C.H.M. van Moorsel

Linkage between Toll-like receptor (TLR) 2 promotor and intron polymorphisms: functional effects and relevance to sarcoidosis

The intracellular pathogens Propionibacterium acnes and Mycobacterium tuberculosis have been leading suspects as the cause of sarcoidosis, a systemic disorder characterized by the formation of non‐caseating granulomas. Toll‐like receptor (TLR) 2 is important in the innate immune response against both pathogens, and is therefore of interest in sarcoidosis research. In the present study, three single nucleotide polymorphisms and one dinucleotide repeat polymorphism in the TLR‐2 gene were genotyped in 419 sarcoidosis patients, divided into a study cohort and a validation cohort, and 196 healthy controls. In the study cohort we found a significant increase in prevalence of the AA‐genotype at promotor location −16934 in patients with chronic disease compared to patients with acute/self‐remitting sarcoidosis (34·5% versus 15·9%, respectively, P = 0·006, Pc = 0·019). These results could not be confirmed in our validation cohort, implicating a possible role for TLR‐2 genetics in only a small percentage of sarcoidosis patients. Furthermore, linkage was found between the promotor polymorphism −16934 A/T and the number of GT repeats in intron 1 (P < 0·0001). After in vitro stimulation of peripheral blood mononuclear cells (PMBCs) with different TLR‐2 agonists, a correlation between induction of TNF‐α (P = 0·008), interleukin (IL)‐12 (P = 0·008) as well as IL‐6 (P = 0·02), and the number of GT repeats was observed. In conclusion, the data show that polymorphisms in TLR‐2 might be important in a small group of sarcoidosis patients and that their functional consequences explain partly some of the variance in cytokine pattern observed in different clinical phenotypes of this disease.

Systemic cytokine response in patients with community-acquired pneumonia.

The role of individual cytokines and polymorphisms in pneumonia has been described, but the relationship between different cytokines and polymorphisms in relation to causative microorganisms, antibiotics, corticosteroids and clinical course has not. This study questions the relationship between cytokines, polymorphisms and clinical characteristics of pneumonia. Patients diagnosed with pneumonia were included in the study. Serum cytokine levels were measured during hospital stay, genotyping was performed, causative microorganisms were identified and patients were monitored throughout the hospital stay. In 201 patients with pneumonia interleukin (IL)-1 receptor antagonist (IL-1RA), IL-6, IL-8 and IL-10 acted as acute phase proteins. After admission, the levels of these cytokines decreased rapidly. Single nucleotide polymorphisms did not influence cytokine production and were not associated with clinical outcome. Cytokine serum levels were significantly higher in patients with pneumococcal pneumonia. The decrease in levels of cytokines was independently influenced by the start of corticosteroid therapy. IL-1RA, IL-6, IL-8 and IL-10 are acute phase proteins, independent of genotype. Their levels are influenced by the nature of the causative microorganism and the start of corticosteroids therapy.

Toll-like receptor (TLR) 4 polymorphism Asp299Gly is not associated with disease course in Dutch sarcoidosis patients.

The aetiology of sarcoidosis, a systemic disorder characterized by the formation of non‐caseating granulomas in variable organs, remains enigmatic. Clarification is hampered by heterogeneity in disease phenotypes and course, due partly to the influence of a variety of genetic and environmental factors. Multiple studies have pointed towards bacteria as possible causative agents. Toll‐like receptors (TLR) are innate immunity receptors important in the immune response against pathogens. TLR‐4, together with CD14 and MD‐2, is an essential receptor for the recognition of lipopolysaccharide (LPS), unique to the cell wall of Gram‐negative bacteria. Recently, an association between TLR‐4 polymorphism Asp299Gly, leading to a change in the extracellular domain of the receptor and possible hyporesponsiveness to LPS, and a chronic course of sarcoidosis was found in German patients. In the present study this polymorphism was genotyped in 156 Dutch sarcoidosis patients and 200 healthy Dutch controls using dual‐labelled fluorescent oligonucleotides. No differences were found in allelic distributions between patients and controls (P = 0·79) or within the different clinical entities of the sarcoidosis group (P = 0·44). Importantly, there were no differences between the Dutch and German sarcoidosis patients (P = 0·62). However, the allelic distribution of the Asp299Gly polymorphism differed significantly between both control groups (P = 0·04). This study highlights the importance of testing a reported gene association in a distinct population when performing genetic association studies.

Genetic variation in the Toll-like receptor gene cluster (TLR10-TLR1-TLR6) influences disease course in sarcoidosis

Sarcoidosis is an inflammatory disease of unknown etiology. Various microorganisms have been proposed as etiologic agent suggesting a role for pattern-recognition receptors such as Toll-like receptors (TLRs) in disease pathogenesis, with a special interest in TLR-2. TLR-10, TLR-1, and TLR-6 act as co-receptors for TLR-2 and the genes encoding these receptors are located in a gene cluster on chromosome 4. The aim of our study was to assess differences in genetic variation in the TLR10-TLR1-TLR6 gene cluster between patients and controls. A total of eight single nucleotide polymorphisms were genotyped in 447 healthy controls and 533 patients, divided in 425 with sarcoidosis and 108 with Löfgrens syndrome. Comparison of the total patient cohort with controls showed that the allele frequencies of rs1109695, rs7658893 (TLR-10), and rs5743604 as well as rs5743594 (TLR-1) differed significantly. Haplotype analysis showed that the most common haplotype found was significantly decreased in patients with chronic sarcoidosis. Furthermore, a less common haplotype was found to be significantly increased in patients with Löfgrens syndrome as well as sarcoidosis patients with self-remitting disease, indicating that it could act as a disease modifying haplotype. In conclusion, our study suggests that absence of the common haplotype in the TLR10-TLR1-TLR6 gene cluster increases the risk of developing chronic disease in patients already affected by sarcoidosis. Based on their role as co-receptors for TLR-2, this study supports the growing evidence that aberrant TLR-2 function is important in sarcoidosis disease pathogenesis.

Genetic variability in the IL1RN gene and the balance between interleukin (IL)‐1 receptor agonist and IL‐1β in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a rapidly progressive interstitial lung disease of unknown aetiology. Interleukin (IL)‐1β plays an important role in inflammation and has been associated with fibrotic remodelling. We investigated the balance between IL‐1β and IL‐1 receptor antagonist (IL‐1Ra) in bronchoalveolar lavage fluid (BALF) and serum as well as the influence of genetic variability in the IL1B and IL1RN gene on disease susceptibility and cytokine levels. In 77 IPF patients and 349 healthy controls, single nucleotide polymorphisms (SNPs) in the IL1RN and IL1B genes were determined. Serum and BALF IL‐1Ra and IL‐1β levels were measured using a multiplex suspension bead array system and were correlated with genotypes. Both in serum and BALF a significantly decreased IL‐1Ra/IL‐1β ratio was found in IPF patients compared to healthy controls. In the IL1RN gene, one SNP was associated with both the susceptibility to IPF and reduced IL‐1Ra/IL‐1β ratios in BALF. Our results show that genetic variability in the IL1RN gene may play a role in the pathogenesis of IPF and that this role may be more important than thought until recently. The imbalance between IL‐1Ra and IL‐1β might contribute to a proinflammatory and pro‐fibrotic environment in their lungs.

Toll-like receptor (TLR)-9 genetics and function in sarcoidosis.

Sarcoidosis is a systemic disorder characterized by the formation of non‐caseating granulomas in variable organs. Toll‐like receptor (TLR)‐9 is important in the innate immune response against both Mycobacterium tuberculosis and Propionibacterium acnes, candidate causative agents in sarcoidosis. The aim of our study was to investigate possible genetic and functional differences in TLR‐9 between patients and controls. TLR‐9 single nucleotide polymorphisms were genotyped in 533 patients and divided into a study cohort and validation cohort and 185 healthy controls. Furthermore, part of the promotor as well as the entire coding region of the TLR‐9 gene were sequenced in 20 patients in order to detect new mutations. No genetic differences were found between patients and controls. In order to test TLR‐9 function, peripheral blood mononuclear cells (PBMCs) of 12 healthy controls and 12 sarcoidosis patients were stimulated with a TLR‐9 agonist and the induction of interleukin (IL)‐6, interferon (IFN)‐γ and IL‐23 was measured. Sarcoidosis patients produce significantly less IFN‐γ upon stimulation with different stimuli. Regarding IL‐23 production, a significant difference between patients and controls was found only after stimulation with the TLR‐9 agonist. In conclusion, we did not find genetic differences in the TLR‐9 gene between sarcoidosis patients and controls. Sarcoidosis patients produce less IFN‐γ regardless of the stimulating agent, probably reflecting the anergic state often seen in their peripheral blood T lymphocytes. The differences in TLR‐9‐induced IL‐23 production could indicate that functional defects in the TLR‐9 pathway of sarcoidosis patients play a role in disease susceptibility or evolution.

Variation in IL7R predisposes to sarcoid inflammation

Sarcoidosis is a chronic granulomatous disorder characterized by a massive influx of Th1 lymphocytes. Both naive and memory T cells express high levels of interleukin 7 receptor-α (IL7Rα), encoded by the IL7R gene. The purpose of this study was to investigate the role of the IL7R gene region in susceptibility to sarcoidosis. Six common single-nucleotide polymorphisms (SNPs) spanning IL7R were genotyped and analyzed in 475 sarcoidosis patients and 465 healthy controls. Replication of one significant associated SNP was carried out in 206 independent sarcoidosis patients, 127 controls and 126 patients with Löfgrens disease. The rs10213865 SNP was associated with sarcoidosis (P=0.008), and in silico analysis showed a complete linkage (r2=1, D′=1) with a functional nonsynonymous coding SNP in exon 6 (rs6897932, T244I). Combined analysis of 663 individuals with sarcoidosis and 586 controls (homozygous carriers of risk allele, P=5 × 10−4, odds ratio=1.49 (1.19–1.86)) provided strong statistical support for a genuine association of IL7R with the risk of sarcoidosis. In addition, we report the same trend between variation in the IL7R gene and patients with Löfgrens disease, suggesting that variation in IL7R may confer general risk for developing granulomatous lung disease.

MRP14 is elevated in the bronchoalveolar lavage fluid of fibrosing interstitial lung diseases

Pulmonary fibrosis is defined by an overgrowth of fibroblasts and extracellular matrix deposition, and results in respiratory dysfunction that is often fatal. It is the end stage in many chronic inflammatory interstitial lung diseases (ILD) such as sarcoidosis and idiopathic pulmonary fibrosis (IPF). The myeloid‐related proteins (MRPs) belong to the S100 family of calcium‐binding proteins and are highly expressed by neutrophils, macrophages and epithelial cells during chronic inflammation. MRP14 stimulates fibroblast proliferation in vitro and is expressed in granulomas from sarcoidosis patients. We hypothesized that MRP14 may be a biomarker for fibrotic interstitial lung diseases. The objective of this study was to investigate whether levels of MRP14 in the bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis and IPF correlate with clinical parameters. We used an enzyme‐linked immunosorbent assay (ELISA) to measure MRP14 in BALF of 74 sarcoidosis patients, 54 IPF patients and 19 controls. Mean BALF levels of MRP14 were elevated significantly in IPF (P < 0·001) and sarcoidosis (P < 0·05) patients compared to controls. MRP14 levels were associated linearly with sarcoidosis disease severity based on chest radiographic stage. Moreover, BALF MRP14 levels were correlated inversely with diffusion capacity and forced vital capacity in sarcoidosis patients. In IPF patients, a correlation with BALF neutrophil percentage was found. In conclusion, BALF MRP14 levels are elevated in IPF and sarcoidosis and are associated with disease severity in sarcoidosis. The results support the need for further studies into the role of MRP14 in the pathogenesis of lung fibrosis.

Genetic variation in GREM1 is a risk factor for fibrosis in pulmonary sarcoidosis

Sarcoidosis is a granulomatous systemic disorder most often affecting the lung. Pulmonary fibrosis develops in approximately 10%-15% of patients with sarcoidosis. The human gene GREM1 encodes gremlin, a member of the bone morphogenetic protein antagonist family. Bone morphogenetic proteins are essential for the maintenance of tissue homeostasis and regeneration after injury. We examined associations between genetic variation in GREM1 and pulmonary disease outcome in patients with pulmonary sarcoidosis. Four common tag single nucleotide polymorphisms spanning GREM1 were genotyped in 483 controls and in 237 sarcoidosis patients with radiographic data at pulmonary disease outcome, defined by chest X-ray after a minimum of 4 years follow-up. Highly significant differences were found between GREM1 genotype frequencies in sarcoidosis patients without chest X-ray abnormalities (stage 0) (n = 116) versus patients who had fibrosis on chest X-ray (stage IV) (n = 59) at pulmonary disease outcome. The most significant association was with GREM1 rs1919364. The recessive model resulted in an increased risk of fibrosis development for homozygous carriers of the C allele at GREM1 rs1919364 versus carriers of the G allele [P = 9.3 × 10⁻⁷, χ² = 24.1, odds ratio (OR) = 6.37 (2.89-14.1)]. This study is the first to suggest that genetic variation of GREM1 predisposes to pulmonary fibrosis in sarcoidosis patients. Carriers of the GREM1 CC genotype at position rs1919364 were at 6.4 times greater risk for developing fibrosis.

Effect of variation in ITGAE on risk of sarcoidosis, CD103 expression, and chest radiography

The integrin alpha(E)beta(7) is believed to play a key role in retention of lymphocytes in mucosal tissues of gut, urogenital tract and lung. Five common single nucleotide polymorphisms spanning ITGAE, the gene encoding the alpha(E) (CD103) unit, were genotyped in 556 sarcoidosis patients and 465 controls. The -1088 A/G polymorphism was associated with sarcoidosis (P=0.004). An increased risk of disease was found for homozygous carriers of the A allele vs. carriers of the G allele (P=0.001, odds ratio=1.63 [1.22-2.17]). Analysis of lymphocytes from bronchoalveolar lavage and in vitro functional tests showed higher percentages of CD103+CD4+ T cells for the sarcoidosis risk genotype. Radiographic staging at disease outcome revealed prevalence of -1088 AA genotype in patients with fibrosis (P=0.01). A higher proportion of CD103+CD4+ T cells and ITGAE -1088 AA genotype might be associated with fibrosis formation in pulmonary sarcoidosis.