IL6 and CRPhaplotypes are associated with COPD risk and systemic inflammation: a case-control study

Chronic obstructive pulmonary disease (COPD) is a multi-component respiratory disease with recognized systemic impact [1]. Numerous studies performed in recent years provide overwhelming evidence of COPD as a condition characterized by an abnormal inflammatory response beyond the lungs with evidence of low-grade systemic inflammation [2‐5]. Raised levels of acute phase proteins like C-reactive protein (CRP), fibrinogen and pro-inflammatory cytokines such as interleukin (IL)-6 were found in circulation of stable COPD patients [3, 6] and have been shown to be associated with impaired functional capacity [7], reduced daily physical activity [8] and decreased health status [5, 7, 9]. However, given the cross-sectional nature of most studies performed so far and possible confounding by a number of lifestyle factors associated with levels of inflammatory biomarkers [10], it is not clear whether these proteins are simply markers of the inflammatory process accompanying chronic diseases such as COPD or key players in the pathogenesis of disease.

Genome-wide scans, twin and family studies have shown that circulating levels of CRP, fibrinogen and IL-6 are heritable (estimated as 25%–40%) [11‐15]. Furthermore, recently CRP and FGB polymorphisms/haplotypes have been described that may partly explain heritability of acute-phase protein and cytokine levels [16‐19]. Genetic association testing of genotypes, which influence circulating levels of proteins and directly relate to the outcome of interest, was suggested as more accurate unconfounded estimate of whether systemic inflammation levels causally influence outcome [20].

In the present study we investigate whether common haplotypes in CRP, IL6 and FGB (encoding fibrinogen β chain) genes influence systemic inflammatory status in COPD, the risk for COPD and, eventually, different disease-related phenotypes. Some of the results of this study have been previously reported in the form of an abstract [21].

Similar to previous studies [4, 9], we have shown that circulating levels of CRP, IL-6 and fibrinogen are increased in clinically stable COPD patients comparing to healthy (ex)smokers. Importantly, inflammatory markers distribution has shown a striking heterogeneity of COPD population in relation to the systemic inflammatory response. Furthermore, we observed a significant association between IL6 and CRP haplotypes and CRP levels in COPD patients as well as in the total study group, similar to the previous population-based and clinical studies [16, 26, 27]. Surprisingly, in our study those haplotypes were not related to any of COPD functional phenotype including BMI and 6MWD, which were the most strongly correlated with CRP plasma levels similar to the previous reports [4, 5, 9]. However, when compared to the controls, a new association of IL6 gene and COPD was identified. In particular, IL6 haplotype H2 (6.2% of cases) was associated with 5 times higher risk of COPD and significant decrease in CRP levels in COPD patients. To our knowledge, the present study is the first haplotype-based association analysis examining common genetic variation in CRP, IL6 and FGB genes on COPD risk and circulating levels of inflammatory markers in COPD patients and healthy smokers.

Although presence of systemic inflammation has been widely accepted in the last years as an essential characteristic of COPD [3, 28], our data clearly show that levels of CRP and 2 other widely studied in COPD markers, IL-6 and fibrinogen, vary significantly across the COPD population. This gives us strong evidence that not all COPD patients have a manifested low-grade systemic inflammatory response. In fact, comparable findings was reported in our previous study of CRP in relation to COPD phenotypes [7]. Further evidence of heterogeneity in relation to CRP comes from a recent intervention study [29], which has shown that COPD patients segregate for provastatin-responders (CRP > 1 mg/uL) and non-responders (CRP < 1 mg/L) based on their baseline CRP levels. Those non-responders may represent the distinct COPD subpopulation with low CRP levels which has impaired immune response to bacteria, viruses and other agents. Interestingly, our findings also suggest that low CRP levels may be associated with significantly higher COPD risk in carriers of a specific IL6 haplotype. Given that lung-expressed CRP could be up regulated by cytokines and has shown cyto-protective effect in innate immune response against bacteria and particulate matter [30‐32], CRP and IL6 haplotypes should be further studied in relation to the local impairment in COPD.

In previous reports there seems to be inconsistency about the relationship involving biomarkers levels, corresponding genes and COPD phenotypes. On the one hand, significant associations between the plasma levels of CRP, IL-6 and fibrinogen and COPD phenotypes have been observed [2, 7, 8, 33]. Yet, on the other, no relationship between the polymorphisms influencing the protein levels and COPD phenotypes could be demonstrated in the current and some other smaller studies [34, 35]. Interestingly, three recent mRNA/protein profiling studies in quadriceps and vastus lateralis of COPD patients [36‐38] failed to confirm a common hypothesis that increased expression of pro-inflammatory cytokines (IL-6, IL-8, IL-1 and TNF-α) induce muscle atrophy in COPD patients with muscle weakness. Several phenomena might explain differences between studies of inflammatory genes, mRNA and proteins in COPD. It is well-known that results of observational studies of inflammatory markers might be confounded because of residual association with numerous non-diseases related factors (i.e. lifestyle and socioeconomic) [10, 39]. Thus, association of CRP or IL6 variation with disease phenotype may suggest a causal relationship between CRP levels and disease [20]. However, genetic variation in CRP actually only explains a relatively small proportion of CRP levels variation (< 3%), compared with other environmental or genetic factors [16, 26, 40]. Moreover, given the fact that COPD might share systemic inflammatory phenotype with cardiovascular disease, cancer and diabetes [41, 42], which are known co morbidities of COPD, conflicting association data may be caused by different composition of the COPD populations used across different studies. Lastly, acute-phase and pro-inflammatory cytokine genes may be only modifiers of other, not widely studied, inflammatory genes, which are critically involved in COPD pathogenesis. Further hypothesis-free genome-wide high-throughput screening and gene expression profiling studies are needed to validate existing and find new candidate markers characterizing distinct clinical profiles of multi-faced COPD population.

Our study has several strengths. It is the first one that has been specifically designed to evaluate relationship between systemic inflammation and COPD on both genetic and protein level. For this purpose, both standard outcomes (COPD diagnosis and lung function) as well as functional disease-related phenotypes (exercise capacity and body composition) were taken into account. We used a tagging SNP based approach capturing all common variability across the genes of interest to investigate whether genetic variation in the genes of interest influences the risk of COPD. Next this study is based on a well-characterized ethnically homogeneous cohort of stable COPD patients and spirometry-proved healthy smokers collected from the same geographic region.

In our analysis of 3 genes in relation to 4 primary outcomes it is possible but unlikely that results were false positives obtained due to a type I error inflation. It is worth mentioning, two associations (IL6 haplotypes and COPD, global P = 0.003, CRP haplotypes and ln(CRP), global P = 0.0004) were still significant after applying the conservative Bonferroni correction. In addition, these associations remained also significant at P < 0.002 after permutation tests with at least 10000 simulations. Nevertheless, all reported associations between COPD and CRP and IL6 genes require replication in other independent studies. Similar to our results, three previous studies of the IL6 gene could not find any association between COPD and the SNPs rs1800795 and rs1800797 [43‐45]. Interestingly, a new association was found with the IL6 SNP rs1800796 in COPD patients from Spain [44]. Because of a low allele frequency in Europeans (4%), this SNP was not selected for current study; however, further studies using large samples size of Dutch COPD patients are required to replicate this new association.

There are also some limitations of the present study. First, the patients with COPD included in the study were assessed to enter pulmonary rehabilitation and had predominantly moderate-to-severe stages of COPD. This could affect generalizability of our results. Next, a relatively small sample size could have underpowered some observed genetic associations. Namely, IL6 association with CRP levels and CRP association with COPD were marginally significant in the overall testing but have shown strong haplotype-specific effects in further analysis. Furthermore, as a result of the failure of 2 FGB tag SNPs for genotyping we were not able to discriminate one common (defined by rs2227439) and one rare (carrying the minor allele of rs2227432) haplotypes of the FGB gene [19]. They were non-discriminative from the FGB haplotypes 1111 and 1121, respectively.

It is also noticeable, that, although we found an increased risk of COPD for the IL6 H2 carriers, the functional SNP causing this risk still has to be identified. IL6 haplotype H2 is tagged by the minor allele of rs1818879. However, it is unlikely, that this SNP located in 3'UTR region of IL6, is functional because no association between haplotype H4 (tagged by the combination of rs2069840 and rs1818879) and COPD risk was found. Most likely, the functional SNP lies on the IL6 H2 haplotype and was not genotyped in our study. Future resequencing of IL6 haplotype H2 carriers may help to identify functional SNP(s). Given that a recent genome-wide association study [46] has found a SNP in IL-6 receptor gene as one of the top associated with six lung function phenotypes and the association we found between COPD and IL6 gene, it would be worthwhile to study IL-6 pathway further [47].

In conclusion, CRP and IL6 haplotypes were shown to be associated with systemic inflammation and COPD but not with exercise capacity, dysnoea, BMI and BODE index. Future studies are needed to replicate the observed associations in large well-defined COPD cohorts.