal volunteers, and of patients with asthma, COPD and chronic cough. The pro-inflammatory cytokine, interleukin (IL)-1β, induced a >100-fold increase in MMP-12 gene expression and a >10-fold enhancement in MMP-12 activity of primary airway smooth muscle cell cultures. Selective inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase and phosphatidylinositol 3-kinase reduced the activity of IL-1β on MMP-12, indicating a role for these kinases in IL-1β-induced induction and release of MMP-12. IL-1β-induced MMP-12 activity and gene expression was down-regulated by the corticosteroid dexamethasone but up-regulated by the inflammatory cytokine tumour necrosis factor (TNF)-α through enhancing activator protein-1 activation by IL-1β. Transforming growth factor-β had no significant effect on MMP-12 induction.

Conclusion

Our findings indicate that human airway smooth muscle cells express and secrete MMP-12 that is up-regulated by IL-1β and TNF-α. Bronchial smooth muscle cells may be an important source of elastolytic activity, thereby participating in remodeling in airway diseases such as COPD and chronic asthma.     

Regulation of Porcine Plasmacytoid Dendritic Cells by Cytokines

Plasmacytoid dendritic cells (pDC) are the most potent producers of type-I interferon (IFN) and represent the main interferon (IFN)-α source in response to many viruses. Considering the important roles played by type I IFN’s, not only as antiviral effectors but also as potent alarming cytokine of the immune system, we investigated how such responses are regulated by various cytokines. To this end, we stimulated enriched pDC in the presence or absence of particular cytokines with a strong activator, CpG DNA, or a weak activator of pDC, foot-and-mouth disease virus (FMDV). Alternatively, we pre-incubated pDC for 16 h before stimulation. The pro-inflammatory cytokines tested Interleukin (IL)-6, IL17A, tumour necrosis factor (TNF)-α did not influence IFN-α responses except TNF-α, which promoted responses induced by FMDV. The haematopoietic cytokines Fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) had enhancing effects on pDC activation at least in one of the protocols tested. IFN-β and IFN-γ were the most potent at enhancing FMDV-induced IFN-α, up to 10-fold. Interestingly, also the Th2 cytokine IL-4 was an efficient promoter of pDC activity, while IL-10 was the only negative regulator of IFN-α in pDC identified. The cytokines enhancing IFN-α responses also promoted pDC survival in cell culture with the exception of GM-CSF. Taken together this work illustrates how the cytokine network can influence pDC activation, a knowledge of relevance for improving vaccines and therapeutic interventions during virus infections, cancers and autoimmune diseases in which pDC play a role.     

Multiplex Genotyping of Cytokine Gene SNPs Using Fluorescence Bead Array

Single nucleotide polymorphisms (SNPs) of genes that affect cytokine production and function are known to influence the susceptibility and progression of immune-related conditions such as infection, autoimmune diseases, transplantation, and cancer. We established a multiplex genotyping method to analyze the SNPs of cytokine genes by combining the multiplex PCR and bead array platform. Thirteen cytokine gene regions, including 20 SNPs, were amplified, and allele-specific primer extension was performed in a single tube. High-quality allele-specific primers were selected for signals greater than 1000 median fluorescence intensity (MFI) for positive alleles, and less than 500 MFI for negative alleles. To select and improve the extension primers, modifications for the reverse direction, length or refractory were performed. 24 primers in the forward or reverse direction step and 12 primers in length or refractory modifications were selected and showed high concordance with results by nucleotide sequencing. Among the 13 candidate cytokine genes, the SNPs of 12 cytokine genes, including IL-1α, IL-1R, IL-1RA, IL-1β, IL-2, IL-4, IL-4Rα, IL-6, IL-10, IL-12, TGF-β1, and TNF-α, were successfully defined with the selected allele-specific primers in healthy Korean subjects. Our genotyping system provides a fast and accurate detection for SNPs of multiple cytokine genes to investigate their association with immune-related diseases and transplantation outcomes.     

Differential induction of innate memory in porcine monocytes by β-glucan or bacillus Calmette-Guerin

Innate immunomodulation via induction of innate memory is one mechanism to alter the host’s innate immune response to reduce or prevent disease. Microbial products modulate innate responses with immediate and lasting effects. Innate memory is characterized by enhanced (training) or depressed (tolerance) innate immune responses, including pro-inflammatory cytokine production, to secondary exposure following a priming event. To investigate the ability of β-glucans and bacillus Calmette-Guerin to induce innate training or tolerance in pig cells, porcine monocytes were cultured with priming agonist (β-glucans or bacillus Calmette-Guerin) then re-stimulated 5 d later with a heterologous microbial agonist to determine induction of innate memory. Priming with β-glucan from Saccharomyces cerevisiae depressed IL-1β and TNF-α cytokine responses to re-stimulation with LPS, indicative of a tolerized state. However, bacillus Calmette-Guerin priming induced a trained state in porcine monocytes, as LPS re-stimulation enhanced IL-1β and TNF-α gene expression and protein production. We present the first evidence of innate memory in pig monocytes, with bacillus Calmette-Guerin (training) or Saccharomyces cerevisiae β-glucan (tolerance). Induction of a trained or tolerized state in vitro is a first step to identify agonists to alter the innate immune system at the animal level with the intent of enhancing disease resistance.     

Interleukin-19 Acts as a Negative Autocrine Regulator of Activated Microglia

Activated microglia can exert either neurotoxic or neuroprotective effects, and they play pivotal roles in the pathogenesis and progression of various neurological diseases. In this study, we used cDNA microarrays to show that interleukin-19 (IL-19), an IL-10 family cytokine, is markedly upregulated in activated microglia. Furthermore, we found that microglia are the only cells in the nervous system that express the IL-19 receptor, a heterodimer of the IL-20Rα and IL-20Rβ subunits. IL-19 deficiency increased the production of such pro-inflammatory cytokines as IL-6 and tumor necrosis factor-α in activated microglia, and IL-19 treatment suppressed this effect. Moreover, in a mouse model of Alzheimer’s disease, we observed upregulation of IL-19 in affected areas in association with disease progression. Our findings demonstrate that IL-19 is an anti-inflammatory cytokine, produced by activated microglia, that acts negatively on microglia in an autocrine manner. Thus, microglia may self-limit their inflammatory response by producing the negative regulator IL-19.     

S100-β aggravates spinal cord injury via activation of M1 macrophage phenotype

Objectives:S100-β has been identified as a sensitive biomarker in central nervous system injuries. However, the functions and mechanisms of S100-β are unknown in spinal cord injury.Methods:Spinal cord injury (SCI) mouse model was generated by surgical operation, microglia activation model was established by inducing BV-2 cells with LPS. The SCI model was evaluated by Basso-Beattie-Bresnahan (BBB) behavioral score, HE staining, and Nissl staining. The expression level of S100-β was detected by qRT-PCR, western blot, and immunofluorescence. qRT-PCR and western blot were used to detect the expression of iNOS and CD16. Pro-inflammatory cytokines TNF-α and IL-1β levels were detected by qRT-PCR and ELISA.Results:The expression of IL-1β, TNF-α, iNOS, and CD16 increased at 3^rd day after SCI. In BV2 microglia, LPS treatment promoted the expression of S100-β, IL-1β, TNF-α, iNOS, and CD16. Knockdown of S100-β reduced the expression of iNOS stimulated by LPS. Over-expression of S100-β increased IL-1β and TNF-α, and S100-β inhibition suppressed IL-1β and TNF-α. In SCI mice, knockdown of S100-β attenuated the spinal cord injury and inhibited the expression of iNOS, IL-1β, and TNF-α.Conclusions:Down-regulation of S100-β could inhibit the pathogenesis of SCI and inhibit the activation of M1 macrophages. S100-β may be a useful diagnostic biomarker or therapeutic target for SCI.     

Zearalenone Mycotoxin Affects Immune Mediators,MAPK Signalling Molecules,Nuclear Receptors and Genome-Wide Gene Expression in Pig Spleen

The toxicity of zearalenone (ZEA) was evaluated in swine spleen, a key organ for the innate and adaptative immune response. Weaned pigs were fed for 18 days with a control or a ZEA contaminated diet. The effect of ZEA was assessed on wide genome expression, pro- (TNF-α, IL-8, IL-6, IL-1β, IFN-γ) and anti-inflammatory (IL-10, IL-4) cytokines, other molecules involved in inflammatory processes (MMPs/TIMPs), as well as signaling molecules, (p38/JNK1/JNK2-MAPKs) and nuclear receptors (PPARγ/NFkB/AP-1/STAT3/c-JUN). Microarray analysis showed that 46% of total number of differentially expressed genes was involved in cellular signaling pathway, 13% in cytokine network and 10% in the inflammatory response. ZEA increased expression and synthesis of pro- inflammatory (TNF-α, IL-8, IL-6, IL-1β) and had no effect on IFN-γ, IL-4 and IL-10 cytokines in spleen. The inflammatory stimulation might be a consequence of JNK pathway activation rather than of p-38MAPK and NF-kB involvement whose gene and protein expression were suppressed by ZEA action. In summary, our findings indicated the role of ZEA as an immune disruptor at spleen level.     

Cyclosporine A Enhances Th2 Bias at the Maternal-Fetal Interface in Early Human Pregnancy with Aid of the Interaction between Maternal and Fetal Cells

Our previous study has demonstrated that cyclosporine A (CsA) administration in vivo induces Th2 bias at the maternal-fetal interface, leading to improved murine pregnancy outcomes. Here, we investigated how CsA treatment in vitro induced Th2 bias at the human maternal-fetal interface in early pregnancy. The cell co-culture in vitro in different combination of component cells at the maternal-fetal interface was established to investigate the regulation of CsA on cytokine production from the interaction of these cells. It was found that interferon (IFN)-γ was produced only by decidual immune cells (DICs), and not by trophoblasts or decidual stromal cells (DSCs); all these cells secreted interleukin (IL)-4, IL-10, and tumor necrosis factor (TNF)-α. Treatment with CsA completely blocked IFN-γ production in DICs and inhibited TNF-α production in all examined cells. CsA increased IL-10 and IL-4 production in trophoblasts co-cultured with DSCs and DICs although CsA treatment did not affect IL-10 or IL-4 production in any of the cells when cultured alone. These results suggest that CsA promotes Th2 bias at the maternal-fetal interface by increasing Th2-type cytokine production in trophoblasts with the aid of DSCs and DICs, while inhibiting Th1-type cytokine production in DICs and TNF-α production in all investigated cells. Our study might be useful in clinical therapeutics for spontaneous pregnancy wastage and other pregnancy complications.     

Cytokine Profile in Human Eyes: Contribution of a New Cytokine Combination for Differential Diagnosis between Intraocular Lymphoma or Uveitis

Primary intraocular lymphoma (PIOL), also called primary vitreoretinal lymphomas, often masquerades as uveitis. This misdiagnosis can result in subsequent brain involvement and oculocerebral lymphoma (OCL). In this study, we sought to characterize the helper T-cell type 1 (Th1)/Th2 cytokine profile in vitreous samples from patients with PIOL, OCL, uveitis and controls with non-inflammatory disease. Vitreous and aqueous humor samples from 87 patients with PIOL (n = 30), OCL (n = 12), uveitis (n = 34), and retinal detachment (RD) without hemorrhage (n = 11) were analyzed and their concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were determined by flow cytometric bead arrays (CBA). The IL-10 levels determined by CBA were compared with those by ELISA. IL-10 concentrations measured by CBA and ELISA were highly correlated. IL-2, IL-4, and TNFα were not detected in any sample. The only cytokine detected at a significant level in samples from RD vitreous was IL-6. The IL-10/IL-6 ratio, as previously reported, was slightly higher in PIOL than in uveitis samples, but not for all patients. Cytokine profiles from PIOL and OCL samples did not differ. The combination of the IL-10/IL-6 and IL-10/IFNγ ratios was highly informative for discriminating PIOL/OCL from uveitis samples and for therapeutic follow up of PIOL. This strategy might be very helpful as an initial screening to rule out PIOL in patients thought to have uveitis.     

Dendritic Cell IL-1α and IL-1β Are Polyubiquitinated and Degraded by the Proteasome

IL-1α and β are key players in the innate immune system. The secretion of these cytokines by dendritic cells (DC) is integral to the development of proinflammatory responses. These cytokines are not secreted via the classical secretory pathway. Instead, 2 independent processes are required; an initial signal to induce up-regulation of the precursor pro-IL-1α and -β, and a second signal to drive cleavage and consequent secretion. Pro-IL-1α and -β are both cytosolic and thus, are potentially subject to post-translational modifications. These modifications may, in turn, have a functional outcome in the context of IL-1α and -β secretion and hence inflammation. We report here that IL-1α and -β were degraded intracellularly in murine bone marrow-derived DC and that this degradation was dependent on active cellular processes. In addition, we demonstrate that degradation was ablated when the proteasome was inhibited, whereas autophagy did not appear to play a major role. Furthermore, inhibition of the proteasome led to an accumulation of polyubiquitinated IL-1α and -β, indicating that IL-1α and -β were polyubiquitinated prior to proteasomal degradation. Finally, our investigations suggest that polyubiquitination and proteasomal degradation are not continuous processes but instead are up-regulated following DC activation. Overall, these data highlight that IL-1α and -β polyubiquitination and proteasomal degradation are central mechanisms in the regulation of intracellular IL-1 levels in DC.     

Cytokines in Acute Chikungunya

Introduction

Acute chikungunya (CHIKV) is predominantly an acute onset of excruciatingly painful, self-limiting musculoskeletal (MSK) arbovirus illness and this was further reported by us during the 2006 Indian epidemic [Chopra et al. Epidemiol Infect 2012]. Selected serum cytokines profile in subjects within one month of onset of illness is being presented.

Methods

Out of 509 clinical CHIKV cases (43% population) identified during a rural population survey, 225 subjects consented blood investigations. 132 examined within 30 days of febrile onset are the study cohort. Anti-CHIKV IgM and IgG antibodies tested by immunochromatography and indirect immunofluorescence respectively. Interferons (IFN)-α, -β and -γ, Interferon Gamma-Induced Protein-10 (CXCL-10/IP-10), Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Interleukin-13 (IL-13), Monocyte Chemoattractant Protein-1 (MCP-1), Interleukin–4 (IL-4) and Interleukin–10 (IL-10) performed by ELISA. Samples collected from neighboring community a year prior to the epidemic used as healthy controls.

Results

Seropositivity for anti-CHIKV IgM and IgG was 65% and 52% respectively. IFN-α, IFN-β, IFN-γ, CXCL10/IP-10 and IL-1β showed intense response in early acute phase. Cytokines (particularly TNF-α, MCP-1, IL-4, IL-6 and IL-10) was maximum in extended symptomatic phase and remained elevated in recovered subjects. Higher (p<0.05) IFN and IL-4 seen in patients seropositive for anti-CHIKV IgG. Elderly cases (≥65 years) showed elevated cytokines (except IFN) and anti-CHIKV antibodies near similar to younger subjects. Significant correlations (p<0.05) found between cytokines and clinical features (fatigue, low back ache, myalgia) and anti-CHIKV antibodies.

Conclusion

An intense cytokine milieu was evident in the early and immediate persistent symptomatic phase and in recovered subjects. Early persistent IgM and lower IgG to anti-CHKV and intense Th2 cytokine phenotype seem to be associated with delay in resolution of MSK symptoms. Intriguingly, maximum TNF-α, IL-6 and IL-13 with low anti-CHIKV IgM response found in subjects recovered from CHIKV within one month of illness.     

IL-17A is involved in diabetic inflammatory pathogenesis by its receptor IL-17RA

Interleukin (IL)-17A, a proinflammatory cytokine produced by T-helper (Th)17 cells, has been associated with autoimmune diseases. Type 1 diabetes (T1D) is caused either due to mutation of insulin gene or developed as an autoimmune disease. Studies have shown that IL-17A expression is upregulated in the pancreas in T1D patients and animal models. However, role or importance of IL-17A in T1D pathogenesis needs elucidation. Particularly, evidence for a direct injury of IL-17A to pancreatic β cells through activating IL-17 receptor A (IL-17RA) is lacking. Ins2^Akita (Akita) mouse, a T1D model with spontaneous mutation in insulin 2 gene leading to β-cell apoptosis, was crossed with IL-17A-knockout mouse and male IL-17A-deficient Akita mice were used. Streptozotocin, a pancreatic β-cell-specific cytotoxin, was employed to induce a diabetic model in MIN6 cells, a mouse insulinoma cell line. IL-17A expression in the pancreas was upregulated in both Akita and streptozotocin-induced diabetic mice. IL-17A-knockout Akita mice manifested reduced blood glucose concentration and raised serum insulin level. IL-17A deficiency also decreased production of the proinflammatory cytokines tumor necrosis factor (TNF)-α, IL-1β, and interferon (IFN)-γ in Akita mice. IL-17RA expression in MIN6 cells was upregulated by IL-17A. IL-17A enhanced expression of TNF-α, IL-1β, IFN-γ, and inducible nitric oxide synthase (iNOS) and further increased streptozotocin-induced expression of the inflammatory factors in MIN6 cells. IL-17A exacerbated streptozotocin-induced MIN6 cell apoptosis and insulin secretion impairment. Blocking IL-17RA with anti-IL-17RA-neutralizing antibody reduced all these deleterious effects of IL-17A on MIN6 cells. Collectively, IL-17A deficiency alleviated hyperglycemia, hypoinsulinemia, and inflammatory response in Akita mice that are characteristic for T1D. IL-17A exerted an alone and synergistic destruction with streptozotocin to pancreatic β cells through IL-17RA pathway. Thus, the data suggest that targeting IL-17A and/or IL-17RA is likely to preserve remaining β-cell function and treat T1D.Impact statementThe participation of interleukin (IL)-17A in diabetic pathogenesis is suggested in animal models of autoimmune diabetes and in patients with type 1 diabetes (T1D), but with some contradictory results. Particularly, evidence for a direct injury of IL-17A to pancreatic β cells is lacking. We showed that IL-17A deficiency alleviated diabetic signs including hyperglycemia, hypoinsulinemia, and inflammatory response in Ins2^Akita (Akita) mice, a T1D model with spontaneous mutation in insulin 2 gene leading to β-cell apoptosis. IL-17A enhanced inflammatory reaction, oxidative stress, and cell apoptosis but attenuated insulin level in mouse insulin-producing MIN6 cells. IL-17A had also a synergistic destruction to MIN6 cells with streptozotocin (STZ), a pancreatic β-cell-specific cytotoxin. Blocking IL-17 receptor A (IL-17RA) reduced all these deleterious effects of IL-17A on MIN6 cells. The results demonstrate the role and the importance of IL-17A in T1D pathogenesis and suggest a potential therapeutic strategy for T1D targeting IL-17A and/or IL-17RA.     

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.     

IL-1β in atherosclerotic vascular calcification: From bench to bedside

Atherosclerotic vascular calcification contributes to increased risk of death in patients with cardiovascular diseases. Assessing the type and severity of inflammation is crucial in the treatment of numerous cardiovascular conditions. IL-1β, a potent proinflammatory cytokine, plays diverse roles in the pathogenesis of atherosclerotic vascular calcification. Several large-scale, population cohort trials have shown that the incidence of cardiovascular events is clinically reduced by the administration of anti-IL-1β therapy. Anti-IL-1β therapy might reduce the incidence of cardiovascular events by affecting atherosclerotic vascular calcification, but the mechanism underlying this effect remains unclear. In this review, we summarize current knowledge on the role of IL-1β in atherosclerotic vascular calcification, and describe the latest results reported in clinical trials evaluating anti-IL-1β therapies for the treatment of cardiovascular diseases. This review will aid in improving current understanding of the pathophysiological roles of IL-1β and mechanisms underlying its activity.     

Anti-Inflammatory Activity of S. Marianum and N. Sativa Extracts on Macrophages

Background:Nigella sativa (N. sativa) and Silybum marianum (S. marianum) are used to regulate macrophage polarization in lipopolysaccharide-induced RAW 264.7 cells and thioglycollate-elicited peritoneal inflammation.Methods:Cytotoxicity assays and acute toxicity tests were performed to investigate the safe dose and toxicity of the prepared extracts. Also, nitric oxide production was determined by Griess assay on RAW264.7 and peritoneal macrophage supernatants. After RNA extraction from macrophages, real-time PCR was performed to measure the relative gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, transforming growth factor (TGF)-β, and IL-10. Finally, regulatory T cells (Treg cells) were counted by flow cytometry.Results:S. marianum methanolic extract (SME), N. sativa ethanolic extract (NEE), and their mixture (SME+NEE) decreased NO levels significantly in RAW264.7 and peritoneal murine macrophages. N. sativa ethanolic extract significantly increased IL-10 gene expression and significantly decreased IL-6 and TNF-α expression in RAW264.7 cells. In mixture-treated peritoneal macrophages, IL-10 and TGF-β expression were significantly increased, while IL-6 and TNF-α were significantly decreased. Also, the percentage of Treg cells was significantly greater in the mixture-treated cells than in controls.Conclusion:These results suggest that an SME and NEE mixture has anti-inflammatory and immunomodulatory activities and may be useful in the treatment of diseases of immunopathologic origin characterized by macrophage hyperactivation.Key Words: Cytokine, Inflammation, Nigella sativa, Nitric oxide, Silybum marianum     

Adjuvant-dependent regulation of interleukin-17 expressing γδ T cells and inhibition of Th2 responses in allergic airways disease

Background

Th2 immune responses are linked primarily to mild and moderate asthma, while Th17 cells, Interleukin-17A (IL-17) and neutrophilia have been implicated in more severe forms of disease. How Th2-dependent allergic reactions are influenced by Th17 and IL-17-γδ T cells is poorly understood. In murine models, under some conditions, IL-17 promotes Th2-biased airway inflammatory responses. However, IL-17-γδ T cells have been implicated in the inhibition and resolution of allergic airway inflammation and hyperresponsiveness (AHR).

Methods

We compared airway responses in Balb/c mice sensitized to OVA with (and without) a Th2-skewing aluminum-based adjuvant and the IL-17 skewing, complete Freund’s adjuvant (CFA). AHR was measured invasively by flexiVent, while serum OVA-IgE was quantified by an enzyme immunoassay. Airway inflammatory and cytokine profiles, and cellular sources of IL-17 were assessed from bronchoalveolar lavage and/or lungs. The role of γδ T cells in these responses was addressed in OVA/CFA sensitized mice using a γδ T cell antibody.

Results

Following OVA challenge, all mice exhibited mixed eosinophilic/neutrophilic airway inflammatory profiles and elevated serum OVA-IgE. Whereas OVA/alum sensitized mice had moderate inflammation and AHR, OVA/CFA sensitized mice had significantly greater inflammation but lacked AHR. This correlated with a shift in IL-17 production from CD4⁺ to γδ T cells. Additionally, OVA/CFA sensitized mice, given a γδ TCR stimulatory antibody, showed increased frequencies of IL-17-γδ T cells and diminished airway reactivity and eosinophilia.

Conclusions

Thus, the conditions of antigen sensitization influence the profile of cells that produce IL-17, the balance of which may then modulate the airway inflammatory responses, including AHR. The possibility for IL-17-γδ T cells to reduce AHR and robust eosinophilic inflammation provides evidence that therapeutic approaches focused on stimulating and increasing airway IL-17-γδ T cells may be an effective alternative in treating steroid resistant, severe asthma.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0090-5) contains supplementary material, which is available to authorized users.     

IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

Interleukin (IL-) 36 cytokines (previously designated as novel IL-1 family member cytokines; IL-1F5– IL-1F10) constitute a novel cluster of cytokines structurally and functionally similar to members of the IL-1 cytokine cluster. The effects of IL-36 cytokines in inflammatory lung disorders remains poorly understood. The current study sought to investigate the effects of IL-36α (IL-1F6) and test the hypothesis that IL-36α acts as a pro-inflammatory cytokine in the lung in vivo. Intratracheal instillation of recombinant mouse IL-36α induced neutrophil influx in the lungs of wild-type C57BL/6 mice and IL-1αβ^−/− mice in vivo. IL-36α induced neutrophil influx was also associated with increased mRNA expression of neutrophil-specific chemokines CXCL1 and CXCL2 in the lungs of C57BL/6 and IL-1αβ^−/− mice in vivo. In addition, intratracheal instillation of IL-36α enhanced mRNA expression of its receptor IL-36R in the lungs of C57BL/6 as well as IL-1αβ^−/− mice in vivo. Furthermore, in vitro incubation of CD11c⁺ cells with IL-36α resulted in the generation of neutrophil-specific chemokines CXCL1, CXCL2 as well as TNFα. IL-36α increased the expression of the co-stimulatory molecule CD40 and enhanced the ability of CD11c⁺ cells to induce CD4⁺ T cell proliferation in vitro. Furthermore, stimulation with IL-36α activated NF-κB in a mouse macrophage cell line. These results demonstrate that IL-36α acts as a pro-inflammatory cytokine in the lung without the contribution of IL-1α and IL-1β. The current study describes the pro-inflammatory effects of IL-36α in the lung, demonstrates the functional redundancy of IL-36α with other agonist cytokines in the IL-1 and IL-36 cytokine cluster, and suggests that therapeutic targeting of IL-36 cytokines could be beneficial in inflammatory lung diseases.     

Protection against Chemotaxis in the Anti-Inflammatory Effect of Bioactives from Tomato Ketchup

The consumption of tomato products has been associated with a decreased risk for chronic inflammatory diseases. In this study, the anti-inflammatory potential of tomato ketchup was evaluated by studying the effect of tomato ketchup extracts and bioactives from tomato ketchup on human monocytes and vascular endothelial cells (HUVEC). HUVEC were pre-treated for 1 h with either individual bioactives (7.5 µM lycopene, 1.4 µM α-tocopherol or 55 µM ascorbic acid) or a combination of these three compounds, or with the hydrophilic or lipophilic tomato ketchup extracts or with the two extracts combined. After the pretreatment, the cells were washed and challenged with TNF-α (10 ng/ml) for 6 h. The medium was used for the determination of the release of cytokines and the chemotaxis of monocytes. Inflammatory protein expression and production were assayed with real-time RT-PCR and ELISA. It was found that tomato ketchup extracts significantly reduced gene expression and release of the pro-inflammatory cytokines TNF-α and IL-8 in HUVEC after the inflammatory challenge, whereas the release of the anti-inflammatory cytokine IL-10 was increased. Chemotaxis was effectively impeded as demonstrated by a reduced monocyte migration. This effect correlated with the reduction of IL-8 production in the presence of the test compounds and extracts. The results consistently emphasize the contribution of lycopene to the anti-inflammatory effect of tomato ketchup. Other compounds in tomato ketchup such as α-tocopherol and ascorbic acid appeared to strengthen the anti-inflammatory effect of lycopene. The tomato ketchup extracts subtly interfered with several inflammatory phases that inhibit chemotaxis. Such a pleotropic mode of action exemplifies its potential mitigation of diseases characterized by prolonged low grade inflammation.     

Plasma Cytokine Profile in Tropical Endomyocardial Fibrosis: Predominance of TNF-a,IL-4 and IL-10

Background

The participation of immune/inflammatory mechanisms in the pathogenesis of tropical endomyocardial fibrosis (EMF) has been suggested by the finding of early blood and myocardial eosinophilia. However, the inflammatory activation status of late-stage EMF patients is still unknown.

Methodology/Principal findings

We evaluated pro- and anti-inflammatory cytokine levels in plasma samples from late stage EMF patients. Cytokine levels of Tumor Necrosis Factor (TNF)-α, Interferon (IFN)-γ, Interleukin (IL)-2, IL-4, IL-6, and IL-10 were assayed in plasma samples from 27 EMF patients and compared with those of healthy control subjects. All EMF patients displayed detectable plasma levels of at least one of the cytokines tested. We found that TNF-α, IL-6, IL-4, and IL-10 were each detected in at least 74% of tested sera, and plasma levels of IL-10, IL-4, and TNF-α were significantly higher than those of controls. Plasma levels of such cytokines positively correlated with each other.

Conclusions/Significance

The mixed pro- and anti-inflammatory/Th2circulating cytokine profile in EMF is consistent with the presence of a persistent inflammatory stimulus. On the other hand, the detection of increased levels of TNF-α may be secondary to the cardiovascular involvement observed in these patients, whereas IL-4 and IL-10 may have been upregulated as a homeostatic mechanism to buffer both production and deleterious cardiovascular effects of pro-inflammatory cytokines. Further studies might establish whether these findings play a role in disease pathogenesis.