Gene Expression Profile of Endotoxin-stimulated Leukocytes of the Term New Born: Control of Cytokine Gene Expression by Interleukin-10

Introduction

Increasing evidence now supports the association between the fetal inflammatory response syndrome (FIRS) with the pathogenesis of preterm labor, intraventricular hemorrhage and bronchopulmonary dysplasia. Polymorphonuclear leukocyte (PMNs) and mononuclear cell (MONOs) infiltration of the placenta is associated with these disorders. The aim of this study was to reveal cell-specific differences in gene expression and cytokine release in response to endotoxin that would elucidate inflammatory control mechanisms in the newly born.

Methods

PMNs and MONOs were separately isolated from the same cord blood sample. A genome-wide microarray screened for gene expression and related pathways at 4 h of LPS stimulation (n = 5). RT-qPCR and ELISA were performed for selected cytokines at 4 h and 18 h of LPS stimulation.

Results

Compared to PMNs, MONOs had a greater diversity and more robust gene expression that included pro-inflammatory (PI) cytokines, chemokines and growth factors at 4 h. Only MONOs had genes changing expression (all up regulated including interleukin-10) that were clustered in the JAK/STAT pathway. Pre-incubation with IL-10 antibody, for LPS-stimulated MONOs, led to up regulated PI and IL-10 gene expression and release of PI cytokines after 4 h.

Discussion

The present study suggests a dominant role of MONO gene expression in control of the fetal inflammatory response syndrome at 4 hrs of LPS stimulation. LPS-stimulated MONOs but not PMNs of the newborn have the ability to inhibit PI cytokine gene expression by latent IL-10 release.     

Macrophage Dysfunction Impairs Resolution of Inflammation in the Wounds of Diabetic Mice

Background

Chronic inflammation is a characteristic feature of diabetic cutaneous wounds. We sought to delineate novel mechanisms involved in the impairment of resolution of inflammation in diabetic cutaneous wounds. At the wound-site, efficient dead cell clearance (efferocytosis) is a pre-requisite for the timely resolution of inflammation and successful healing.

Methodology/Principal Findings

Macrophages isolated from wounds of diabetic mice showed significant impairment in efferocytosis. Impaired efferocytosis was associated with significantly higher burden of apoptotic cells in wound tissue as well as higher expression of pro-inflammatory and lower expression of anti-inflammatory cytokines. Observations related to apoptotic cell load at the wound site in mice were validated in the wound tissue of diabetic and non-diabetic patients. Forced Fas ligand driven elevation of apoptotic cell burden at the wound site augmented pro-inflammatory and attenuated anti-inflammatory cytokine response. Furthermore, successful efferocytosis switched wound macrophages from pro-inflammatory to an anti-inflammatory mode.

Conclusions/Significance

Taken together, this study presents first evidence demonstrating that diabetic wounds suffer from dysfunctional macrophage efferocytosis resulting in increased apoptotic cell burden at the wound site. This burden, in turn, prolongs the inflammatory phase and complicates wound healing.     

IL-17A expression is localised to both mononuclear and polymorphonuclear synovial cell infiltrates

Introduction

This study examines the expression of IL-17A-secreting cells within the inflamed synovium and the relationship to in vivo joint hypoxia measurements.

Methods

IL-17A expression was quantified in synovial tissue (ST), serum and synovial fluid (SF) by immunohistochemistry and MSD-plex assays. IL-6 SF and serum levels were measured by MSD-plex assays. Dual immunofluorescence for IL-17A was quantified in ST CD15+ cells (neutrophils), Tryptase+ (mast cells) and CD4+ (T cells). Synovial tissue oxygen (tpO₂) levels were measured under direct visualisation at arthroscopy. Synovial infiltration was assessed using immunohistochemistry for cell specific markers. Peripheral blood mononuclear and polymorphonuclear cells were isolated and exposed to normoxic or 3% hypoxic conditions. IL-17A and IL-6 were quantified as above in culture supernatants.

Results

IL-17A expression was localised to mononuclear and polymorphonuclear (PMN) cells in inflamed ST. Dual immunoflourescent staining co-localised IL-17A expression with CD15+ neutrophils Tryptase+ mast cells and CD4+T cells. % IL-17A positivity was highest on CD15+ neutrophils, followed by mast cells and then CD4+T-cells. The number of IL-17A-secreting PMN cells significantly correlated with sublining CD68 expression (r = 0.618, p<0.01). IL-17A SF levels correlated with IL-6 SF levels (r = 0.675, p<0.01). Patients categorized according to tp0₂< or >20mmHg, showed those with low tp0₂<20mmHg had significantly higher IL-17A+ mononuclear cells with no difference observed for PMNs. Exposure of mononuclear and polymorphonuclear cells to 3% hypoxia, significantly induced IL-6 in mononuclear cells, but had no effect on IL-17A expression in mononuclear and polymorphonuclear cells.

Conclusion

This study demonstrates IL-17A expression is localised to several immune cell subtypes within the inflamed synovial tissue, further supporting the concept that IL-17A is a key mediator in inflammatory arthritis. The association of hypoxia with Il-17A expression appears to be indirect, probably through hypoxia-induced pro-inflammatory pathways and leukocyte influx within the joint microenvironment.     

A differential concentration-dependent effect of IVIg on neutrophil functions: relevance for anti-microbial and anti-inflammatory mechanisms

Background

Polymorphonuclear neutrophils (PMN) play a key role in host defences against invading microorganisms but can also potentiate detrimental inflammatory reactions in case of excessive or misdirected responses. Intravenous immunoglobulins (IVIg) are used to treat patients with immune deficiencies and, at higher doses, in autoimmune, allergic and systemic inflammatory disorders.

Methodology/Principal Findings

We used flow cytometry to examine the effects of IVIg on PMN functions and survival, using whole-blood conditions in order to avoid artifacts due to isolation procedures. IVIg at low concentrations induced PMN activation, as reflected by decreased L-selectin and increased CD11b expression at the PMN surface, oxidative burst enhancement, and prolonged cell survival. In contrast, IVIg at higher concentrations inhibited LPS-induced CD11b degranulation and oxidative burst priming, and counteracted LPS-induced PMN lifespan prolongation.

Conclusions/Significance

IVIg appears to have differential, concentration-dependent effects on PMN, possibly supporting the use of IVIg as either an anti-microbial or an anti-inflammatory agent.     

Mesenchymal Stem Cells Augment the Anti-Bacterial Activity of Neutrophil Granulocytes

Background

Mesenchymal stem cells (MSCs) participate in the regulation of inflammation and innate immunity, for example by responding to pathogen-derived signals and by regulating the function of innate immune cells. MSCs from the bone-marrow and peripheral tissues share common basic cell-biological functions. However, it is unknown whether these MSCs exhibit different responses to microbial challenge and whether this response subsequently modulates the regulation of inflammatory cells by MSCs.

Methodology/Principal Findings

We isolated MSCs from human bone-marrow (bmMSCs) and human salivary gland (pgMSCs). Expression levels of TLR4 and LPS-responsive molecules were determined by flow cytometry and quantitative PCR. Cytokine release was determined by ELISA. The effect of supernatants from unstimulated and LPS-stimulated MSCs on recruitment, cytokine secretion, bacterial clearance and oxidative burst of polymorphonuclear neutrophil granulocytes (PMN) was tested in vitro. Despite minor quantitative differences, bmMSCs and pgMSCs showed a similar cell biological response to bacterial endotoxin. Both types of MSCs augmented anti-microbial functions of PMNs LPS stimulation, particularly of bmMSCs, further augmented MSC-mediated activation of PMN.

Conclusions/Significance

This study suggests that MSCs may contribute to the resolution of infection and inflammation by promoting the anti-microbial activity of PMNs. This property is exerted by MSCs derived from both the bone-marrow and peripheral glandular tissue.     

Expression of human beta-defensins in children with chronic inflammatory bowel disease

Background

Human beta-defensins (hBDs) are antimicrobial peptides known to play a major role in intestinal innate host defence. Altered mucosal expression of hBDs has been suggested to be implicated in chronic inflammatory bowel disease pathogenesis. However, little is known about expression of these peptides in children.

Methods

Intestinal biopsies were obtained from the duodenum (n = 88), terminal ileum (n = 90) and ascending colon (n = 105) of children with Crohn''s disease (n = 26), ulcerative colitis (n = 11) and healthy controls (n = 16). Quantitative real-time (RT) PCR was performed and absolute mRNA copy numbers analyzed for hBD1-3 as well as inflammatory cytokines IL-8 and TNF-alpha.

Results

Significant induction of hBD2 and hBD3 was observed in the inflamed terminal ileum and ascending colon of IBD children. In the ascending colon induction of hBD2 was found to be significantly lower in children with Crohn''s disease compared to ulcerative colitis. A strong correlation was found between inducible defensins hBD2 and 3 and the inflammatory cytokines IL-8 and TNF-alpha, both in the terminal ileum and ascending colon.

Conclusion

Our study demonstrates distinct changes in hBD expression throughout the intestinal tract of children with IBD, lending further support for their potential role in disease pathogenesis.     

A self-propagating matrix metalloprotease-9 (MMP-9) dependent cycle of chronic neutrophilic inflammation

Background

Chronic neutrophilic inflammation is a poorly understood feature in a variety of diseases with notable worldwide morbidity and mortality. We have recently characterized N-acetyl Pro-Gly-Pro (Ac-PGP) as an important neutrophil (PMN) chemoattractant in chronic inflammation generated from the breakdown of collagen by the actions of MMP-9. MMP-9 is present in the granules of PMNs and is differentially released during inflammation but whether Ac-PGP contributes to this ongoing proteolytic activity in chronic neutrophilic inflammation is currently unknown.

Methodology/Principal Findings

Utilizing isolated primary blood PMNs from human donors, we found that Ac-PGP induces significant release of MMP-9 and concurrently activates the ERK1/2 MAPK pathway. This MMP-9 release is attenuated by an inhibitor of ERK1/2 MAPK and upstream blockade of CXCR1 and CXCR2 receptors with repertaxin leads to decreased MMP-9 release and ERK 1/2 MAPK activation. Supernatants obtained from PMNs stimulated by Ac-PGP generate more Ac-PGP when incubated with intact collagen ex vivo; this effect is inhibited by an ERK1/2 pathway inhibitor. Finally, clinical samples from individuals with CF demonstrate a notable correlation between Ac-PGP (as measured by liquid chromatography-tandem mass spectrometry) and MMP-9 levels even when accounting for total PMN burden.

Conclusions/Significance

These data indicate that ECM-derived Ac-PGP could result in a feed-forward cycle by releasing MMP-9 from activated PMNs through the ligation of CXCR1 and CXCR2 and subsequent activation of the ERK1/2 MAPK, highlighting for the first time a matrix-derived chemokine (matrikine) augmenting its generation through a discrete receptor/intracellular signaling pathway. These findings have notable implications to the development unrelenting chronic PMN inflammation in human disease.     

Two Chromogranin A-Derived Peptides Induce Calcium Entry in Human Neutrophils by Calmodulin-Regulated Calcium Independent Phospholipase A2

Background

Antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides.

Methodology/Principal Findings

PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity.

Conclusions/Significance

For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.     

IL-27-Induced Gene Expression Is Downregulated in HIV-Infected Subjects

Objective

To characterize the effect of HIV infection on IL-27-induced gene expression.

Design

During HIV infection, cytokine expression and function become deregulated. IL-27 is an important modulator of inflammatory responses. Interestingly, IL-27 can inhibit HIV replication in T cells and monocytes, implicating IL-27 as a potential adjunct to anti-viral treatment. Our previous work demonstrated that circulating HIV may suppress IL-27 expression, therefore, this study, in continuation of our previous work, aimed to understand how HIV affects expression levels of the IL-27 receptor and downstream functions of IL-27.

Methods

Peripheral blood mononuclear cells (PBMC) were isolated from whole blood of HIV negative and HIV positive (viremic) individuals to assess IL-27-induced gene expression by flow cytometry and ELISA. PBMC were also processed for monocyte enrichment to assess IL-27 receptor expression by flow cytometry and real-time PCR.

Results

Expression of the IL-27 receptor subunit, gp130, was upregulated in response to IL-27 in HIV negative individuals, however, in HIV positive individuals, this IL-27 response was diminished. Furthermore, we observed downregulation of IL-27-induced IL-6, TNF-α, and IL-10 expression in HIV positive subjects.

Conclusion

In HIV infection, IL-27-induced gene expression was impaired, indicating HIV-mediated dysregulation of IL-27 functions occurs during HIV infection. This study provides evidence for new viral pathogenic mechanisms contributing to the widespread impairment of immune responses observed in HIV pathogenesis.     

Flagellin-Induced Corneal Antimicrobial Peptide Production and Wound Repair Involve a Novel NF-κB–Independent and EGFR-Dependent Pathway

Background

The bacterial protein flagellin plays a major role in stimulating mucosal surface innate immune response to bacterial infection and uniquely induces profound cytoprotection against pathogens, chemicals, and radiation. This study sought to determine signaling pathways responsible for the flagellin-induced inflammatory and cytoprotective effects on human corneal epithelial cells (HCECs).

Methodology/Principal Findings

Flagellin purified from Pseudomonas aeruginosa (strain PAK) or live bacteria were used to challenge cultured HCECs. The activation of signaling pathways was assessed with Western blot, and the secretion of cytokine/chemokine and production of antimicrobial peptides (AMPs) were measured with ELISA and dot blot, respectively. Effects of flagellin on wound healing were assessed in cultured porcine corneas. L94A (a site mutation in TLR5 binding region) flagellin and PAK expressing L94A flagellin were unable to stimulate NF-κB activation, but were potent in eliciting EGFR signaling in a TGF-α–related pathway in HCECs. Concomitant with the lack of NF-κB activation, L94A flagellin was ineffective in inducing IL-6 and IL-8 production in HCECs. Surprisingly, the secretion of two inducible AMPs, LL-37 and hBD2, was not affected by L94A mutation. Similar to wild-type flagellin, L94A induced epithelial wound closure in cultured porcine cornea through maintaining EGFR-mediated signaling.

Conclusions/Significance

Our data suggest that inflammatory response mediated by NF-κB can be uncoupled from epithelial innate defense machinery (i.e., AMP expression) and major epithelial proliferation/repair pathways mediated by EGFR, and that flagellin and its derivatives may have broad therapeutic applications in cytoprotection and in controlling infection in the cornea and other mucosal tissues.     

Utility of Intraoperative Frozen Section in the Diagnosis of Periprosthetic Joint Infection

Purpose

Intraoperative frozen section (FS) is an effective diagnostic test for periprosthetic joint infection (PJI). We evaluated the diagnostic characteristics of single- and multiplex-site intraoperative FS, and evaluated the results of single-site FS combined with those of C-reactive protein (CRP) level and erythrocyte sedimentation rate (ESR) for assessing PJI.

Methods

We studied 156 painful joint arthroplasties in 152 consecutive patients presenting for revision total joint arthroplasty due to PJI. Receiver operating characteristic analysis was used to determine the optimal cutoff values for CRP level, ESR, and intraoperative FS histopathology. Sensitivity, specificity, positive and negative predictive values, and accuracy of the diagnostic tests were assessed using a 2×2 table.

Results

We investigated the diagnostic utility of polymorphonuclear leukocyte number (PMN) per high-power field (HPF) on FS. Our data showed that 5 PMNs per HPF is a suitable diagnostic threshold, with a high accuracy in single- and multiplex-site FS. Five PMNs in any 1 of 5 sites had the highest sensitivity of 0.86 and a specificity of 0.96. Five PMNs in every 1 of 5 sites had greater diagnostic utility, with a specificity of 1; however, the sensitivity of this measure fell to 0.62. Five PMNs in single-site FS had a sensitivity of 0.70 and a specificity of 0.94. Five PMNs in single-site FS or CRP level ≥15 mg/L increased the sensitivity to 0.92; however, the specificity decreased to 0.79.

Conclusion

Compared with single-site FS, any 1 positive site on multiplex-site FS may improve sensitivity, while every 1 positive site on multiplex-site FS may improve specificity. Five PMNs in any 1 of 5 sites on FS has excellent utility for the diagnosis of PJI. Additional systematic large-scale studies are needed to verify this result.     

Coenzyme Q10 Ameliorates Pain and Cartilage Degradation in a Rat Model of Osteoarthritis by Regulating Nitric Oxide and Inflammatory Cytokines

Objective

To investigate the effect of CoenzymeQ10 (CoQ10) on pain severity and cartilage degeneration in an experimental model of rat osteoarthritis (OA).

Materials and Methods

OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA) to the knee. Oral administration of CoQ10 was initiated on day 4 after MIA injection. Pain severity was assessed by measuring secondary tactile allodynia using the von Frey assessment test. The degree of cartilage degradation was determined by measuring cartilage thickness and the amount of proteoglycan. The mankin scoring system was also used. Expressions of matrix metalloproteinase-13 (MMP-13), interleukin-1β (IL-1β), IL-6, IL-15, inducible nitric oxide synthase (iNOS), nitrotyrosine and receptor for advanced glycation end products (RAGE) were analyzed using immunohistochemistry.

Results

Treatment with CoQ10 demonstrated an antinociceptive effect in the OA animal model. The reduction in secondary tactile allodynia was shown by an increased pain withdrawal latency and pain withdrawal threshold. CoQ10 also attenuated cartilage degeneration in the osteoarthritic joints. MMP-13, IL-1β, IL-6, IL-15, iNOS, nitrotyrosine and RAGE expressions were upregulated in OA joints and significantly reduced with CoQ10 treatment.

Conclusion

CoQ10 exerts a therapeutic effect on OA via pain suppression and cartilage degeneration by inhibiting inflammatory mediators, which play a vital role in OA pathogenesis.     

Functional characterization of cultured keratinocytes after acute cutaneous burn injury

Background

In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable - keratinocyte-derived - cytokine and growth factor milieu.

Methods

In this study the morphology and cytokine expression profile of keratinocytes from skin after acute burn injury was compared to non-burned skin. Skin samples were obtained from patients after severe burn injury and healthy controls. Cells were cultured and secretion of selected inflammatory mediators was quantified using Bioplex Immunoassays. Immunohistochemistry was performed to analyse further functional and morphologic parameters.

Results

Histology revealed increased terminal differentiation of keratinocytes (CK10, CK11) in allografts from non-burned skin compared to a higher portion of proliferative cells (CK5, vimentin) in acute burn injury. Increased levels of IL-1α, IL-2, IL-4, IL-10, IFN-γ and TNFα could be detected in culture media of burn injury skin cultures. Both culture groups contained large amounts of IL-1RA. IL-6 and GM-CSF were increased during the first 15 days of culture of burned skin compared to control skin. Levels of VEGF, FGF-basic, TGF-ß und G-CSF were high in both but not significantly different. Cryoconservation led to a diminished mediator synthesis except for higher levels of intracellular IL-1α and IL-1ß.

Conclusion

Skin allografts from non-burned skin show a different secretion pattern of keratinocyte-derived cytokines and inflammatory mediators compared to keratinocytes after burn injury. As these secreted molecules exert auto- and paracrine effects and subsequently contribute to healing and barrier restoration after acute burn injury therapies affecting this specific cytokine/growth factor micromilieu could be beneficial in burned patients.     

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.     

Endogenous Morphine Levels Are Increased in Sepsis: A Partial Implication of Neutrophils

Background

Mammalian cells synthesize morphine and the respective biosynthetic pathway has been elucidated. Human neutrophils release this alkaloid into the media after exposure to morphine precursors. However, the exact role of endogenous morphine in inflammatory processes remains unclear. We postulate that morphine is released during infection and can be determined in the serum of patients with severe infection such as sepsis.

Methodology

The presence and subcellular immunolocalization of endogenous morphine was investigated by ELISA, mass spectrometry analysis and laser confocal microscopy. Neutrophils were activated with Interleukin-8 (IL-8) or lipopolysaccharide (LPS). Morphine secretion was determined by a morphine-specific ELISA. μ opioid receptor expression was assessed with flow cytometry. Serum morphine concentrations of septic patients were determined with a morphine-specific ELISA and morphine identity was confirmed in human neutrophils and serum of septic patients by mass spectrometry analysis. The effects of the concentration of morphine found in serum of septic patients on LPS-induced release of IL-8 by human neutrophils were tested.

Principal Findings

We confirmed the presence of morphine in human neutrophil extracts and showed its colocalisation with lactoferrin within the secondary granules of neutrophils. Morphine secretion was quantified in the supernatant of activated human polymorphonuclear neutrophils in the presence and absence of Ca²⁺. LPS and IL-8 were able to induce a significant release of morphine only in presence of Ca²⁺. LPS treatment increased μ opioid receptor expression on neutrophils. Low concentration of morphine (8 nM) significantly inhibited the release of IL-8 from neutrophils when coincubated with LPS. This effect was reversed by naloxone. Patients with sepsis, severe sepsis and septic shock had significant higher circulating morphine levels compared to patients with systemic inflammatory response syndrome and healthy controls. Mass spectrometry analysis showed that endogenous morphine from serum of patient with sepsis was identical to poppy-derived morphine.

Conclusions

Our results indicate that morphine concentrations are increased significantly in the serum of patients with systemic infection and that morphine is, at least in part, secreted from neutrophils during sepsis. Morphine concentrations equivalent to those found in the serum of septic patients significantly inhibited LPS-induced IL-8 secretion in neutrophils.     

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.     

Pharmacological characterisation of anti-inflammatory compounds in acute and chronic mouse models of cigarette smoke-induced inflammation

Background

Candidate compounds being developed to treat chronic obstructive pulmonary disease are typically assessed using either acute or chronic mouse smoking models; however, in both systems compounds have almost always been administered prophylactically. Our aim was to determine whether the prophylactic effects of reference anti-inflammatory compounds in acute mouse smoking models reflected their therapeutic effects in (more clinically relevant) chronic systems.

Methods

To do this, we started by examining the type of inflammatory cell infiltrate which occurred after acute (3 days) or chronic (12 weeks) cigarette smoke exposure (CSE) using female, C57BL/6 mice (n = 7-10). To compare the effects of anti-inflammatory compounds in these models, mice were exposed to either 3 days of CSE concomitant with compound dosing or 14 weeks of CSE with dosing beginning after week 12. Budesonide (1 mg kg^-1; i.n., q.d.), roflumilast (3 mg kg^-1; p.o., q.d.) and fluvastatin (2 mg kg^-1; p.o., b.i.d.) were dosed 1 h before (and 5 h after for fluvastatin) CSE. These dose levels were selected because they have previously been shown to be efficacious in mouse models of lung inflammation. Bronchoalveolar lavage fluid (BALF) leukocyte number was the primary endpoint in both models as this is also a primary endpoint in early clinical studies.

Results

To start, we confirmed that the inflammatory phenotypes were different after acute (3 days) versus chronic (12 weeks) CSE. The inflammation in the acute systems was predominantly neutrophilic, while in the more chronic CSE systems BALF neutrophils (PMNs), macrophage and lymphocyte numbers were all increased (p < 0.05). In the acute model, both roflumilast and fluvastatin reduced BALF PMNs (p < 0.01) after 3 days of CSE, while budesonide had no effect on BALF PMNs. In the chronic model, therapeutically administered fluvastatin reduced the numbers of PMNs and macrophages in the BALF (p ≤ 0.05), while budesonide had no effect on PMN or macrophage numbers, but did reduce BALF lymphocytes (p < 0.01). Roflumilast''s inhibitory effects on inflammatory cell infiltrate were not statistically significant.

Conclusions

These results demonstrate that the acute, prophylactic systems can be used to identify compounds with therapeutic potential, but may not predict a compound''s efficacy in chronic smoke exposure models.