Lymphocyte senescence in COPD is associated with loss of glucocorticoid receptor expression by pro-inflammatory/cytotoxic lymphocytes

Background

Glucocorticoid (GC) resistance is a major barrier in COPD treatment. We have shown increased expression of the drug efflux pump, Pgp1 in cytotoxic/pro-inflammatory lymphocytes in COPD. Loss of lymphocyte co-stimulatory molecule CD28 (lymphocyte senescence) was associated with a further increase in their pro-inflammatory/cytotoxic potential and resistance to GC. We hypothesized that lymphocyte senescence and increased Pgp1 are also associated with down-regulation of the GC receptor (GCR).

Methods

Blood was collected from 10 COPD and 10 healthy aged-matched controls. Flow cytometry was applied to assess intracellular pro-inflammatory cytokines, CD28, Pgp1, GCR, steroid binding and relative cytoplasm/nuclear GCR by CD28+ and CD28null T, NKT-like cells. GCR localization was confirmed by fluorescent microscopy.

Results

COPD was associated with increased numbers of CD28nullCD8+ T and NKT-like cells. Loss of CD28 was associated with an increased percentage of T and NKT-like cells producing IFNγ or TNFα and associated with a loss of GCR and Dex-Fluor staining but unchanged Pgp1. There was a significant loss of GCR in CD8 + CD28null compared with CD8 + CD28+ T and NKT-like cells from both COPD and controls (eg, mean ± SEM 8 ± 3% GCR + CD8 + CD28null T-cells vs 49 ± 5% GCR + CD8 + CD28+ T-cells in COPD). There was a significant negative correlation between GCR expression and IFNγ and TNFα production by T and NKT-like cells(eg, COPD: T-cell IFNγ R = −.615; ) and with FEV1 in COPD (R = −.777).

Conclusions

COPD is associated with loss of GCR in senescent CD28null and NKT-like cells suggesting alternative treatment options to GC are required to inhibit these pro-inflammatory/cytotoxic cells.     

Increased Peripheral Blood Pro-Inflammatory/Cytotoxic Lymphocytes in Children with Bronchiectasis

Objective

Bronchiectasis (BE) in children is common in some communities including Indigenous children in Australia. Relatively little is known about the nature of systemic inflammation in these children, especially the contribution of specific pro-inflammatory and cytotoxic lymphocyte subsets: T-cells, natural killer (NK) cells and NKT-like cells. We have shown that these cells produce increased cytotoxic (granzyme b and perforin) and inflammatory (IFNγ and TNFα) mediators in several adult chronic lung diseases and hypothesised that similar changes would be evident in children with BE.

Methods

Intracellular cytotoxic mediators perforin and granzyme b and pro-inflammatory cytokines were measured in T cell subsets, NKT-like and NK cells from blood and bronchoalveolar samples from 12 children with BE and 10 aged-matched control children using flow cytometry.

Results

There was a significant increase in the percentage of CD8+ T cells and T and NKT-like subsets expressing perforin/granzyme and IFNγ and TNFα in blood in BE compared with controls. There was a further increase in the percentage of pro-inflammatory cytotoxic T cells in Indigenous compared with non-Indigenous children. There was no change in any of these mediators in BAL.

Conclusions

Childhood bronchiectasis is associated with increased systemic pro-inflammatory/cytotoxic lymphocytes in the peripheral blood. Future studies need to examine the extent to which elevated levels of pro-inflammatory cytotoxic cells predict future co-morbidities.     

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.     

Enhanced effector function of cytotoxic cells in the induced sputum of COPD patients

Background

We have previously shown that NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells are reduced in both numbers and cytotoxicity in peripheral blood. The aim of the present study was to investigate their numbers and function within induced sputum.

Methods

Induced sputum cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD56⁺ cells (NK and NKT-like cells) were used in an LDH release assay to determine cytotoxicity.

Results

The proportion of NK cells and NKT-like cells in smokers with COPD (COPD subjects) was significantly higher (12.7% and 3%, respectively) than in healthy smokers (smokers) (5.7%, p < 0.01; 1%, p < 0.001) and non-smoking healthy subjects (HNS) (4.2%, p < 0.001; 0.8%, p < 0.01). The proportions of NK cells and NKT-like cells expressing both perforin and granzyme B were also significantly higher in COPD subjects compared to smokers and HNS. CD56⁺ cells from COPD subjects were significantly more cytotoxic (1414 biological lytic activity) than those from smokers (142.5; p < 0.01) and HNS (3.8; p < 0.001) and were inversely correlated to FEV₁. (r = -0.75; p = 0.0098).

Conclusion

We have shown an increased proportion of NK and NKT-like cells in the induced sputum of COPD subjects and have demonstrated that these cells are significantly more cytotoxic in COPD subjects than smokers and HNS.     

Altered effector function of peripheral cytotoxic cells in COPD

Background

There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8⁺ T lymphocytes, natural killer (NK; CD56⁺CD3^-) cells and NKT-like (CD56⁺CD3⁺) cells.

Methods

Peripheral blood mononuclear cells (PBMCs) were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies.

Results

The proportion of peripheral blood NKT-like (CD56⁺CD3⁺) cells in smokers with COPD (COPD subjects) was significantly lower (0.6%) than in healthy smokers (smokers) (2.8%, p < 0.001) and non-smoking healthy participants (HNS) (3.3%, p < 0.001). NK (CD56⁺CD3^-) cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p < 0.001) as were NKT-like (CD56⁺CD3⁺) cells (16.7% vs 52.4% specific lysis, p < 0.001). Both cell types had lower proportions expressing both perforin and granzyme B. Blocking the action of perforin and granzyme B reduced the cytotoxic activity of NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells from smokers and HNS.

Conclusion

In this study, we show that the relative numbers of peripheral blood NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells in COPD subjects are reduced and that their cytotoxic effector function is defective.     

Cigarette smoke increases TLR4 and TLR9 expression and induces cytokine production from CD8+ T cells in chronic obstructive pulmonary disease

Background

Cigarette smoke is a major risk factor for chronic obstructive pulmonary disease (COPD), an inflammatory lung disorder. COPD is characterized by an increase in CD8⁺ T cells within the central and peripheral airways. We hypothesized that the CD8⁺ T cells in COPD patients have increased Toll-like receptor (TLR) expression compared to control subjects due to the exposure of cigarette smoke in the airways.

Methods

Endobronchial biopsies and peripheral blood were obtained from COPD patients and control subjects. TLR4 and TLR9 expression was assessed by immunostaining of lung tissue and flow cytometry of the peripheral blood. CD8⁺ T cells isolated from peripheral blood were treated with or without cigarette smoke condensate (CSC) as well as TLR4 and TLR9 inhibitors. PCR and western blotting were used to determine TLR4 and TLR9 expression, while cytokine secretion from these cells was detected using electrochemiluminescence technology.

Results

No difference was observed in the overall expression of TLR4 and TLR9 in the lung tissue and peripheral blood of COPD patients compared to control subjects. However, COPD patients had increased TLR4 and TLR9 expression on lung CD8⁺ T cells. Exposure of CD8⁺ T cells to CSC resulted in an increase of TLR4 and TLR9 protein expression. CSC exposure also caused the activation of CD8⁺ T cells, resulting in the production of IL-1β, IL-6, IL-10, IL-12p70, TNFα and IFNγ. Furthermore, inhibition of TLR4 or TLR9 significantly attenuated the production of TNFα and IL-10.

Conclusions

Our results demonstrate increased expression of TLR4 and TLR9 on lung CD8⁺ T cells in COPD. CD8⁺ T cells exposed to CSC increased TLR4 and TLR9 levels and increased cytokine production. These results provide a new perspective on the role of CD8⁺ T cells in COPD.     

Adalimumab regulates intracellular TNFα production in patients with rheumatoid arthritis

Introduction

Adalimumab is a fully human anti–tumor necrosis factor α (anti-TNFα) monoclonal antibody that specifically blocks the interaction of TNFα with its receptors. It binds both soluble and transmembrane TNFα. We hypothesized that blocking these TNFα signals regulates the altered TNFα production in rheumatoid arthritis (RA) patients.

Methods

We compared, by flow cytometry, Toll-like receptor induction levels of membrane and intracellular TNFα in monocytes (iTNFα + CD14+ cells) from 12 patients before and after adalimumab treatment with those from 5 healthy donors.

Results

Before starting the treatment, the percentage of iTNFα+ CD14+ cells in the RA patients was significantly lower than that in healthy donors (mean ± SEM = 33.16 ± 4.82% vs 66.51 ± 2.4%, P < 0.001). When we added in vitro TNFα to healthy donor culture cells, levels of iTNFα+ CD14+ cells decreased, suggesting that the TNFα signal was responsible for the iTNFα+ CD14+ cell downregulation observed in the RA patients. After 2, 6 and 12 adalimumab injections, we observed significant blocking of membrane and soluble TNFα and a progressive increase in iTNFα+ CD14+ cells in ten patients with a good to moderate response as defined by the European League Against Rheumatism (EULAR) criteria. Levels of iTNFα+ CD14+ cells after 12 injections in these 10 patients were comparable to levels in healthy donors. In two patients, iTNFα+ CD14+ cell upregulation was not observed, and their EULAR-defined responses had not improved. The first patient developed antiadalimumab antibodies, explaining why adalimumab was not able to block membrane and soluble TNFα. In the second patient, adalimumab was discontinued because of adverse effects, which led to a decrease in iTNFα+ CD14+ cells to levels measured before treatment.

Conclusions

Our findings suggest that adalimumab treatment in RA patients can return iTNFα levels to those of healthy donors. This effect was not observed in the presence of neutralizing antiadalimumab antibodies.     

Healthy Neonates Possess a CD56-Negative NK Cell Population with Reduced Anti-Viral Activity

Background

Neonatal Natural Killer (NK) cells show functional impairment and expansion of a CD56 negative population of uncertain significance.

Methods

NK cells were isolated from cord blood and from adult donors. NK subpopulations were identified as positive or negative for the expression of CD56 and characterized for expression of granzyme B and surface markers by multi-parameter flow cytometry. Cell function was assessed by viral suppression and cytokine production using autologous lymphocytes infected with HIV. Activating (NKp30, NKp46) and inhibitory (Siglec-7) markers in healthy infants and adults were compared with viremic HIV-infected adults.

Results

Cord blood contained increased frequencies of CD56 negative (CD56neg) NK cells with reduced expression of granzyme B and reduced production of IFNγ and the CC-class chemokines RANTES, MIP1α and MIP1β upon stimulation. Both CD56pos and CD56neg NK subpopulations showed impaired viral suppression in cord blood, with impairment most marked in the CD56neg subset. CD56neg NK cells from cord blood and HIV-infected adults shared decreased inhibitory and activating receptor expression when compared with CD56pos cells.

Conclusions

CD56neg NK cells are increased in number in normal infants and these effectors show reduced anti-viral activity. Like the expanded CD56neg population described in HIV-infected adults, these NK cells demonstrate functional impairments which may reflect inadequate development or activation.     

The Vitamin D Receptor Agonist BXL-01-0029 as a Potential New Pharmacological Tool for the Treatment of Inflammatory Myopathies

Objective

This study aims to investigate in vitro the effect of the VDR agonist BXL-01-0029 onto IFNγ/TNFα-induced CXCL10 secretion by human skeletal muscle cells compared to elocalcitol (VDR agonist), methylprednisolone, methotrexate, cyclosporin A, infliximab and leflunomide; to assess in vivo circulating CXCL10 level in subjects at time of diagnosis with IMs, before therapy, together with TNFα, IFNγ, IL-8, IL-6, MCP-1, MIP-1β and IL-10, vs. healthy subjects.

Methods

Human fetal skeletal muscle cells were used for in vitro studies; ELISA and Bio-Plex were used to measure cell supernatant and IC₅₀ determination or serum cytokines; Western blot and Bio-Plex were for cell signaling analysis.

Results

BXL-01-0029 decreased with the highest potency IFNγ/TNFα-induced CXCL10 protein secretion and targeted cell signaling downstream of TNFα in human skeletal muscle cells; CXCL10 level was the highest in sera of subjects diagnosed with IMs before therapy and the only one significantly different vs. healthy controls.

Conclusions

Our in vitro and in vivo data, while confirm the relevance of CXCL10 in IMs, suggested BXL-01-0029 as a novel pharmacological tool for IM treatment, hypothetically to be used in combination with the current immunosuppressants to minimize side effects.     

Coincident Pre-Diabetes Is Associated with Dysregulated Cytokine Responses in Pulmonary Tuberculosis

Background

Cytokines play an important role in the pathogenesis of pulmonary tuberculosis (PTB) - Type 2 diabetes mellitus co-morbidity. However, the cytokine interactions that characterize PTB coincident with pre-diabetes (PDM) are not known.

Methods

To identify the influence of coincident PDM on cytokine levels in PTB, we examined circulating levels of a panel of cytokines in the plasma of individuals with TB-PDM and compared them with those without PDM (TB-NDM).

Results

TB-PDM is characterized by elevated circulating levels of Type 1 (IFNγ, TNFα and IL-2), Type 17 (IL-17A and IL-17F) and other pro-inflammatory (IL-1β, IFNβ and GM-CSF) cytokines. TB-PDM is also characterized by increased systemic levels of Type 2 (IL-5) and regulatory (IL-10 and TGFβ) cytokines. Moreover, TB antigen stimulated whole blood also showed increased levels of pro-inflammatory (IFNγ, TNFα and IL-1β) cytokines as well. However, the cytokines did not exhibit any significant correlation with HbA1C levels or with bacterial burdens.

Conclusion

Our data reveal that pre-diabetes in PTB individuals is characterized by heightened cytokine responsiveness, indicating that a balanced pro and anti - inflammatory cytokine milieu is a feature of pre-diabetes - TB co-morbidity.     

The Contribution of Non-Conventional T Cells and NK Cells in the Mycobacterial-Specific IFNγ Response in Bacille Calmette-Guérin (BCG)-Immunized Infants

Background

The Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is given to >120 million infants each year worldwide. Most studies investigating the immune response to BCG have focused on adaptive immunity. However the importance of TCR-gamma/delta (γδ) T cells and NK cells in the mycobacterial-specific immune response is of increasing interest.

Methods

Participants in four age-groups were BCG-immunized. Ten weeks later, in vitro BCG-stimulated blood was analyzed for NK and T cell markers, and intracellular IFNgamma (IFNγ) by flow cytometry. Total functional IFNγ response was calculated using integrated median fluorescence intensity (iMFI).

Results

In infants and children, CD4 and CD4-CD8- (double-negative (DN)) T cells were the main IFNγ-expressing cells representing 43-56% and 27-37% of total CD3+ IFNγ+ T cells respectively. The iMFI was higher in DN T cells compared to CD4 T cells in all age groups, with the greatest differences seen in infants immunized at birth (p=0.002) or 2 months of age (p<0.0001). When NK cells were included in the analysis, they accounted for the majority of total IFNγ-expressing cells and, together with DN Vδ2 γδ T cells, had the highest iMFI in infants immunized at birth or 2 months of age.

Conclusion

In addition to CD4 T cells, NK cells and DN T cells, including Vδ2 γδ T cells, are the key populations producing IFNγ in response to BCG immunization in infants and children. This suggests that innate immunity and unconventional T cells play a greater role in the mycobacterial immune response than previously recognized and should be considered in the design and assessment of novel tuberculosis vaccines.     

Cellular and Cytokine Correlates of Severe Dengue Infection

Background

The occurrence of dengue haemorrhagic fever (DHF) is thought to result from a complex interplay between the virus, host genetics and host immune factors. Existing published data are not consistent, in part related to relatively small sample sizes. We set out to determine possible associations between dengue virus (DEN-V) NS3 specific T cells and cytokine and chemokine levels and the pathogenesis of severe disease in a large cohort of individuals with DHF.

Methodology/Principal Findings

By using ex vivo IFNγ ELISpot assays we determined DENV-NS3 specific responses in patients with varying severity of DHF. Other cytokines produced by DENV-NS3 specific T cells were determined by using multiple bead array analysis (MBAA). We also determined the serum cytokine levels using MBAA, lymphocyte subsets and Annexin V expression of lymphocytes in patients with varying severity of DHF. Of the 112 DHF patients studied, 29 developed shock. Serum IL-10 and IP-10 levels positively and significantly correlated with T cell apoptosis while IL-10 levels inversely correlated with T cell numbers. In contrast, TGFß showed a very significant (P<0.0001) and positive correlation (Spearman’s R = 0.65) with the platelet counts, consistent with platelet release. We found that whilst patients with severe dengue had lower total T cell numbers, the DV-NS3 specific T cells persisted and produced high levels of IFNγ but not TNFα, IL-3, IL-13, IL-2, IL-10 or IL-17.

Conclusions/Significance

Our data suggest that serum IL-10, TNFα and TGFβ differentially associate with dengue disease severity.     

Effect of Different Interferonα2 Preparations on IP10 and ET-1 Release from Human Lung Cells

Background

Alfa-interferons (IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b) are effective treatments for chronic hepatitis C infection. However, their usage has been associated with a variety of adverse events, including interstitial pneumonitis and pulmonary arterial hypertension. Although rare, these adverse events can be severe and potentially life-threatening, emphasizing the need for simple biomarkers of IFN-induced lung toxicity.

Methods

Human lung microvascular endothelial cells (HLMVEC), human pulmonary artery smooth muscle (HPASM) cells and A549 cells were grown under standard conditions and plated into 96- or 6-well plates. Cells were stimulated with various concentrations of different IFNs in hydrocortisone-free medium. After 24 and 48 hours, IP10 and ET-1 were measured by ELISA in conditioned medium. In a second set of experiments, cells were pre-treated with tumour necrosis factor-α (TNF-α) (10 ng/mL).

Results

IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b, but not IFNλ, induced IP10 (CXCL10) release and increased IP10 gene induction in HLMVEC. In addition, all four IFNα preparations induced IP10 release from HPASM cells and A549 cells pre-treated with TNFα. In each of these cell types, 40KDa-PEGIFNα2a was significantly less active than the native forms of IFNα2a, IFNα2b or 12KDa-PEGIFNα2b. Similarly, IFNα2a, IFNα2b and 12KDa-PEGIFNα2b, but not 40KDa-PEGIFNα2a, induced endothelin (ET)-1 release from HPASM cells.

Conclusions

Consistent with other interstitial pulmonary diseases, both IP10 and ET1 may serve as markers to monitor IFN-induced lung toxicity in patients. In addition, both markers may also serve to help characterize the risk associated with IFNα preparations to induce lung toxicity.     

Acquired defects in CFTR-dependent β-adrenergic sweat secretion in chronic obstructive pulmonary disease

Rationale

Smoking-induced chronic obstructive pulmonary disease (COPD) is associated with acquired systemic cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. Recently, sweat evaporimetry has been shown to efficiently measure β-adrenergic sweat rate and specifically quantify CFTR function in the secretory coil of the sweat gland.

Objectives

To evaluate the presence and severity of systemic CFTR dysfunction in smoking-related lung disease using sweat evaporimetry to determine CFTR-dependent sweat rate.

Methods

We recruited a cohort of patients consisting of healthy never smokers (N = 18), healthy smokers (12), COPD smokers (25), and COPD former smokers (12) and measured β-adrenergic sweat secretion rate with evaporative water loss, sweat chloride, and clinical data (spirometry and symptom questionnaires).

Measurements and main results

β-adrenergic sweat rate was reduced in COPD smokers (41.9 ± 3.4, P < 0.05, ± SEM) and COPD former smokers (39.0 ± 5.4, P < 0.05) compared to healthy controls (53.6 ± 3.4). Similarly, sweat chloride was significantly greater in COPD smokers (32.8 ± 3.3, P < 0.01) and COPD former smokers (37.8 ± 6.0, P < 0.01) vs. healthy controls (19.1 ± 2.5). Univariate analysis revealed a significant association between β-adrenergic sweat rate and female gender (β = 0.26), age (−0.28), FEV₁% (0.35), dyspnea (−0.3), and history of smoking (−0.27; each P < 0.05). Stepwise multivariate regression included gender (0.39) and COPD (−0.43) in the final model (R² = 0.266, P < 0.0001).

Conclusions

β-adrenergic sweat rate was significantly reduced in COPD patients, regardless of smoking status, reflecting acquired CFTR dysfunction and abnormal gland secretion in the skin that can persist despite smoking cessation. β-adrenergic sweat rate and sweat chloride are associated with COPD severity and clinical symptoms, supporting the hypothesis that CFTR decrements have a causative role in COPD pathogenesis.     

Compartment differences of inflammatory activity in chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is associated with local and systemic inflammation. The knowledge of interaction and co-variation of the inflammatory responses in different compartments is meagre.

Method

Healthy controls (n = 23), smokers with (n = 28) and without (n = 29) COPD performed spirometry and dental examinations. Saliva, induced sputum, bronchoalveolar lavage (BAL) fluid and serum were collected. Inflammatory markers were assessed in all compartments using ELISA, flow cytometry and RT-PCR.

Results

Negative correlations between lung function and saliva IL-8 and matrix metalloproteinase-9 (MMP-9) were found in smokers with COPD. IL-8 and MMP-9 in saliva correlated positively with periodontal disease as assessed by gingival bleeding in non-smokers.Tumor necrosis factor-α (TNF-α) in saliva, serum and TNF-α mRNA expression on macrophages in BAL-fluid were lower in smokers than in non-smokers. There were positive correlations between soluble TNF-α receptor 1 (sTNFR1) and soluble TNF-α receptor 2 (sTNFR2) in sputum, BAL-fluid and serum in all groups. Sputum interleukin-8 (IL-8) or interleukin-6 (IL-6) was positively correlated with sTNFR1 or sTNFR2 in non-smokers and with sTNFR2 in COPD.

Conclusion

Saliva which is convenient to collect and analyse, may be suitable for biomarker assessment of disease activity in COPD. An attenuated TNF-α expression was demonstrated by both protein and mRNA analyses in different compartments suggesting that TNF-α response is altered in moderate and severe COPD. Shedding of TNFR1 or TNFR2 is similarly regulated irrespective of airflow limitation.     

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.     

Myofibroblast expression in airways and alveoli is affected by smoking and COPD

Background

Chronic obstructive pulmonary disease (COPD) is characterized by structural changes in alveoli and airways. Our aim was to analyse the numbers of alpha-smooth muscle actin (α-SMA) positive cells, as a marker of myofibroblasts, in different lung compartments in non-smokers and smokers with normal lung function or COPD.

Methods

α-SMA, tenascin-C (Tn-C) and EDA-fibronectin in alveolar level and airways were assayed by immunohistochemistry and quantified by image analysis. Immunohistochemical findings were correlated with clinical data. α-SMA protein was also analysed by Western blotting from fibroblastic cells cultured from peripheral lung of non-smokers, smokers without COPD and smokers with COPD.

Results

In many cases, the endings of the detached alveolar walls were widened, the structures of which were named as widened alveolar tips. Widened alveolar tips contained α-SMA positive cells, which were obviously myofibroblasts. There were less alveolar tips containing positive cells for α-SMA in alveoli and α-SMA positive cells in bronchioles in smokers and in COPD compared to non-smokers. The quantity of α-SMA positive cells was increased in bronchi in COPD. Tn-C was elevated in bronchi in COPD and smokers’ lung. The α-SMA protein level was 1.43-fold higher in stromal cells cultured from non-smokers than in those of smokers.

Conclusions

Myofibroblasts are localized variably in normal and diseased lung. This indicates that they have roles in both regeneration of lung and pathogenesis of COPD. The widened alveolar tips, these newly characterized histological structures, seemed to be the source of myofibroblasts at the alveolar level.     

Natural killer cells and natural killer T cells in Lyme arthritis

Introduction

Natural killer (NK) and natural killer T (NKT) cells provide a first line of defense against infection. However, these cells have not yet been examined in patients with Lyme arthritis, a late disease manifestation. Lyme arthritis usually resolves with antibiotic treatment. However, some patients have persistent arthritis after spirochetal killing, which may result from excessive inflammation, immune dysregulation and infection-induced autoimmunity.

Methods

We determined the frequencies and phenotypes of NK cells and invariant NKT (iNKT) cells in paired peripheral blood (PB) and synovial fluid (SF) samples from eight patients with antibiotic-responsive arthritis and fifteen patients with antibiotic-refractory arthritis using flow cytometry and cytokine analyses.

Results

In antibiotic-responsive patients, who were seen during active infection, high frequencies of CD56bright NK cells were found in SF, the inflammatory site, compared with PB (P <0.001); at both sites, a high percentage of cells expressed the activation receptor NKG2D and the chaperone CD94, a low percentage expressed inhibitory killer immunoglobulin-like receptors (KIR), and a high percentage produced IFN-γ. In antibiotic-refractory patients, who were usually evaluated near the conclusion of antibiotics when few if any live spirochetes remained, the phenotype of CD56bright cells in SF was similar to that in patients with antibiotic-responsive arthritis, but the frequency of these cells was significantly less (P = 0.05), and the frequencies of CD56dim NK cells tended to be higher. However, unlike typical NKdim cells, these cells produced large amounts of IFN-γ, suggesting that they were not serving a cytotoxic function. Lastly, iNKT cell frequencies in the SF of antibiotic-responsive patients were significantly greater compared with that of antibiotic-refractory patients where these cells were often absent (P = 0.003).

Conclusions

In patients with antibiotic-responsive arthritis, the high percentage of activated, IFN-γ-producing CD56bright NK cells in SF and the presence of iNKT cells suggest that these cells still have a role in spirochetal killing late in the illness. In patients with antibiotic-refractory arthritis, the frequencies of IFN-γ-producing CD56bright and CD56dim NK cells remained high in SF, even after spirochetal killing, suggesting that these cells contribute to excessive inflammation and immune dysregulation in joints, and iNKT cells, which may have immunomodulatory effects, were often absent.     

Linking Power Doppler Ultrasound to the Presence of Th17 Cells in the Rheumatoid Arthritis Joint

Background

Power Doppler ultrasound (PDUS) is increasingly used to assess synovitis in Rheumatoid Arthritis (RA). Prior studies have shown correlations between PDUS scores and vessel counts, but relationships with T cell immunopathology have not been described.

Methodology/Principal Findings

PBMC were isolated from healthy controls (HC) or RA patients and stimulated ex vivo with PMA and ionomycin for 3 hours in the presence of Golgistop. Paired synovial fluid (SF) or synovial tissue (ST) were analysed where available. Intracellular expression of IL-17, IFNγ, and TNFα by CD4+ T cells was determined by flow cytometry. Synovial blood flow was evaluated by PDUS signal at the knees, wrists and metacarpophalangeal joints of RA patients. Serum, SF and fibroblast culture supernatant levels of vascular endothelial growth factor-A (VEGF-A) were measured by ELISA. The frequency of IL17+IFNγ-CD4+ T cells (Th17 cells) was significantly elevated in peripheral blood (PB) from RA patients vs. HC (median (IQR) 0.5 (0.28–1.59)% vs. 0.32 (0.21–0.54)%, p = 0.005). Th17 cells were further enriched (mean 6.6-fold increase) in RA SF relative to RA PB. Patients with active disease had a higher percentage of IL-17+ T cells in ST than patients in remission, suggesting a possible role for Th17 cells in active synovitis in RA. Indeed, the percentage of Th17 cells, but not Th1, in SF positively correlated with CRP (r = 0.51, p = 0.04) and local PDUS-defined synovitis (r = 0.61, p = 0.002). Furthermore, patients with high levels of IL-17+CD4+ T cells in SF had increased levels of the angiogenic factor VEGF-A in SF. Finally, IL-17, but not IFNγ, increased VEGF-A production by RA synovial fibroblasts in vitro.

Conclusions/Significance

Our data demonstrate a link between the presence of pro-inflammatory Th17 cells in SF and local PDUS scores, and offer a novel immunological explanation for the observation that rapid joint damage progression occurs in patients with persistent positive PDUS signal.     

Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection

Background

Although antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood.

Methodology/Principal findings

Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone.We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus.

Conclusions/significance

The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials.