Increase in TGF-β Secreting CD4+CD25+ FOXP3+ T Regulatory Cells in Anergic Lepromatous Leprosy Patients

Background

Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25⁺FOXP3⁺ regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples.

Methodology/Principle Findings

Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3⁺, TGF-β in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4⁺CD25⁺FOXP3⁺ T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-β which was further confirmed by increase in TGF-β in culture supernatants (p<0.003). Furthermore, TGF-β in iTreg cells was associated with phosphorylation of STAT5A. TGF-β was seen in CD25⁺ cells of the CD4⁺ but not that of CD8⁺ T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients.

Conclusions/Significance

Our results indicate that FOXP3⁺ iTregs with TGF-β may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.     

Macrophage derived chemokine (CCL22), thymus and activation-regulated chemokine (CCL17), and CCR4 in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a chronically progressive interstitial lung disease of unknown etiology. Previously, we have demonstrated the selective upregulation of the macrophage-derived chemokine CCL22 and the thymus activation-regulated chemokine CCL17 among chemokines, in a rat model of radiation pneumonitis/pulmonary fibrosis and preliminarily observed an increase in bronchoalveolar (BAL) fluid CCL22 levels of IPF patients.

Methods

We examined the expression of CCR4, a specific receptor for CCL22 and CCL17, in bronchoalveolar lavage (BAL) fluid cells, as well as the levels of CCL22 and CCL17, to elucidate their pathophysiological roles in pulmonary fibrosis. We also studied their immunohistochemical localization.

Results

BAL fluid CCL22 and CCL17 levels were significantly higher in patients with IPF than those with collagen vascular diseases and healthy volunteers, and there was a significant correlation between the levels of CCL22 and CCL17 in patients with IPF. CCL22 levels in the BAL fluid did not correlate with the total cell numbers, alveolar lymphocytes, or macrophages in BAL fluid. However, the CCL22 levels significantly correlated with the numbers of CCR4-expressing alveolar macrophages. By immunohistochemical and immunofluorescence analysis, localization of CCL22 and CCR4 to CD68-positive alveolar macrophages as well as that of CCL17 to hyperplastic epithelial cells were shown. Clinically, CCL22 BAL fluid levels inversely correlated with DLco/VA values in IPF patients.

Conclusion

We speculated that locally overexpressed CCL22 may induce lung dysfunction through recruitment and activation of CCR4-positive alveolar macrophages.     

CCL20 Secretion from the Nucleus Pulposus Improves the Recruitment of CCR6-Expressing Th17 Cells to Degenerated IVD Tissues

Background

Studies elucidated that Th17 cells are important contributors to the pathogenesis of many immune-mediated diseases, and IL-17A is present in pathologic intervertebral disc (IVD) tissues. However, the mechanisms, how these cells traffic into the degenerate discs are not clear.

Materials and Methods

The samples collected from 53 patients had been divided into 3 groups: Group P (annulus fibrosus was intact), Group E (annulus fibrosus was reptured) and normal control. Immunohistochemistry was used to detect the expression of CCL20, CCR6, IL-17A, TNF–α and CD4 in IVD tissues. Moreover, nucleus pulposus (NP) cells had been cultured in the presence and absence of Th17 associated cytokines. The supernatants were detected for CCL20 concentrations by ELISA, and the NP cells for the expression of CCL20 mRNA. Additionally, peripheral blood (PB) samples had undergone detection for the expression of CCR6 mRNA and the proportion of IL-17-producing cells, including the surface expression of CCR6.

Results

Immunohistochemistry revealed that CCL20 and TNF-α were expressed in degenerated NP cells. Double-labeled immunofluorescence elaborated, IL-17-producing cells (CD4⁺IL-17A⁺ and CD4⁺CCR6⁺) appeared in the Group E samples, but no traces or expression in Group P and normal control. IL-17A and TNF-α, alone or combined, could enhance CCL20 secretion in a dose-dependent manner, which was obtained through RT-PCR results. There was a notable difference of CCR6 mRNA expression between patients and normal controls. In comparison to controls, flow cytometry data indicated that the proportion of IL-17-producing cells and the CCR6 expression in PB were significantly increased.

Conclusion

Our results provide a potential explanation for involvement of the CCL20-CCR6 system in the trafficking of IL-17-producing cells to degenerated IVD tissues. Additionally, our results explain the contribution of Th17 associated cytokines to the development of degenerated discs via the up-regulation of CCL20 secretion from NP cells, which forms a positive chemotactic feedback loop.     

Hemolytic Streptococcus May Exacerbate Kidney Damage in IgA Nephropathy through CCL20 Response to the Effect of Th17 Cells

Background

The exacerbation of IgA nephropathy (IgAN) is related to respiratory tract infection with hemolytic streptococcus (HS), but the mechanism is unknown. In this study we investigated the role of chemokine ligand 20 (CCL20) in response to the effect of T helper 17 (Th17) cells in the pathogenesis of IgAN associated with HS.

Methods

Thirty mice were randomly divided into five groups: control mice (control), IgAN mice (IgAN), HS-infected IgAN mice (HS-IgAN), CCL20-treated IgAN mice (CCL20-IgAN), and CCL20-treated HS infected IgAN mice (CCL20-HS-IgAN). IgAN mice were induced with lipopolysaccharide, carbon tetrachloride and bovine serum albumin. Then the mice were sensitized with CCL20 antibody and infected with alpha-hemolytic streptococcus (α-HS) isolated from tonsils in sequence. Urine Albumin-Creatinine ratio and sediments were measured. The pathological changes in kidney and lung tissues were observed under microscopy. Th17 cells and regulatory T cells (Tregs) in kidneys were tested by flow cytometry. CCL20, IL-17A, IL-6 and IL-21 in the kidneys were detected by ELISA.

Results

The IgAN mice had albuminuria and microscopic hematuria, renal mesangial proliferation, IgA deposition, high electron dense deposition in glomerular mesangial region, decreased frequency of Tregs, increased frequency of Th17 and Th17-Treg ratio. Furthermore, Th17-related cytokines CCL20, IL-17A, IL-6 and IL-21 were all increased in the kidneys of IgAN mice. Compared with IgAN mice, the manifestations in HS-IgAN mice were more severe, but alleviated in CCL20-treated groups.

Conclusion

α-HS may exacerbate kidney damage in IgAN through CCL20 response to the effect of Th17 cells.     

Involvement of CCR6/CCL20/IL-17 axis in NSCLC disease progression

Objectives

Autocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.

Methods

A specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation.

Results

CCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52–15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells.

Conclusion

Our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.     

Higher expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 chemokines in the skin associated with parasite density in canine visceral leishmaniasis

Background

The immune response in the skin of dogs infected with Leishmania infantum is poorly understood, and limited studies have described the immunopathological profile with regard to distinct levels of tissue parasitism and the clinical progression of canine visceral leishmaniasis (CVL).

Methodology/Principal Findings

A detailed analysis of inflammatory cells (neutrophils, eosinophils, mast cells, lymphocytes, and macrophages) as well as the expression of chemokines (CCL2, CCL4, CCL5, CCL13, CCL17, CCL21, CCL24, and CXCL8) was carried out in dermis skin samples from 35 dogs that were naturally infected with L. infantum. The analysis was based on real-time polymerase chain reaction (PCR) in the context of skin parasitism and the clinical status of CVL. We demonstrated increased inflammatory infiltrate composed mainly of mononuclear cells in the skin of animals with severe forms of CVL and high parasite density. Analysis of the inflammatory cell profile of the skin revealed an increase in the number of macrophages and reductions in lymphocytes, eosinophils, and mast cells that correlated with clinical progression of the disease. Additionally, enhanced parasite density was correlated with an increase in macrophages and decreases in eosinophils and mast cells. The chemokine mRNA expression demonstrated that enhanced parasite density was positively correlated with the expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. In contrast, there was a negative correlation between parasite density and CCL24 expression.

Conclusions/Significance

These findings represent an advance in the knowledge about skin inflammatory infiltrates in CVL and the systemic consequences. Additionally, the findings may contribute to the design of new and more efficient prophylactic tools and immunological therapies against CVL.     

Leprosy Reactions Show Increased Th17 Cell Activity and Reduced FOXP3+ Tregs with Concomitant Decrease in TGF-β and Increase in IL-6

Background

50% of leprosy patients suffer from episodes of Type 1/ reversal reactions (RR) and Type 2/ Erythema Nodosum Leprosum (ENL) reactions which lead to morbidity and nerve damage. CD4⁺ subsets of Th17 cells and CD25⁺FOXP3⁺ regulatory T cells (Tregs) have been shown to play a major role in disease associated immunopathology and in stable leprosy as reported by us and others. The aim of our study was to analyze their role in leprosy reactions.

Methodology and Principle Findings

Quantitative reverse transcribed PCR (qPCR), flowcytometry and ELISA were used to respectively investigate gene expression, cell phenotypes and supernatant levels of cytokines in antigen stimulated PBMC cultures in patients with stable disease and those undergoing leprosy reactions. Both types of reactions are associated with significant increase of Th17 cells and associated cytokines IL-17A, IL-17F, IL-21, IL-23 and chemokines CCL20, CCL22 as compared to matching stable forms of leprosy. Concurrently patients in reactions show reduction in FOXP3⁺ Treg cells as well as reduction in TGF-β and increase in IL-6. Moreover, expression of many T cell markers, cytokines, chemokines and signaling factors were observed to be increased in RR as compared to ENL reaction patients.

Conclusions

Patients with leprosy reactions show an imbalance in Th17 and Treg populations. The reduction in Treg suppressor activity is associated withhigherTh17cell activity. The combined effect of reduced TGF-β and enhanced IL-6, IL-21 cytokines influence the balance between Th17 or Treg cells in leprosy reactions as reported in the murine models and autoimmune diseases. The increase in Th17 cell associated cytokines may contribute to lesional inflammation.     

Higher serum CCL17 may be a promising predictor of acute exacerbations in chronic hypersensitivity pneumonitis

Background

Recent research has suggested that the Th1 and Th2 chemokine/cytokine axis contributes to the development of chronic hypersensitivity pneumonitis (HP). Acute exacerbations (AE) are significant factors in the prognosis of chronic HP. Little is known, however, about these biomarkers in association with AE in chronic HP patients.

Methods

Fifty-six patients with chronic HP were evaluated, including 14 patients during episodes of AE. Th1 mediators (C-X-C chemokine ligand [CXCL]10 and interferon [IFN]-γ), Th2 mediators (C-C chemokine ligand [CCL]17, interleukin-4, and interleukin-13), and pro-fibrotic mediator (transforming growth factor [TGF]-β) were measured to evaluate the mediators as predictors of AE. C-C chemokine receptor (CCR)4 (receptor for CCL17)-positive lymphocytes were quantified in lung specimens.

Results

Serum CCL17 levels at baseline independently predicted the first episode of AE (HR, 72.0; 95% CI, 5.03-1030.23; p = 0.002). AE was significantly more frequent in the higher-CCL17 group (≥285 pg/ml) than in the lower-CCL17 group (<285 pg/ml) (log-rank test, p = 0.0006; 1-year incidence: higher CCL17 vs. lower CCL17, 14.3% vs. 0.0%). Serum CCL17 levels and CCR4-positive cells during episodes of AE were increased from the baseline (p = 0.01 and 0.031).

Conclusions

Higher serum concentrations of CCL17 at baseline may be predictive of AE in patients with chronic HP, and CCL17 may contribute to the pathology of AE by inducing the accumulation of CCR4-positive lymphocytes in the lungs.     

Increased levels of soluble interleukin-6 receptor and CCL3 in COPD sputum

Background

COPD patients have increased numbers of macrophages and neutrophils in the lungs. Interleukin-6 (IL-6) trans-signaling via its soluble receptor sIL-6R, governs the influx of innate immune cells to inflammatory foci through regulation of the chemokine CCL3. We hypothesized that there would be enhanced levels of IL-6, sIL-6R and CCL3 in COPD sputum.

Methods

59 COPD patients, 15 HNS and 15 S underwent sputum induction and processing with phosphate buffered saline to obtain supernatants for IL-6, sIL-6R and CCL3 analysis. Cytoslides were produced for differential cell counting and immunocytochemistry (COPD; n = 3) to determine cell type surface expression of the CCL3 receptors CCR5 and CCR1.

Results

COPD patients expressed higher levels (p < 0.05) of sIL-6R and CCL3 compared to controls (sIL-6R medians pg/ml: COPD 166.4 vs S 101.1 vs HNS 96.4; CCL3 medians pg/ml: COPD 117.9 vs S 0 vs HNS 2.7). COPD sIL-6R levels were significantly correlated with sputum neutrophil (r = 0.5, p < 0.0001) and macrophage (r = 0.3, p = 0.01) counts. Immunocytochemical analysis revealed that CCR5 and CCR1 were exclusively expressed on airway macrophages.

Conclusion

Enhanced airway generation of sIL-6R may promote IL-6 trans-signaling in COPD. Associated upregulation of CCL3 may facilitate the recruitment of macrophages into the airways by ligation of CCR1 and CCR5.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0103-4) contains supplementary material, which is available to authorized users.     

CCL25/CCR9 interactions regulate large intestinal inflammation in a murine model of acute colitis

Background & Aims

CCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine.

Methods

Acute inflammation and recovery in wild-type (WT) and CCR9^−/− mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level.

Results

CCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9^−/− mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9^−/− colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9^−/− animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNFα, IFNγ, IL2, IL-6 and IL-17A while down modulating IL-10 production.

Conclusions

Our results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation.     

Cytokine and protein markers of leprosy reactions in skin and nerves: baseline results for the North Indian INFIR cohort

Background

Previous studies investigating the role of cytokines in the pathogenesis of leprosy have either been on only small numbers of patients or have not combined clinical and histological data. The INFIR Cohort study is a prospective study of 303 new multibacillary leprosy patients to identify risk factors for reaction and nerve damage. This study characterised the cellular infiltrate in skin and nerve biopsies using light microscopic and immunohistochemical techniques to identify any association of cytokine markers, nerve and cell markers with leprosy reactions.

Methodology/Principal Findings

TNF-α, TGF-β and iNOS protein in skin and nerve biopsies were detected using monoclonal antibody detection immunohistochemistry techniques in 299 skin biopsies and 68 nerve biopsies taken from patients at recruitment. The tissues were stained with hematoxylin and eosin, modified Fite Faraco, CD68 macrophage cell marker and S100.

Conclusions/Significance

Histological analysis of the biopsies showed that 43% had borderline tuberculoid (BT) leprosy, 27% borderline lepromatous leprosy, 9% lepromatous leprosy, 13% indeterminate leprosy types and 7% had no inflammation. Forty-six percent had histological evidence of a Type 1 Reaction (T1R) and 10% of Erythema Nodosum Leprosum. TNF-α was detected in 78% of skin biopsies (181/232), iNOS in 78% and TGF-β in 94%. All three molecules were detected at higher levels in patients with BT leprosy. TNF-α was localised within macrophages and epithelioid cells in the granuloma, in the epidermis and in dermal nerves in a few cases. TNF-α, iNOS and TGF-β were all significantly associated with T1R (p<0.001). Sixty-eight nerve biopsies were analysed. CD68, TNF-α and iNOS staining were detectable in 88%, 38% and 28% of the biopsies respectively. The three cytokines TNF-α, iNOS and TGF-β detected by immunohistochemistry showed a significant association with the presence of skin reaction. This study is the first to demonstrate an association of iNOS and TGF-β with T1R.     

Macrophage Activation in Acute Exacerbation of Idiopathic Pulmonary Fibrosis

Background

Acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF) is a common cause of disease acceleration in IPF and has a major impact on mortality. The role of macrophage activation in AE of IPF has never been addressed before.

Methods

We evaluated BAL cell cytokine profiles and BAL differential cell counts in 71 IPF patients w/wo AE and in 20 healthy volunteers. Twelve patients suffered from AE at initial diagnosis while sixteen patients developed AE in the 24 months of follow-up. The levels of IL-1ra, CCL2, CCL17, CCL18, CCL22, TNF-α, IL-1β, CXCL1 and IL-8 spontaneously produced by BAL-cells were analysed by ELISA.

Results

In patients with AE, the percentage of BAL neutrophils was significantly increased compared to stable patients. We found an increase in the production rate of the pro-inflammatory cytokines CXCL1 and IL-8 combined with an increase in all tested M2 cytokines by BAL-cells. An increase in CCL18 levels and neutrophil counts during AE was observed in BAL cells from patients from whom serial lavages were obtained. Furthermore, high baseline levels of CCL18 production by BAL cells were significantly predictive for the development of future AE.

Conclusions

BAL cell cytokine production levels at acute exacerbation show up-regulation of pro-inflammatory as well as anti-inflammatory/ M2 cytokines. Our data suggest that AE in IPF is not an incidental event but rather driven by cellular mechanisms including M2 macrophage activation.     

Smoking in Asthma Is Associated with Elevated Levels of Corticosteroid Resistant Sputum Cytokines—An Exploratory Study

Background

Current cigarette smoking is associated with reduced acute responses to corticosteroids and worse clinical outcomes in stable chronic asthma. The mechanism by which current smoking promotes this altered behavior is currently unclear. Whilst cytokines can induce corticosteroid insensitivity in-vitro, how current and former smoking affects airway cytokine concentrations and their responses to oral corticosteroids in stable chronic asthma is unclear.

Objectives

To examine blood and sputum cytokine concentrations in never, ex and current smokers with asthma before and after oral corticosteroids.

Methods

Exploratory study utilizing two weeks of oral dexamethasone (equivalent to 40 mg/day prednisolone) in 22 current, 21 never and 10 ex-smokers with asthma. Induced sputum supernatant and plasma was obtained before and after oral dexamethasone. 25 cytokines were measured by multiplex microbead system (Invitrogen, UK) on a Luminex platform.

Results

Smokers with asthma had elevated sputum cytokine interleukin (IL) -6, -7, and -12 concentrations compared to never smokers with asthma. Few sputum cytokine concentrations changed in response to dexamethasone IL-17 and IFNα increased in smokers, CCL4 increased in never smokers and CCL5 and CXCL10 reduced in ex-smokers with asthma. Ex-smokers with asthma appeared to have evidence of an ongoing corticosteroid resistant elevation of cytokines despite smoking cessation. Several plasma cytokines were lower in smokers with asthma compared to never smokers with asthma.

Conclusion

Cigarette smoking in asthma is associated with a corticosteroid insensitive increase in multiple airway cytokines. Distinct airway cytokine profiles are present in current smokers and never smokers with asthma and could provide an explanatory mechanism for the altered clinical behavior observed in smokers with asthma.     

Tumor-associated macrophages recruit CCR6+ regulatory T cells and promote the development of colorectal cancer via enhancing CCL20 production in mice

Background

Tumor-associated macrophages (TAMs) remodel the colorectal cancer (CRC) microenvironment. Yet, findings on the role of TAMs in CRC seem to be contradictory compared with other cancers. FoxP3⁺ regulatory T (Treg)-cells dominantly infiltrate CRC. However, the underlying molecular mechanism in which TAMs may contribute to the trafficking of Treg-cells to the tumor mass remains unknown.

Methodology/Principal Findings

CRC was either induced by N-methyl-N-nitrosourea (MNU) and H. pylori or established by subcutaneous injection of mouse colorectal tumor cell line (CMT93) in mice. CMT93 cells were co-cultured with primary macrophages in a transwell apparatus. Recruitment of FoxP3 green fluorescence protein positive (FoxP3^GFP+) Treg-cells was assessed using the IVIS Imaging System or immunofluorescence staining. A role for macrophages in trafficking of Treg-cells and in the development of CRC was investigated in CD11b diphtheria toxin receptor (CD11b-DTR) transgenic C57BL/6J mice in which macrophages can be selectively depleted. Treg-cells remarkably infiltrated solid tumor, and predominantly expressed the homing chemokine receptor (CCR) 6 in the induced CRC model. Both CMT93 cancer cells and macrophages produced a large amount of CCL20, the sole ligand of CCR6 in vitro and in vivo. Injection of recombinant mouse CCL20 into tumor sites promoted its development with a marked recruitment of Treg-cells in the graft CRC model. Conditional macrophage ablation decreased CCL20 levels, blocked Treg-cell recruitment and inhibited tumor growth in CD11b-DTR mice grafted with CMT93.

Conclusions/Significance

TAMs recruit CCR6⁺ Treg-cells to tumor mass and promote its development via enhancing the production of CCL20 in a CRC mouse model.     

Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

Introduction

Altered phenotypes of circulating monocytes of patients with systemic sclerosis (SSc) have been reported, but the role of these alterations in the pathogenesis of SSc remains unclear. This study was undertaken to identify molecules that are preferentially expressed by SSc monocytes, and to investigate the roles of these molecules in the pathogenic process of SSc.

Methods

We analyzed circulating CD14⁺ monocytes isolated from 36 patients with SSc and 32 healthy control subjects. The monocytes'' gene expression profiles were assessed by Oligo GEArray^® (SABiosciences, Frederic, MA, USA) and semiquantitative or quantitative PCR; their protein expression was evaluated in culture supernatants of unstimulated monocytes by immunoblotting or ELISA, and by immunocytostaining. Monocyte chemoattractant activity of CCL2 was assessed in a TransWell^® system (Corning Incorporated, Corning, NY, USA) in the presence or absence of chondroitin sulfate (CS).

Results

A step-wise approach to profiling gene expression identified that versican and CCL2 were upregulated in SSc monocytes. Subsequent analysis of proteins expressed in monocyte culture supernatants confirmed enhanced production of versican and CCL2 in SSc monocytes compared with control monocytes. CCL2 bound to CS chains of versican and colocalized with versican in the monocytes'' Golgi apparatus. Finally, CCL2 had a greater ability to mediate monocyte migration when bound to CS chains, because this binding provided efficient formation of CCL2 gradients and protection from protease attack.

Conclusion

Circulating monocytes with elevated versican and CCL2 levels may contribute to the fibrotic process in a subset of SSc patients by amplifying a positive feedback loop consisting of versican, CCL2, and the influx of monocytes.     

High Glucose Induces CCL20 in Proximal Tubular Cells via Activation of the KCa3.1 Channel

Background

Inflammation plays a key role in the development and progression of diabetic nephropathy (DN). KCa3.1, a calcium activated potassium channel protein, is associated with vascular inflammation, atherogenesis, and proliferation of endothelial cells, macrophages, and fibroblasts. We have previously demonstrated that the KCa3.1 channel is activated by TGF-β1 and blockade of KCa3.1 ameliorates renal fibrotic responses in DN through inhibition of the TGF-β1 pathway. The present study aimed to identify the role of KCa3.1 in the inflammatory responses inherent in DN.

Methods

Human proximal tubular cells (HK2 cells) were exposed to high glucose (HG) in the presence or absence of the KCa3.1 inhibitor TRAM34 for 6 days. The proinflammatory cytokine chemokine (C-C motif) ligand 20 (CCL20) expression was examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The activity of nuclear factor-κB (NF-κB) was measured by nuclear extraction and electrophoretic mobility shift assay (EMSA). In vivo, the expression of CCL20, the activity of NF-κB and macrophage infiltration (CD68 positive cells) were examined by real-time PCR and/or immunohistochemistry staining in kidneys from diabetic or KCa3.1-/- mice, and in eNOS-/- diabetic mice treated with the KCa3.1 channel inhibitor TRAM34.

Results

In vitro data showed that TRAM34 inhibited CCL20 expression and NF-κB activation induced by HG in HK2 cells. Both mRNA and protein levels of CCL20 significantly decreased in kidneys of diabetic KCa3.1-/- mice compared to diabetic wild type mice. Similarly, TRAM34 reduced CCL20 expression and NF-κB activation in diabetic eNOS-/- mice compared to diabetic controls. Blocking the KCa3.1 channel in both animal models led to a reduction in phosphorylated NF-κB.

Conclusions

Overexpression of CCL20 in human proximal tubular cells is inhibited by blockade of KCa3.1 under diabetic conditions through inhibition of the NF-κB pathway.     

Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M

Background

Regulation of human airway smooth muscle cells (HASMC) by cytokines contributes to chemotactic factor levels and thus to inflammatory cell accumulation in lung diseases. Cytokines such as the gp130 family member Oncostatin M (OSM) can act synergistically with Th2 cytokines (IL-4 and IL-13) to modulate lung cells, however whether IL-17A responses by HASMC can be altered is not known.

Objective

To determine the effects of recombinant OSM, or other gp130 cytokines (LIF, IL-31, and IL-6) in regulating HASMC responses to IL-17A, assessing MCP-1/CCL2 and IL-6 expression and cell signaling pathways.

Methods

Cell responses of primary HASMC cultures were measured by the assessment of protein levels in supernatants (ELISA) and mRNA levels (qRT-PCR) in cell extracts. Activation of STAT, MAPK (p38) and Akt pathways were measured by immunoblot. Pharmacological agents were used to assess the effects of inhibition of these pathways.

Results

OSM but not LIF, IL-31 or IL-6 could induce detectable responses in HASMC, elevating MCP-1/CCL2, IL-6 levels and activation of STAT-1, 3, 5, p38 and Akt cell signaling pathways. OSM induced synergistic action with IL-17A enhancing MCP-1/CCL-2 and IL-6 mRNA and protein expression, but not eotaxin-1 expression, while OSM in combination with IL-4 or IL-13 synergistically induced eotaxin-1 and MCP-1/CCL2. OSM elevated steady state mRNA levels of IL-4Rα, OSMRβ and gp130, but not IL-17RA or IL-17RC. Pharmacologic inhibition of STAT3 activation using Stattic down-regulated OSM, OSM/IL-4 or OSM/IL-13, and OSM/IL-17A synergistic responses of MCP-1/CCL-2 induction, whereas, inhibitors of Akt and p38 MAPK resulted in less reduction in MCP-1/CCL2 levels. IL-6 expression was more sensitive to inhibition of p38 (using SB203580) and was affected by Stattic in response to IL-17A/OSM stimulation.

Conclusions

Oncostatin M can regulate HASMC responses alone or in synergy with IL-17A. OSM/IL-17A combinations enhance MCP-1/CCL2 and IL-6 but not eotaxin-1. Thus, OSM through STAT3 activation of HASMC may participate in inflammatory cell recruitment in inflammatory airway disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0164-4) contains supplementary material, which is available to authorized users.