Chlamydia pneumoniae-Mediated Inflammation in Atherosclerosis: A Meta-Analysis

Several studies have attempted to relate the C. pneumoniae-mediated inflammatory state with atherosclerotic cardiovascular diseases, providing inconsistent results. Therefore, we performed a meta-analysis to clarify whether C. pneumoniae may contribute to the pathogenesis of atherosclerosis by enhancing inflammation. 12 case-control, 6 cross-sectional, and 7 prospective studies with a total of 10,176 patients have been included in this meta-analysis. Odds Ratio (OR) with a 95% confidence interval was used to assess the seroprevalence of C. pneumoniae and differences between levels of inflammatory markers were assessed by standard mean differences. Publication bias was performed to ensure the statistical power. hsCRP, fibrinogen, interleukin- (IL-) 6, TNF-α, and IFN-γ showed a significant increase in patients with atherosclerosis compared to healthy controls (P < 0.05), along with a higher seroprevalence of C. pneumoniae (OR of 3.11, 95% CI: 2.88–3.36, P < 0.001). More interestingly, hsCRP, IL-6, and fibrinogen levels were significantly higher in C. pneumoniae IgA seropositive compared to seronegative atherosclerotic patients (P < 0.0001). In conclusion, the present meta-analysis suggests that C. pneumoniae infection may contribute to atherosclerotic cardiovascular diseases by enhancing the inflammatory state, and, in particular, seropositivity to C. pneumoniae IgA, together with hsCRP, fibrinogen, and IL-6, may be predictive of atherosclerotic cardiovascular risk.


Introduction
Atherosclerosis, a chronic inflammatory disease of multifactorial aetiology, typically begins with endothelial dysfunction followed by low density lipoprotein (LDL) infiltration of the arterial intima, mononuclear cell recruitment into vascular wall, and the differentiation of macrophages in foam cells [1,2]. Macrophages and foam cells, within the evolving atherosclerotic lesion, secrete various proinflammatory cytokines, such as interleukin-(IL-) 6, interferon-(IFN-) , tumor necrosis factor-(TNF-) , and chemokines, including intercellular adhesion molecule-(ICAM-) 1 and vascular cell adhesion molecule-(VCAM-) 1. In response to proinflammatory cytokines, endothelial cells express high levels of leukocyte adhesion molecules on their surface, leading to further mononuclear cell recruitment and, hence, to a chronic inflammatory state [3,4]. As a result, the formation, progression, and destabilisation of atherosclerotic plaque occur, leading to cardiovascular diseases, a major public health problem in developed countries, accounting for one-third of all deaths worldwide [5].
In the last decades, several infectious agents have been related to the pathogenesis of atherosclerotic cardiovascular diseases, and current opinion is that the most implicated pathogen is Chlamydia pneumoniae, an obligate intracellular microorganism, known as being responsible for respiratory tract infections [6,7].
Specifically, C. pneumoniae persistent form seems to be responsible for chronic infection and, hence, for the inflammatory process underlying atherosclerosis, since it is able to endure for a long time inside host cells [41,42].
In addition to the direct effect on vascular cells, previously described, C. pneumoniae has also shown to contribute to the systemic inflammation involved in the pathogenesis of atherosclerotic cardiovascular diseases, as evidenced by high levels of IL-6 and high sensitivity c-reactive protein (hsCRP) [7,43].
Given the central role of inflammation in the atherosclerotic process and the inflammatory effects of C. pneumoniae, several studies have attempted to relate the C. pneumoniaemediated inflammatory state with atherosclerotic cardiovascular diseases, providing inconsistent results.
We performed, therefore, a meta-analysis to clarify whether C. pneumoniae may contribute to the pathogenesis of atherosclerotic cardiovascular diseases by the means of enhanced inflammatory state.

Search Strategy.
We performed a systematic search of all articles in journals indexed on the electronic databases PubMed and Scopus up to December 2014. The search terms used were "chlamydia pneumoniae" or "chlamydophila pneumoniae," "inflammatory markers" or "inflammation," "atherosclerosis," and "cardiovascular disease." The reference lists of reviews and retrieved articles were hand-searched simultaneously. When more than one of the same patient population was included in several publications, only the most recent or complete study was included in this metaanalysis.

Inclusion and Exclusion
Criteria. The inclusion criteria in this meta-analysis were as follows: (i) studies comparing atherosclerotic patients with healthy subjects (control group) in relation to the seropositivity to C. pneumoniae IgG or IgA, (ii) studies comparing atherosclerotic patients in relation to the seropositivity to C. pneumoniae IgG or IgA, and (iii) studies analysing the levels of inflammatory markers.
Case reports, reviews, letters, and studies which did not present their results in a correct and/or explicit manner, animal studies, and studies where the control groups were made up of patients with other chronic inflammatory disorders were excluded from our meta-analysis. We did not define any minimum number of patients to include a study in our metaanalysis.

Data Extraction.
Information was carefully extracted from all eligible publications independently by two authors according to the inclusion and exclusion criteria listed above. Disagreement was resolved by discussion between the two authors. The following data were collected from each study: first author's surname, year of publication, study design, type of cases, type of controls, total numbers of cases and controls, prevalence of traditional risk factors for CVD (obesity, diabetes, smoking, dyslipidemia, and hypertension), the prevalence of C. pneumoniae antibodies (IgA or IgG), determined by microimmunofluorescence or ELISA, and the levels of inflammatory markers (CRP, IL-6, fibrinogen, IFN-, TNF-, ICAM-1, and VCAM-1). Only inflammatory markers analysed by at least two studies were included in our meta-analysis.

Statistical Analysis.
Differences between groups were assessed by means of differences in averages and standard deviations of the difference, as markers assessed were all continuous. Standard errors were computed after a normal approximation. Pooled effects were computed by means of a meta-analysis. The meta-analysis conducted was strictly under heterogeneity among studies, by means of a hierarchical Bayesian model. Effect sizes were assumed to be normally distributed. Each study effect was assumed to arise from a Gaussian centered on a study-specific effect and the extracted standard error, inflated by 25% to obtain a conservative statement. The study-specific effect was assumed to be Gaussian, centered on an unknown pooled effect, which is the main object of interest. An informative prior was used for the variance of the pooled effect, as an inverse Gamma centered on an estimator obtained with a moment-based approach (inflated by 25% to obtain a conservative statement). We also estimated posterior probabilities of no difference ( ). of less than 0.05 was considered as statistically significant. Potential publication bias was estimated using Egger's linear regression test and funnel plots. Sensitivity analyses were assessed by deleting each study; in all cases, pooled estimates were very stable.
All data were collected using Microsoft Office Excel 2007; statistical analyses were performed using R software.

Study
Characteristics. 25 studies concerning the association of C. pneumoniae and atherosclerosis through inflammation met the inclusion criteria and comprised 12 casecontrol, 6 cross-sectional, and 7 prospective studies . Overall, a total of 3633 cases and 2781 controls for the case-control analysis, 1593 seropositive and 915 seronegative      in patients with atherosclerosis compared to health controls ( < 0.05), whereas the adhesion molecules ICAM-1 and VCAM-1 did not show any significant difference ( > 0.05) (Table 3, Figure 1).
For the C. pneumoniae IgA analysis, hsCRP, IL-6, and fibrinogen were markedly increased in seropositive patients ( < 0.01), whereas IFN-did not show any significant difference between seropositive and seronegative patients ( > 0.05) (Figure 3). Results are independent of the main confounding factors as the meta-analysis was computed under heterogeneity. We also have verified that they could not form a relationship with mean age and study proportions of males, patients with diabetes, hypertension, obesity, smoking, dyslipidemia, and risk factors for CVDs.

Publication Bias.
Funnel plots and Egger's test were performed to assess publication bias. Publication bias was detected only for meta-analysis of fibrinogen with IgA ( = 0.012). In particular no publication bias was detected for hsCRP for IgA ( = 0.200), IgG ( = 0.890), and case-control data ( = 0.122).

Discussion
Atherosclerosis, precursor to cardiovascular diseases, is recognised as a chronic inflammatory disease of large arteries, involving cytokines, such as IFN-and TNF-, adhesion molecules, such as ICAM-1 and VCAM-1, and several plasma inflammatory markers, such as hsCRP, IL-6, and fibrinogen [2,3]. The latter have been demonstrated to better correlate with the chronic inflammation underlying atherosclerotic cardiovascular diseases; in fact, IL-6, a proinflammatory cytokine that plays a role in the instability of a vulnerable plaque, and hsCRP, an acute-phase protein, appear to be strong independent predictors of cardiovascular events [3]. Lastly, fibrinogen, a substrate leading to the generation of thrombin, the end point of the hemostatic process, is involved in the early formation and growth of the atherosclerotic plaque [69].
As previously described, C. pneumoniae has been suggested to contribute to the chronic inflammation underlying the atherosclerotic process, as evidenced by elevated levels of circulating inflammatory markers in atherosclerotic patients. Gattone et al., in 2001, have suggested, for the first time, the association of C. pneumoniae seropositivity and high hsCRP levels, with increased risk of myocardial infarction [46]. Since then, other studies [47,49,50,52,57,61,64,67] have supported the relationship between C. pneumoniae infection and atherogenesis through systemic inflammation. On the other hand, some studies have failed to demonstrate such association [44,48,55,68], thus making it difficult to draw conclusions.
To the best of our knowledge, our study is the first metaanalysis to evaluate whether C. pneumoniae may contribute to the pathogenesis of atherosclerotic cardiovascular diseases by the means of enhanced inflammation, assessed by levels of inflammatory markers.
The first result of our meta-analysis showed a significant increase in hsCRP, fibrinogen, IL-6, TNF-, and IFNin atherosclerotic patients compared to healthy controls. These data, together with the higher seroprevalence of C. pneumoniae in atherosclerotic patients compared to healthy controls ( < 0.001), suggest that C. pneumoniae infection may contribute to the chronic inflammation underlying the development and progression of atherosclerosis, even though it is not possible to establish a causal relationship.
Consequently, in order to better define the contribution of C. pneumoniae on the inflammation underlying atherosclerosis, we performed a further analysis on studies comparing the levels of inflammatory markers in atherosclerotic patients seropositive to C. pneumoniae IgG or IgA with seronegative patients.
The more relevant data is that C. pneumoniae IgA seropositivity was overall more strongly related to the inflammatory state than IgG seropositivity. Indeed, our results showed an increase of inflammatory marker levels, such as hsCRP, fibrinogen, and IL-6, in IgA seropositive patients. Specifically, the standard mean differences of hsCRP, fibrinogen, and IL-6 were, respectively, 7.17, 7.79, and 1.37 times higher in the IgA compared to the IgG analysis, thus showing that seropositivity to C. pneumoniae IgA might better correlate to a chronic inflammatory state in patients with atherosclerosis. This is not particularly surprising since C. pneumoniae IgA is considered a marker of chronic infection, known to be involved in chronic disorders.
The main strength of our meta-analysis is the statistical robustness of our data, since the results were not affected by exclusion of any specific study from the pooled analysis.
However, our meta-analysis has potential limitations. First, since multiple infectious agents, labeled as infectious burden, rather than any single pathogen, have been shown to contribute to the pathogenesis of atherosclerosis, the inflammatory effect of C. pneumoniae might be overestimated. Of note, in all the studies included in our metaanalysis, data regarding other infectious agents were missing or incomplete and, hence, we were not able to quantify their influence on chronic inflammation. Second, potential bias might be introduced in that not all the included studies reported data concerning the confounding factors. Lastly, two different diagnostic tests, along with different cutoffs, were used for detecting C. pneumoniae seropositivity in the included studies and, hence, may partly account for further bias.

Conclusions
In conclusion, despite some limitations, our study suggests that C. pneumoniae infection may contribute to atherosclerotic cardiovascular diseases by enhancing the inflammatory state, as demonstrated by increased levels of systemic inflammatory markers. Furthermore, seropositivity to C. pneumoniae IgA, together with hsCRP, fibrinogen, and IL-6, may be predictive of atherosclerotic cardiovascular risk.
In the future, large scale, prospective, and well-designed studies should be needed to deepen our knowledge concerning the causative role of C. pneumoniae in inflammation in atherosclerotic cardiovascular diseases.