Lung T cells in inflammatory disorders : an approach to interstitial lung disease, multiple sclerosis and smoking induced inflammation

The lungs are constantly exposed to microorganisms and environmental irritants. Pulmonary inflammation is the result of an immune process to protect the body, and may sometimes eventually result in disease. T cells including various subsets are of major importance for orchestrating the protection of the lung as well as for inflammatory reactions. Activated pulmonary T cells not only have the potential to affect the lungs themselves, but they could contribute to immune responses in other organs as well. The overall aim of the study presented in this thesis was to investigate the potential effect of T cell immune responses for chronic lung inflammation from two different aspects. We thus first investigated antigen- specific T cell responses in patients with pulmonary sarcoidosis, and in the second part determined how smoking affected lung T-cell immunity, with regard to the influence of smoking in the development of autoimmunity. Sarcoidosis is a granulomatous systemic inflammatory disorder which commonly affects the lungs. T cells and particularly activated CD4+ T cells are considered to be involved in the pathogenesis of the disease. A subgroup of sarcoidosis patients known as Löfgren’s syndrome differs strikingly from other patients by particular clinical symptoms. Spontaneous recovery within two years is particularly common in Löfgren ́s syndrome patients who are HLA-DRB1*0301positive, and these patients virtually always have accumulations of T cells expressing a particular T cell receptor (TCR) V gene segment, termed AV2S3, in the lungs. The aetiology of sarcoidosis is still not known. However, recently a specific mycobacterial protein, M. tuberculosis catalase-peroxidase (mKatG) was identified in sarcoidosis tissues. BAL CD4+ T cells from HLA-DRB1*0301positive Löfgren’s syndrome responded to mKatG with a more pronounced multifunctional cytokine profile, i.e. with simultaneous production of IFNγ and TNF compared to non-Löfgren’s syndrome patients. Non-Löfgren’s syndrome patients instead responded with a higher proportion of cells producing single cytokines, i.e. production of IFNγ alone. Moreover, AV2S3+ CD4+ T cells from both BAL and blood had a higher IFNγ production in response to mKatG compared to AV2S3- CD4+ T cells, while the opposite was found for BAL AV2S3+ CD4+ cells in response to PPD. Furthermore, BAL T cells from Löfgren ́s syndrome patients had compared to T cells of non Löfgren ́s syndrome higher frequencies of IL-17-producing cells in response to mKatG. Löfgren ́s syndrome HLA- DRB1*03 positive patients clearly had higher levels of IL-17 in BAL fluid compared to healthy controls and to patients without Löfgren’s syndrome. Our results indicate that the quality of the T cell response in sarcoidosis patients may play a key role in disease presentation and clinical outcome. These findings imply that the presence of multifunctional BAL CD4+ T cells, higher activities of TCR AV2S3+ CD4+ T cells, and more pronounced IL-17 production in particular subgroups of sarcoidosis patients are involved in antigen elimination at the site of inflammation and may play a role in spontaneous recovery, typical for patients with Löfgren ́s syndrome (in particular DRB1*03 positive). Cigarette smoking is a well-known risk factor for several inflammatory and autoimmune disorders. The risk of developing multiple sclerosis (MS) is strongly increased by smoking in people with genetic susceptibility. Smoking is associated with both release and inhibition of pro-inflammatory and anti- inflammatory mediators that influence different T cell subsets. Our results indicate that cigarette smoke induces a decline in lung Th17 cells and alters the phenotype of T regulatory cells by decreasing the proportion of IL-10 producing Foxp3+ CD4+ cells and increasing the fraction of lung Foxp3+ Helios negative T cells. Thus, an imbalance between Th17/Tregs may be caused by cigarette smoking, which could result in an increased risk for infection and may also have consequences for autoimmune processes postulated to be initiated in the lung. Furthermore we studied the effect of smoking and conventional treatment in the lungs and blood of MS patients compared to healthy individuals. We found that the frequency of Foxp3+Helios+ regulatory T cells, important in the context of autoimmunity, was reduced in BAL of MS patients. However, the frequencies of both this subset of Tregs and of total Foxp3+ CD4+ BAL Treg cells was increased after treatment particularly in IFNβ treated MS patients. If the lungs are involved in initiation and propagation of inflammatory processes in MS, the observed effects in IFNβ- treated patients may be involved in disease amelioration in MS patients following such treatment

Distinctive Regulatory T Cells and Altered Cytokine Profile Locally in the Airways of Young Smokers with Normal Lung Function.

Smoking influences the immune system in different ways and, hypothetically, effects on pulmonary effector and regulatory T cells emerge as potentially detrimental. Therefore, we characterized the frequencies and characteristics of CD4+ and CD8+ T cell subsets in the blood and lungs of young tobacco smokers. Bronchoalveolar lavage (BAL) and peripheral blood were obtained from healthy moderate smokers (n = 18; 2-24 pack-years) and never-smokers (n = 15), all with normal lung function. Cells were stimulated ex vivo and key intracellular cytokines (IFNγ, IL-17, IL-10 and TNFα) and transcription factors (Foxp3, T-bet and Helios) were analyzed using flow cytometry. Our results indicate that smoking is associated with a decline in lung IL-17+ CD4+ T cells, increased IFNγ+ CD8+ T cells and these alterations relate to the history of daily cigarette consumption. There is an increased fraction of Foxp3+ regulatory T cells being Helios- in the lungs of smokers. Cytokine production is mainly confined to the Helios- T cells, both in regulatory and effector subsets. Moreover, we detected a decline of Helios+Foxp3- postulated regulatory CD8+ T cells in smokers. These alterations in the immune system are likely to increase risk for infection and may have implications for autoimmune processes initiated in the lungs among tobacco smokers

Analysis of regulatory T cells.

<p>(a) Representative flow cytometry plot to detect the combination of Foxp3+ and/or Helios+ BAL CD4+ T cells. (b) The fraction of BAL CD4+ Foxp3+ T cells that are Helios- compared in smokers and non-smokers. (c) Comparison of IFNγ and IL-10 production between Foxp3+Helios+ and Foxp3+Helios- BAL CD4+ T cells in non-smokers. (d) Comparison of the percentages of Foxp3- Helios+ BAL CD8+ T cells between smokers and non-smokers and correlation of this population with BAL CD8+ IFNγ producing cells in non-smokers, *(<i>p</i><0.05) and **(<i>p</i><0.01).</p

Percentage of IFNγ, IL-17, IL-10 and TNF producing cells in BAL CD4+ and CD8+ T cells of healthy smokers and never-smokers.

<p>T cells were stimulated with anti-CD3/CD28 and following intracellular cytokine staining the frequency of cells expressing each cytokine was determined by flow cytometry. The statistical analyses were done after background deduction *(<i>p</i><0.05) and **(<i>p</i><0.01).</p

Cytokine production by Foxp3- effector T cells.

<p>(a) The fraction of BAL CD4+ Foxp3- T cells that are Helios+ compared in smokers and non-smokers; and the fraction of BAL CD4+ Helios+ T cells that are Foxp3- compared in smokers and non-smokers (b) Comparison of IFNγ production between Foxp3-Helios+ and Foxp3-Helios- BAL CD4+ T cells in non-smokers and smokers.</p

CD4/CD8 ratio and percentage of live lymphocytes in BAL and blood from healthy smokers and non-smokers (in unstimulated sample).

<p>Cells were stained and characterized by flow cytometry to characterize CD4/CD8 ratio. Aqua fluorescent reactive dye (Live/Dead fixable dead cell stain kits) was used for all samples in order to gate on live cells, *(<i>p</i><0.05) and **(<i>p</i><0.01).</p

Transcription factors analysis.

<p>(a) Representative flow cytometry plots of BAL CD4+ T cells to detect Foxp3, T-bet and Helios. Foxp3+ and Helios+ T cells constitute distinct populations, while for gating on T-bet+ cells we set it according to isotype control and also matching with IFNγ producing cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164751#pone.0164751.s003" target="_blank">S3 Fig</a>). The percentages of cells expressing each transcription factor was compared between smokers and non-smokers in (b) BAL CD4+ and (c) BAL CD8+ T cells, *(<i>p</i><0.05).</p

Features and BALF characteristics of the subjects.

<p>Features and BALF characteristics of the subjects.</p

A Proline/Arginine-Rich End Leucine-Rich Repeat Protein (PRELP) Variant Is Uniquely Expressed in Chronic Lymphocytic Leukemia Cells

<div><p>Proline/arginine-rich end leucine-rich repeat protein (PRELP) belongs to the small leucine-rich proteoglycan (SLRP) family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1). As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR) in all CLL patients (30/30), as well as in some patients with mantle cell lymphoma (3/5), but not in healthy donor leukocytes (0/20) or tumor samples from other hematological malignancies (0/35). PRELP was also detected in CLL cell-lines (4/4) but not in cell-lines from other hematological tumors (0/9). PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies.</p></div

Chemical deglycosylation by TMFS of a yeast-derived recombinant mature PRELP protein.

<p>Western blot was performed using the anti- C-terminal polyclonal antibody. <b>Lane 1</b>: Untreated (0 h) yeast-derived recombinant PRELP showing a 100 kDa band. <b>Lanes 2 and 3</b>: Yeast-derived recombinant PRELP treated with TFMS for 2 and 4 h, respectively. After complete removal of the carbohydrate structures, a 38 kDa band was seen.</p