Sarcoidosis : expression of cell regulatory markers and the influence of patient phenotype on bronchoalveolar lavage cell differential counts

Sarcoidosis is a systemic inflammatory disease that can affect almost any organ, but the respiratory system is affected in more than 90% of the cases. To elicit an immune response, an antigen(s) is processed by antigen-presenting cells (APCs), e.g. alveolar macrophages (AMs), and is presented in association with HLAmolecules to specific T cells, using their T cell receptor (TCR). In sarcoidosis, this interaction between the innate and adaptive immune system leads to an exaggerated immune response and formation of non-caseating granulomas in affected organs. The causative antigen remains elusive. To generate the sarcoid inflammatory process, the genetic background as well as exposure for antigens, endogenous or exogenous, is it of importance. Clinically, sarcoidosis patients can be divided into two major groups, i.e. patients with Löfgren’s syndrome (LS) or with non-Löfgren’s syndrome (non-LS). LS is a clinically distinct and well-defined phenotype that is characterized by an acute onset and is associated with specific HLA molecules, i.e. HLA-DRB1*03. In most of the LS patients, the disease resolves within two years. On the other hand, non-LS patients constitute a heterogeneous group and are prone to develop a chronic disease course. Collecting cells from the deep lung compartment via bronchoalveolar lavage (BAL) enabled many researchers to explore immunological mechanisms in the alveolar space. In a healthy individual, BAL fluid (BALF) are mainly macrophages, some lymphocytes, and fewer neutrophils, and eosinophils; basophils and mast cells are rare. BALF from sarcoidosis patients contains an increased number of all these cell types, especially lymphocytes. The first two studies (I, II) aimed to shed some light on the expression of cell regulatory markers in LS and non-LS patients. Macrophages are classically subdivided into two major subtypes, i.e. M1 – known as proinflammatory macrophages – and M2 – known as remodeling macrophages. We found in the first study (I) reduced gene expression of toll-like receptor 2 (TLR2: M1 associated marker) – mainly in LS patients – and increased expression of CCL18 (M2 associated marker) in AM of sarcoidosis patients. This finding could indicate a shift toward M2-like macrophages in sarcoidosis. The reduced TLR2 expression in LS patients might allow for a more effective immune response leading to resolution of granulomas. The CCL18 chemokine is known to act as T cell chemoattractant and can also induce collagen production in fibroblasts. Hence, the increased expression of CCL18 in AM of patients might attract T cells to the lung in the early stages of the disease and exhibit a profibrotic role in more advanced disease. The second study (II) explored the expression of specific transcription factors/nuclear receptors known to have regulatory roles in inflammatory diseases, i.e. the peroxisome proliferator-activated receptors (PPARs): PPARα, PPARβ/δ and PPARγ. Compared to LS patients, PPARα expression was downregulated in BALF and blood CD4 and CD8 T cells in non-LS patients. Thus, CCL18 and PPARα could be used as biomarkers and might help in identifying patients at increased risk of developing more advanced lung disease. The third study (III) aimed to explore the influence of patient phenotypes on BALF cell differential counts. We found that genetic variants associated with risk of LS and clustered in the extended MHC region were associated with the quantitative levels of BALF macrophages, lymphocytes, and neutrophils. Genetic variants associated with non-LS and located in the MHC II region associated with the quantitative levels of BALF macrophages only. In addition, these genetic variants exhibit regulatory effects on other genes in the lung, blood, T cells, B cells, macrophages and neutrophils. The fourth study (IV) aimed to utilize data from a BALF registry of pulmonary sarcoidosis patients (LS and non-LS) to identify BALF cells that could predict disease severity (defined as advanced chest radiographs, reduced pulmonary function, or necessity for treatment) and/or chronicity (non-resolving course after two years). Compared with LS-resolving patients, LS-chronic patients exhibited higher BALF lymphocytes, neutrophils, and eosinophils. Additionally, in newly diagnosed LS patients, increased BALF neutrophils and basophils were more likely to associate with more severe disease; and increased BALF lymphocytes count was more likely to associate with a chronic disease course. In non-LS patients, increased BALF mast cells associated with a more severe and a chronic (nonresolving) disease, and increased BALF lymphocytes, neutrophils, eosinophils, and basophils associated with a more severe disease. In summary, searching for biomarkers, we identified two possible markers for severe and/or chronic disease, i.e. PPARα and CCL18. In study III and IV, we showed that genetic variants associated with LS and non-LS can influence BALF cell counts and that increased BALF neutrophils, eosinophils, lymphocytes, basophils and notably mast cells have prognostic implication in newly diagnosed sarcoidosis patients. LIST OF SCIENTIFIC PAPERS I. Maria Wikén, Farah Idali, MUNTASIR ABO AL HAYJA, Johan Grunewald, Anders Eklund, and Jan Wahlström. No evidence of altered alveolar macrophage polarization, but reduced expression of TLR2, in bronchoalveolar lavage cells in sarcoidosis. Respiratory Research, 2010, 11:121. II. MUNTASIR ABO AL HAYJA, Anders Eklund, Johan Grunewald, and Jan Wahlström. Reduced expression of peroxisome proliferator-activated receptor alpha in BAL and blood T cells of non-Löfgren’s sarcoidosis patients. Journal of Inflammation, 2015, 12:28. III. MUNTASIR ABO AL HAYJA, Susanna Kullberg, Anders Eklund, Leonid Padyukov, Johan Grunewald, and Natalia V. Rivera. Influence of sarcoidosis risk genetic variants on the quantitative level of bronchoalveolar lavage fluid (BALF) cell populations. Submitted to Genes & Immunity. IV. MUNTASIR ABO AL HAYJA, Jan Wahlström, Susanna Kullberg, Pernilla Darlington, Anders Eklund, and Johan Grunewald. Bronchoalveolar lavage fluid cell subsets associate with the disease course in Löfgren’s and non-Löfgren’s sarcoidosis patients. Manuscript