The effects of corticosteroids on COPD lung macrophages: a pooled analysis

Background

There is large variation in the therapeutic response to inhaled corticosteroids (ICS) in COPD patients. We present a pooled analysis of our previous studies investigating the effects of corticosteroids on lung macrophages, in order to robustly determine whether corticosteroid sensitivity in COPD cells is reduced compared to controls, and also to evaluate the degree of between individual variation in drug response.

Methods

Data from 20 never smokers (NS), 27 smokers (S) and 45 COPD patients was used. Lung macropahges had been stimulated with lipopolysaccharide (LPS), with or without the corticosteroid dexamethasone, and tumour necrosis factor (TNF)-α, interleukin (IL)-6 and chemokine C-X-C motif ligand (CXCL) 8 production was measured.

Results

There was no difference in the anti-inflammatory effects of corticosteroids when comparing group mean data of COPD patients versus controls. The inhibition of TNF-α and IL-6 was greater than CXCL8. The effects of corticosteroids varied considerably between subjects, particularly at lower corticosteroid concentrations.

Conclusions

We confirm that overall corticosteroid sensitivity in COPD lung macrophages is not reduced compared to controls. The varied effect of corticosteroids between subjects suggests that some individuals have an inherently poor corticosteroid response. The limited suppression of lung macrophage derived CXCL8 may promote neutrophilic inflammation in COPD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0260-0) contains supplementary material, which is available to authorized users.     

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.     

No evidence of altered alveolar macrophage polarization,but reduced expression of TLR2, in bronchoalveolar lavage cells in sarcoidosis

Background

Sarcoidosis is a granulomatous inflammatory disease, possibly of infectious aetiology. We aimed to investigate whether the degree of functional polarization of alveolar macrophages (AMs), or Toll-like receptor (TLR) expression, is associated with sarcoidosis or with distinct clinical manifestations of this disease.

Methods

Total BAL cells (cultured four or 24 h in medium, or stimulated 24 h with LPS) from 14 patients and six healthy subjects, sorted AMs from 22 patients (Löfgren''s syndrome n = 11) and 11 healthy subjects, and sorted CD4+ T cells from 26 patients (Löfgren''s syndrome n = 13) and seven healthy subjects, were included. Using real-time PCR, the relative gene expression of IL-10, IL-12p35, IL-12p40, IL-23p19, CCR2, CCR7, iNOS, CXCL10, CXCL11, CXCL16, CCL18, CCL20, CD80, and CD86, and innate immune receptors TLR2, TLR4, and TLR9, was quantified in sorted AMs, and for selected genes in total BAL cells, while IL-17A was quantified in T cells.

Results

We did not find evidence of a difference with regard to alveolar macrophage M1/M2 polarization between sarcoidosis patients and healthy controls. TLR2 gene expression was significantly lower in sorted AMs from patients, particular in Löfgren''s patients. CCL18 gene expression in AMs was significantly higher in patients compared to controls. Additionally, the IL-17A expression was lower in Löfgren''s patients'' CD4+ T cells.

Conclusions

Overall, there was no evidence for alveolar macrophage polarization in sarcoidosis. However, there was a reduced TLR2 mRNA expression in patients with Löfgren''s syndrome, which may be of relevance for macrophage interactions with a postulated sarcoidosis pathogen, and for the characteristics of the ensuing T cell response.     

A Bioplex Analysis of Cytokines and Chemokines in First Trimester Maternal Plasma to Screen for Predictors of Miscarriage

Background

We have previously shown in two independent cohorts that circulating first trimester Macrophage Inhibitory Cytokine-1 (MIC-1) levels are lower in women in early pregnancy who are destined to miscarriage. While promising, the diagnostic performance of measuring MIC-1 alone was not sufficient for it to be a useful predictive test for miscarriage. Besides MIC-1, there are other cytokines, as well as chemokines, involved in facilitating early pregnancy. We reasoned that screening these factors in maternal plasma could uncover other predictive markers of miscarriage.

Methods

This was a nested case control study, of 78 women from a prospective study of 462 attending the Early Pregnancy Assessment Unit in the first trimester (EPAU) with a threatened miscarriage; 34 of these subsequently miscarried (cases) and 44 went on to have a normal delivery (controls) Cytokines IL-1β, IL-6 and IL-10, and the chemokines, CXCL8, CCL2, CCL5, CCL7 and CX3CL1 were measured in plasma from our cohort.

Results

The cytokines IL-1β, IL-6, IL-10 and the chemokine CXCL8 were not detectable in first trimester plasma. The chemokines CCL2, CCL5, CCL7 and CX3CL1 were detectable in all samples but levels did not vary across 5–12 weeks of gestation among controls. Plasma levels of these chemokines were no different in the miscarriage cohort compared to controls.

Conclusion

The chemokines CCL2, CCL5, CCL7 and CX3CL1 were detectable in plasma during the first trimester while IL-1β, IL-6, IL-10 and CXCL8 were not. However, none of the cytokines and chemokines screened were different in maternal plasma in cases or controls. These therefore do not appear to have potential for application as predictive biomarkers of miscarriage.     

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.     

Activation of eosinophils interacting with dermal fibroblasts by pruritogenic cytokine IL-31 and alarmin IL-33: implications in atopic dermatitis

Background

IL-31 is a pruritogenic cytokine, and IL-33 is an alarmin for damaging inflammation. They together relate to the pathogenesis of atopic dermatitis (AD). Eosinophil infiltration into the inner dermal compartment is a predominant pathological feature of AD. We herein investigated the in vitro inflammatory effects of IL-31 and IL-33 on the activation of human eosinophils and dermal fibroblasts.

Methodology/Principal Findings

Receptors, adhesion molecules and signaling molecules were assessed by Western blot or flow cytometry. Chemokines and cytokine were quantitated by multiplex assay. Functional IL-31 receptor component IL-31RA, OSMR-β and IL-33 receptor component ST2 were constitutively expressed on the surface of eosinophils. Co-culture of eosinophils and fibroblasts significantly induced pro-inflammatory cytokine IL-6 and AD-related chemokines CXCL1, CXCL10, CCL2 and CCL5. Such inductions were further enhanced with IL-31 and IL-33 stimulation. IL-31 and IL-33 could significantly provoke the release of CXCL8 from eosinophils and fibroblasts, respectively, which was further enhanced upon co-culture. In co-culture, eosinophils and fibroblasts were the main source for the release of CCL5, and IL-6, CXCL1, CXCL8, CXCL10 and CCL2, respectively. Direct interaction between eosinophils and fibroblasts was required for CXCL1, CXCL10, CXCL8 and CCL5 release. Cell surface expression of intercellular adhesion molecule-1 on eosinophils and fibroblasts was up-regulated in co-culture upon IL-31 and IL-33 stimulation. The interaction between eosinophils and fibroblasts under IL-31 and IL-33 stimulation differentially activated extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, nuclear factor-κB and phosphatidylinositol 3-kinase–Akt pathways. Using specific signaling molecule inhibitors, the differential induction of IL-31 and IL-33-mediated release of cytokines and chemokines such as IL-6 and CXCL8 from co-culture should be related to their distinct activation profile of intracellular signaling pathways.

Conclusions/Significance

The above findings suggest a crucial immunopathological role of IL-31 and IL-33 in AD through the activation of eosinophils-fibroblasts interaction via differential intracellular signaling mechanisms.     

Soluble immune complexes shift the TLR-induced cytokine production of distinct polarized human macrophage subsets towards IL-10

Background

Costimulation of murine macrophages with immune complexes (ICs) and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages.

Materials and Methods

Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN-γ, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs). Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/or RT-qPCR.

Results

HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MΦ_IL-4. In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2). The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6.

Conclusion

HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.     

Dynamics of pro-inflammatory and anti-inflammatory cytokine release during zcute inflammation in chronic obstructive pulmonary disease: an ex vivo study

Background

Exacerbations of Chronic obstructive pulmonary disease (COPD) are an important cause of the morbidity and mortality associated with the disease. Strategies to reduce exacerbation frequency are thus urgently required and depend on an understanding of the inflammatory milieu associated with exacerbation episodes. Bacterial colonisation has been shown to be related to the degree of airflow obstruction and increased exacerbation frequency. The aim of this study was to asses the kinetics of cytokine release from COPD parenchymal explants using an ex vivo model of lipopolysaccharide (LPS) induced acute inflammation.

Methods

Lung tissue from 24 patients classified by the GOLD guidelines (7F/17M, age 67.9 ± 2.0 yrs, FEV₁ 76.3 ± 3.5% of predicted) and 13 subjects with normal lung function (8F,5M, age 55.6 ± 4.1 yrs, FEV₁ 98.8 ± 4.1% of predicted) was stimulated with 100 ng/ml LPS alone or in combination with either neutralising TNFα or IL-10 antibodies and supernatant collected at 1,2,4,6,24, and 48 hr time points and analysed for IL-1β, IL-5, IL-6, CXCL8, IL-10 and TNFα using ELISA. Following culture, explants were embedded in glycol methacrylate and immunohistochemical staining was conducted to determine the cellular source of TNFα, and numbers of macrophages, neutrophils and mast cells.

Results

In our study TNFα was the initial and predictive cytokine released followed by IL-6, CXCL8 and IL-10 in the cytokine cascade following LPS exposure. The cytokine cascade was inhibited by the neutralisation of the TNFα released in response to LPS and augmented by the neutralisation of the anti-inflammatory cytokine IL-10. Immunohistochemical analysis indicated that TNFα was predominantly expressed in macrophages and mast cells. When patients were stratified by GOLD status, GOLD I (n = 11) and II (n = 13) individuals had an exaggerated TNFα responses but lacked a robust IL-10 response compared to patients with normal lung function (n = 13).

Conclusion

We report on a reliable ex vitro model for the investigation of acute lung inflammation and its resolution using lung parenchymal explants from COPD patients. We propose that differences in the production of both TNFα and IL-10 in COPD lung tissue following exposure to bacterial LPS may have important biological implications for both episodes of exacerbation, disease progression and amelioration.     

Effect of recombinant IL-10 on cultured fetal rat alveolar type II cells exposed to 65%-hyperoxia

Background

Hyperoxia plays an important role in the genesis of lung injury in preterm infants. Although alveolar type II cells are the main target of hyperoxic lung injury, the exact mechanisms whereby hyperoxia on fetal alveolar type II cells contributes to the genesis of lung injury are not fully defined, and there have been no specific measures for protection of fetal alveolar type II cells.

Objective

The aim of this study was to investigate (a) cell death response and inflammatory response in fetal alveolar type II cells in the transitional period from canalicular to saccular stages during 65%-hyperoxia and (b) whether the injurious stimulus is promoted by creating an imbalance between pro- and anti-inflammatory cytokines and (c) whether treatment with an anti-inflammatory cytokine may be effective for protection of fetal alveolar type II cells from injury secondary to 65%-hyperoxia.

Methods

Fetal alveolar type II cells were isolated on embryonic day 19 and exposed to 65%-oxygen for 24 h and 36 h. Cells in room air were used as controls. Cellular necrosis was assessed by lactate dehydrogenase-release and flow cytometry, and apoptosis was analyzed by TUNEL assay and flow cytometry, and cell proliferation was studied by BrdU incorporation. Release of cytokines including VEGF was analyzed by ELISA, and their gene expressions were investigated by qRT-PCR.

Results

65%-hyperoxia increased cellular necrosis, whereas it decreased cell proliferation in a time-dependent manner compared to controls. 65%-hyperoxia stimulated IL-8-release in a time-dependent fashion, whereas the anti-inflammatory cytokine, IL-10, showed an opposite response. 65%-hyperoxia induced a significant decrease of VEGF-release compared to controls, and similar findings were observed on IL-8/IL-10/VEGF genes expression. Preincubation of recombinant IL-10 prior to 65%-hyperoxia decreased cellular necrosis and IL-8-release, and increased VEGF-release and cell proliferation significantly compared to hyperoxic cells without IL-10.

Conclusions

The present study provides an experimental evidence that IL-10 may play a potential role in protection of fetal alveolar type II cells from injury induced by 65%-hyperoxia.     

Microarray analysis identifies IL-1 receptor type 2 as a novel candidate biomarker in patients with acute respiratory distress syndrome

Background

Acute respiratory distress syndrome (ARDS) is a disease associated with a high mortality rate. The initial phase is characterized by induction of inflammatory cytokines and chemokines and influx of circulating inflammatory cells, including macrophages which play a pivotal role in the innate and adaptive immune responses to injury. Growing evidence points to phenotypic heterogeneity and plasticity between various macrophage activation states.

Methods

In this study, gene expression in alveolar macrophages and circulating leukocytes from healthy control subjects and patients with ARDS was assessed by mRNA microarray analysis.

Results

Both alveolar macrophages and circulating leukocytes demonstrated up-regulation of genes encoding chemotactic factors, antimicrobial peptides, chemokine receptors, and matrix metalloproteinases. Two genes, the pro-inflammatory S100A12 and the anti-inflammatory IL-1 decoy receptor IL-1R2 were significantly induced in both cell populations in ARDS patients, which was confirmed by protein quantification. Although S100A12 levels did not correlate with disease severity, there was a significant association between early plasma levels of IL-1R2 and APACHE III scores at presentation. Moreover, higher levels of IL-1R2 in plasma were observed in non-survivors as compared to survivors at later stages of ARDS.

Conclusions

These results suggest a hybrid state of alveolar macrophage activation in ARDS, with features of both alternative activation and immune tolerance/deactivation.. Furthermore, we have identified a novel plasma biomarker candidate in ARDS that correlates with the severity of systemic illness and mortality.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0190-x) contains supplementary material, which is available to authorized users.     

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.     

Salivary Inflammatory Mediator Profiling and Correlation to Clinical Disease Markers in Asthma

Rationale

There is a need for a readily available, non-invasive source of biomarkers that predict poor asthma control.

Objectives

We sought to determine if there is an association between the salivary inflammatory profile and disease control in children and adults with asthma.

Methods

In this cross-sectional study, we collected demographic and clinical information from two independent populations at different sites, resulting in convenience samples of 58 pediatric and 122 adult urban asthmatics. Control was assessed by symptom questionnaire (children) and by Asthma Control Questionnaire and current exacerbation (adults). Saliva was collected in all subjects. We applied principal component analysis to a 10-plex panel of relevant inflammatory markers to characterize marker profiles and determined if profiles were associated with asthma control.

Results

There were similar, strong correlations amongst biologically related markers in both populations: eosinophil-related: eotaxin-1/CCL11, RANTES/CCL5, and IL-5 (p<.001); myeloid/innate: IL-1β, IL-6, MCP-1/CCL2, and IL-8/CXCL8 (p<.001). The first three principal components captured ≥74% of variability across all ten analytes in both populations. In adults, the Principal Component 1 score, broadly reflective of all markers, but with greater weight given to myeloid/innate markers, was associated with Asthma Control Questionnaire score and exacerbation. The Principal Component 3 score, reflective of IP-10/CXCL10, was associated with current exacerbation. In children, the Principal Component 1, 2, and 3 scores were associated with recent asthma symptoms. The Principal Component 2 score, reflective of higher eosinophil markers, was inversely correlated with symptoms. The Principal Component 3 score was positively associated with all symptom outcomes.

Conclusion

The salivary inflammatory profile is associated with disease control in children and adults with asthma.     

Vitamin D deficiency exacerbates COPD-like characteristics in the lungs of cigarette smoke-exposed mice

Background

Chronic obstructive pulmonary disease (COPD) is characterized by excessive inflammation and disturbed bacterial clearance in the airways. Although cigarette smoke (CS) exposure poses a major risk, vitamin D deficiency could potentially contribute to COPD progression. Many in vitro studies demonstrate important anti-inflammatory and antibacterial effects of vitamin D, but a direct contribution of vitamin D deficiency to COPD onset and disease progression has not been explored.

Methods

In the current study, we used a murine experimental model to investigate the combined effect of vitamin D deficiency and CS exposure on the development of COPD-like characteristics. Therefore, vitamin D deficient or control mice were exposed to CS or ambient air for a period of 6 (subacute) or 12 weeks (chronic). Besides lung function and structure measurements, we performed an in depth analysis of the size and composition of the cellular infiltrate in the airways and lung parenchyma and tested the ex vivo phagocytic and oxidative burst capacity of alveolar macrophages.

Results

Vitamin D deficient mice exhibited an accelerated lung function decline following CS exposure compared to control mice. Furthermore, early signs of emphysema were only observed in CS-exposed vitamin D deficient mice, which was accompanied by elevated levels of MMP-12 in the lung. Vitamin D deficient mice showed exacerbated infiltration of inflammatory cells in the airways and lung parenchyma after both subacute and chronic CS exposure compared to control mice. Furthermore, elevated levels of typical proinflammatory cytokines and chemokines could be detected in the bronchoalveolar lavage fluid (KC and TNF-α) and lung tissue (IP-10, MCP-1, IL-12) of CS-exposed vitamin D deficient mice compared to control mice. Finally, although CS greatly impaired the ex vivo phagocytic and oxidative burst function of alveolar macrophages, vitamin D deficient mice did not feature an additional defect.

Conclusions

Our data demonstrate that vitamin D deficiency both accelerates and aggravates the development of characteristic disease features of COPD. As vitamin D deficiency is highly prevalent, large randomized trials exploring effects of vitamin D supplementation on lung function decline and COPD onset are needed.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0271-x) contains supplementary material, which is available to authorized users.     

Smoking status and anti-inflammatory macrophages in bronchoalveolar lavage and induced sputum in COPD

Background

Macrophages have been implicated in the pathogenesis of COPD. M1 and M2 macrophages constitute subpopulations displaying pro- and anti-inflammatory properties. We hypothesized that smoking cessation affects macrophage heterogeneity in the lung of patients with COPD. Our aim was to study macrophage heterogeneity using the M2-marker CD163 and selected pro- and anti-inflammatory mediators in bronchoalveolar lavage (BAL) fluid and induced sputum from current smokers and ex-smokers with COPD.

Methods

114 COPD patients (72 current smokers; 42 ex-smokers, median smoking cessation 3.5 years) were studied cross-sectionally and underwent sputum induction (M/F 99/15, age 62 ± 8 [mean ± SD] years, 42 (31-55) [median (range)] packyears, post-bronchodilator FEV₁ 63 ± 9% predicted, no steroids past 6 months). BAL was collected from 71 patients. CD163⁺ macrophages were quantified in BAL and sputum cytospins. Pro- and anti-inflammatory mediators were measured in BAL and sputum supernatants.

Results

Ex-smokers with COPD had a higher percentage, but lower number of CD163⁺ macrophages in BAL than current smokers (83.5% and 68.0%, p = 0.04; 5.6 and 20.1 ×10⁴/ml, p = 0.001 respectively). The percentage CD163⁺ M2 macrophages was higher in BAL compared to sputum (74.0% and 30.3%, p < 0.001). BAL M-CSF levels were higher in smokers than ex-smokers (571 pg/ml and 150 pg/ml, p = 0.001) and correlated with the number of CD163⁺ BAL macrophages (Rs = 0.38, p = 0.003). No significant differences were found between smokers and ex-smokers in the levels of pro-inflammatory (IL-6 and IL-8), and anti-inflammatory (elafin, and Secretory Leukocyte Protease Inhibitor [SLPI]) mediators in BAL and sputum.

Conclusions

Our data suggest that smoking cessation partially changes the macrophage polarization in vivo in the periphery of the lung towards an anti-inflammatory phenotype, which is not accompanied by a decrease in inflammatory parameters.     

A Combined CXCL10, CXCL8 and H-FABP Panel for the Staging of Human African Trypanosomiasis Patients

Background

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic tropical disease. It progresses from the first, haemolymphatic stage to a neurological second stage due to invasion of parasites into the central nervous system (CNS). As treatment depends on the stage of disease, there is a critical need for tools that efficiently discriminate the two stages of HAT. We hypothesized that markers of brain damage discovered by proteomic strategies and inflammation-related proteins could individually or in combination indicate the CNS invasion by the parasite.

Methods

Cerebrospinal fluid (CSF) originated from parasitologically confirmed Trypanosoma brucei gambiense patients. Patients were staged on the basis of CSF white blood cell (WBC) count and presence of parasites in CSF. One hundred samples were analysed: 21 from stage 1 (no trypanosomes in CSF and ≤5 WBC/µL) and 79 from stage 2 (trypanosomes in CSF and/or >5 WBC/µL) patients. The concentration of H-FABP, GSTP-1 and S100β in CSF was measured by ELISA. The levels of thirteen inflammation-related proteins (IL-1ra, IL-1β, IL-6, IL-9, IL-10, G-CSF, VEGF, IFN-γ, TNF-α, CCL2, CCL4, CXCL8 and CXCL10) were determined by bead suspension arrays.

Results

CXCL10 most accurately distinguished stage 1 and stage 2 patients, with a sensitivity of 84% and specificity of 100%. Rule Induction Like (RIL) analysis defined a panel characterized by CXCL10, CXCL8 and H-FABP that improved the detection of stage 2 patients to 97% sensitivity and 100% specificity.

Conclusion

This study highlights the value of CXCL10 as a single biomarker for staging T. b. gambiense-infected HAT patients. Further combination of CXCL10 with H-FABP and CXCL8 results in a panel that efficiently rules in stage 2 HAT patients. As these molecules could potentially be markers of other CNS infections and disorders, these results should be validated in a larger multi-centric cohort including other inflammatory diseases such as cerebral malaria and active tuberculosis.     

Lung CD8+ T cells in COPD have increased expression of bacterial TLRs

Background

Toll-like receptors (TLRs) on T cells can modulate their responses, however, the extent and significance of TLR expression by lung T cells, NK cells, or NKT cells in chronic obstructive pulmonary disease (COPD) is unknown.

Methods

Lung tissue collected from clinically-indicated resections (n = 34) was used either: (a) to compare the expression of TLR1, TLR2, TLR2/1, TLR3, TLR4, TLR5, TLR6 and TLR9 on lung CD8+ T cells, CD4+ T cells, NK cells and NKT cells from smokers with or without COPD; or (b) to isolate CD8+ T cells for culture with anti-CD3ε without or with various TLR ligands. We measured protein expression of IFN-γ, TNF-α, IL-13, perforin, granzyme A, granzyme B, soluble FasL, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL9 in supernatants.

Results

All the lung subsets analyzed demonstrated low levels of specific TLR expression, but the percentage of CD8+ T cells expressing TLR1, TLR2, TLR4, TLR6 and TLR2/1 was significantly increased in COPD subjects relative to those without COPD. In contrast, from the same subjects, only TLR2/1 and TLR2 on lung CD4+ T cells and CD8+ NKT cells, respectively, showed a significant increase in COPD and there was no difference in TLR expression on lung CD56+ NK cells. Production of the Tc1 cytokines IFN-γ and TNF-α by lung CD8+ T cells were significantly increased via co-stimulation by Pam3CSK4, a specific TLR2/1 ligand, but not by other agonists. Furthermore, this increase in cytokine production was specific to lung CD8+ T cells from patients with COPD as compared to lung CD8+ T cells from smokers without COPD.

Conclusions

These data suggest that as lung function worsens in COPD, the auto-aggressive behavior of lung CD8+ T cells could increase in response to microbial TLR ligands, specifically ligands against TLR2/1.     

Atorvastatin Reduces Plasma Levels of Chemokine (CXCL10) in Patients with Crohn's Disease

Background

In Crohn''s disease high tissue expression and serum levels of chemokines and their receptors are known to correlate with disease activity. Because statins can reduce chemokine expression in patients with coronary diseases, we wanted to test whether this can be achieved in patients with Crohn''s disease.

Methodology/Principal Findings

We investigated plasma levels of chemokines (CCL2, CCL4, CCL11, CCL13, CCL17, CCL22, CCL26, CXCL8, CXCL10) and endothelial cytokines (sP-selectin, sE-selectin, sICAM-3, thrombomodulin) in ten Crohn''s disease patients before and after thirteen weeks'' daily treatment with 80 mg atorvastatin. Of the 13 substances investigated, only CXCL10 was found to be significantly reduced (by 34%, p = 0.026) in all of the treated patients. Levels of CXCL10 correlated with C-reactive protein (r = 0.82, p<0.01).

Conclusions/Significance

CXCL10 is a ligand for the CXCR3 receptor, the activation of which results in the recruitment of T lymphocytes and the perpetuation of mucosal inflammation. Hence the reduction of plasma CXCL10 levels by atorvastatin may represent a candidate for an approach to the treatment of Crohns disease in the future.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00454545","term_id":"NCT00454545"}}NCT00454545     

Alveolar macrophages regulate neutrophil recruitment in endotoxin-induced lung injury

Background

Alveolar macrophages play an important role during the development of acute inflammatory lung injury. In the present study, in vivo alveolar macrophage depletion was performed by intratracheal application of dichloromethylene diphosphonate-liposomes in order to study the role of these effector cells in the early endotoxin-induced lung injury.

Methods

Lipopolysaccharide was applied intratracheally and the inflammatory reaction was assessed 4 hours later. Neutrophil accumulation and expression of inflammatory mediators were determined. To further analyze in vivo observations, in vitro experiments with alveolar epithelial cells and alveolar macrophages were performed.

Results

A 320% increase of polymorphonuclear leukocytes in bronchoalveolar lavage fluid was observed in macrophage-depleted compared to macrophage-competent lipopolysaccharide-animals. This neutrophil recruitment was also confirmed in the interstitial space. Monocyte chemoattractant protein-1 concentration in bronchoalveolar lavage fluid was significantly increased in the absence of alveolar macrophages. This phenomenon was underlined by in vitro experiments with alveolar epithelial cells and alveolar macrophages. Neutralizing monocyte chemoattractant protein-1 in the airways diminished neutrophil accumulation.

Conclusion

These data suggest that alveolar macorphages play an important role in early endotoxin-induced lung injury. They prevent neutrophil influx by controlling monocyte chemoattractant protein-1 production through alveolar epithelial cells. Alveolar macrophages might therefore possess robust anti-inflammatory effects.