Chlamydia pneumoniae Heat Shock Protein 60 Enhances Expression of ERK,TLR-4 and IL-8 in Atheromatous Plaques of Coronary Artery Disease Patients

Chlamydia pneumoniae heat shock protein (cHSP) 60 is produced during chronic chlamydial infection and activate innate immune and inflammatory responses thereby contributing to atherogenesis. However, to date there is no apparent signaling cascade delineated in human atherosclerotic plaques in C. pneumoniae positive coronary artery disease (CAD) patients. Atherosclerotic plaques were obtained from 40 CAD patients (28 men, 12 women) attending Department of Cardio Thoracic and Vascular Surgery Safdarjung Hospital, New Delhi. Atherosclerotic plaques were used for gene expression studies at RNA level by real-time PCR and to study expression of ERK1/2, JNK1/2, NF-kB, IkkB and MCP-1 at protein level by immunoblotting. Significantly higher (p < 0.001) RNA expression was found for IL-8, TLR-2/4, TGF-β, ICAM1, VCAM1 and MAPKinase genes, whereas significantly lower (p < 0.001) RNA expression for SMAD4, IkkB, BRCA1 and IL-10 was detected in cHSP60-positive atheromatous plaque of CAD patients. Moreover, at proteins level pERK1/2 (p = 0.05), NF-kB (p = 0.017), MCP-1 (p = 0.011) was higher and IkkB expression was lower (p = 0.038) in cHSP60-positive atheromatous plaque of CAD patients. This study by using human atheromatous plaques at RNA and protein levels demonstrated higher expression of TLR-2/4, IL-8, ICAM1, VCAM1, ERK1/2 and NF-kB in cHSP60-positive CAD patients.     

Antibodies to 60-kilodalton heat shock protein and outer membrane protein 2 of Chlamydia pneumoniae in patients with coronary heart disease.

Evidence linking Chlamydia pneumoniae infection to atherosclerosis and to atherothrombotic events has recently emerged. A primary candidate implicated in these pathogenetic events is the 60-kDa chlamydial heat shock protein (HSP60). Another putative candidate to activate a potential proinflammatory mechanism is the chlamydial outer membrane protein 2 (OMP2). We have generated both HSP60 and OMP2 recombinant antigens in a nondenatured form and shown that (i) the two antigens were highly immunogenic in mice and (ii) murine antisera thus generated recognized the native C. pneumoniae proteins. We measured by enzyme linked immunosorbent assay (ELISA) and immunoblot assay antibody titers to the recombinant antigens in samples from 219 patients with coronary heart disease (CHD), 179 patients with unstable angina (UA), 40 patients with acute myocardial infarction (AMI), and 100 age-, sex-, and risk factor-matched healthy controls. We also examined whether anti-HSP60 and/or anti-OMP2 antibodies correlated with anti-C. pneumoniae antibodies assessed by a commercial microimmunofluorescence (MIF) assay. Immunoglobulin G (IgG), but neither IgA nor IgM, antibodies against the two recombinant proteins were detected by ELISA. In particular, anti-HSP60 antibodies were detected in >99% of CHD patients versus 0% of the controls, whereas the proportions of anti-OMP2 positive subjects were >70 and 27%, respectively. Nonetheless, among CHD patients, similar frequencies of positive subjects and titers of anti-HSP60 or anti-OMP2 antibodies were present in UA and AMI subjects. The anti-OMP2, but not the anti-HSP60, antibodies showed high specificity. Consistently, high serological correlation was observed between IgG MIF titers and IgG ELISA reactivity to OMP2 but not to HSP60. Overall, the results of this study demonstrate a strong correlation between CHD and anti-HSP60 IgG levels, as measured by our in-house ELISA. They also suggest that recombinant OMP2 ELISA, because of its high specificity and strong correlation with MIF assay, could be a candidate diagnostic marker for C. pneumoniae infection, which would be of potential usefulness for its specificity and nonsubjective nature.     

Role of chlamydial heat shock protein 60 in the stimulation of innate immune cells by Chlamydia pneumoniae

Chlamydia pneumoniae stimulates potently maturation of and cytokine secretion by bone marrow-derived dendritic cells (BMDDC). BMDDC responses depend mainly on Toll-like receptor (TLR)2 and to a minor extent on TLR4. We demonstrate here using C. pneumoniae in an infectious model with the replication-permissive epithelial cell line HEp2 that HSP60 is produced in substantial amounts in chlamydial inclusions during infection. Electron microscopy of chlamydial inclusions revealed that HSP60 was mainly associated with reticulate bodies, but was also located in between the different chlamydial developmental forms. Supernatants of permissive HEp2 cells infected with C. pneumoniae contained soluble chlamydial HSP60 as demonstrated by Western blotting and were able to stimulate BMDDC of wild-type mice. The stimulatory capacity of culture supernatants correlated with the presence of chlamydial HSP60. In contrast, BMDDC from TLR4-mutant mice crossed to TLR2-deficient mice were not stimulated by the culture supernatant, indicating that chlamydial HSP60 but not cytokines, possibly secreted by infected HEp2 cells, are responsible for the observed stimulation of BMDDC. Purified recombinant HSP60 from C. pneumoniae stimulated BMDDC in a TLR2- and TLR4-dependent fashion similar to the whole microorganism. In summary, these data suggest chlamydial HSP60 as an important mediator of inflammatory responses during infection with C. pneumoniae.     

Continuous B-cell epitopes in Chlamydia trachomatis heat shock protein 60.

B-cell peptide epitopes in chlamydial heat shock protein 60 (hsp60) were elucidated with antisera from 13 rabbits immunized with Chlamydia trachomatis serovars B, C, and L2 and antisera from eight women with C. trachomatis-associated ectopic pregnancies. Thirteen major epitopes were identified with the human sera, 10 of which were also observed with rabbit antisera. Seven of the 13 epitopes recognized by human antisera exhibited cross-reactive antibody binding to homologous peptide sequences in human hsp60. Self-reactive B-cell immunity to hsp60 may contribute to chlamydial disease pathogenesis.     

肺炎衣原体热休克蛋白10的表达及对炎症因子的诱生作用

目的在大肠杆菌中表达肺炎衣原体(Cpn)热休克蛋白10(Hsp10),并观察该蛋白诱生巨噬细胞(MΦ)分泌TNF-α和IL-6等促炎因子的作用。方法将Hsp10基因克隆于pQE30载体,在大肠杆菌(E.coli)M15中表达重组蛋白rHsp10,经亲和层析获得纯化rHsp10。以不同浓度的rHsp10作用于小鼠MΦRAW264.7,酶联免疫吸附试验(ELISA)检测细胞因子TNF-α和IL-6。结果成功构建了pQF30/Hsp10基因,并在大肠杆菌M15中得到高效表达,该蛋白能诱导小鼠MΦ以时间、剂量依赖方式分泌TNF-α和IL-6。结论表达并纯化的rHsp10蛋白能诱导MΦ分泌TNF-α和IL-6等促炎因子。     

Association of Chlamydia pneumoniae infection with HLA-B*35 in patients with coronary artery disease

The immune system may interplay between Chlamydia pneumoniae infection and coronary artery disease (CAD). Major histocompatibility complex genes regulate innate and adaptive immunity. Patients with CAD (n = 100) and controls (n = 74) were enrolled. Human leukocyte antigens (HLA-A, HLA-B, and HLA-DRB1), four lymphotoxin alpha single-nucleotide polymorphisms, and complement C4A and C4B allotypes were typed, and their haplotypes were inferred. The presence of serum C. pneumoniae immunoglobulin A (IgA) (titer, ≥40) or IgG (titer, ≥128) antibodies or immune complex (IC)-bound IgG antibodies (titer, ≥2) was considered to be a serological marker suggesting chronic C. pneumoniae infection. C. pneumoniae IgA antibodies were found more frequently in patients than in controls (P = 0.04). Among the patients, multiple logistic regression analysis showed the HLA-B*35 allele to be the strongest-risk gene for C. pneumoniae infection (odds ratio, 7.88; 95% confidence interval, 2.44 to 25.43; P = 0.0006). Markers of C. pneumoniae infection were found more frequently in patients with the HLA-A*03-B*35 haplotype than in those without the haplotype (P = 0.007 for IgA; P = 0.008 for IgG; P = 0.002 for IC). Smokers with HLA-B*35 or HLA-A*03-B*35 had markers of C. pneumoniae infection that appeared more often than in smokers without these genes (P = 0.003 and P = 0.001, respectively). No associations were found in controls. In conclusion, HLA-B*35 may be the link between chronic C. pneumoniae infection and CAD.     

Antibodies against the human heat shock protein hsp70 in patients with severe coronary artery disease

Heat shock proteins (hsps) play complex role in the function of the immune system, they can activate both humoral and cellular immune response, as well the complement system. Although autoimmunity to hsp70 was implicated in certain autoimmune diseases and other conditions, the exact role of anti-hsp70 antibodies is not known. It was demonstrated by our previous work and other's findings that antibodies against the 60 kDa hsps are strongly associated with coronary atherosclerosis and carotis disease. It is also known that there is increased hsp70 expression at different sites of atherosclerosis. Therefore our aim was to study whether level of anti-hsp70 antibodies correlate with the presence of severe coronary artery disease (CAD). We measured and compared anti-hsp70 IgG antibody levels in CAD patients (n = 99) and healthy subjects (n = 99) with ELISA. The frequency of these antibodies was high in both groups and there was no significant difference in the median level of anti-hsp70 antibodies between patients with severe CAD and controls (653 (400-1141) vs. 630 (326-1152) AU/mL, P = 0.337). Adjustment for age, sex, BMI and lipid parameters did not change this result. Furthermore we did not find a correlation between anti-hsp70 antibody levels and certain risk factors of CAD (age, lipid parameters, body mass index, C-reactive protein, gender, smoking, diabetes and anti-hsp60 antibodies). By contrast, in accordance with our previous findings, anti-hsp60 and anti-hsp65 antibody levels were significantly higher in CAD patients, compared to this control group (p < 0.0001 for both variables). We did not find any correlation between the levels of anti-hsp70 and anti-hsp60 or anti-hsp65 antibodies either in the patients or the controls. The exact role of hsp70 in atherosclerosis is controversial, but we suggest that humoral immunity against human hsp70 does not contribute to coronary atherosclerosis in contrast to antibodies against 60kDa hsps.     

Chronic Chlamydia pneumoniae infection may promote coronary artery disease in humans through enhancing secretion of interleukin-4

Atherosclerosis is an inflammatory response, probably to a range of initiating causes. Chronic infection with Chlamydia pneumoniae (C.pn) has been suggested as one cause, but the nature of the association is controversial, in large part due to lack of an identified mechanism to link infection with the atherosclerotic process in man. This study examined 139 consecutive subjects with stable chest pain, with the aim of correlating the serological status of C.pn infection with the pattern of secretion of cytokines from CD4(+) T lymphocytes. C.pn seropositive subjects secreted significantly more interleukin (IL)-4 than did those who were C.pn seronegative (P = 0.02). No significant difference was noted for secreted interferon (IFN)-gamma. The amount of secreted IL-4, but not of secreted IFN-gamma, correlated positively with the extent of coronary artery disease (P = 0.006). A similar correlation with secreted IL-4 was not identified with Helicobacter pylori infection. These results support the hypothesis that C.pn infection contributes to the inflammatory process responsible for coronary artery atherosclerosis. The method used to detect cytokine secretion involves ligation of CD40L on blood CD4(+) T cells, which may have relevance to tissue events.     

Cortical senile plaques in coronary artery disease, aging and Alzheimer's disease

Mild alterations in cognitive function are present in normal aging and severe cognitive alterations are a hallmark of Alzheimer's disease (AD). The cognitive change in AD has been correlated to the characteristic pathologic lesions in the brain, senile plaques (SP) and neurofibrillary tangles. Senile plaques are the most consistent correlative marker in AD. We present preliminary data indicating that abundant SP are found in the brains of nondemented patients dying with or as a result of critical coronary artery disease (cCAD) compared to nonheart disease (non-HD) subjects; 15 of 20 cCAD patients contained SP and only two of 16 non-HD patients contained SP.     

Heat shock protein 60 from Chlamydia pneumoniae elicits an unusual set of inflammatory responses via Toll-like receptor 2 and 4 in vivo

Heat shock protein 60 (HSP60) from Chlamydia pneumoniae was described to trigger in vitro inflammatory and cytokine responses including TNF and IL-12p40. Although it can be found in atherosclerotic plaques of patients, the stimulatory potential of chlamydial and other HSP60 in vivo is unclear. We now report that chlamydial HSP60 fails to induce TNF expression in vivo, and significant serum levels of IL-12p40 are only found upon intraperitoneal injection of high doses of HSP60 or after intravenous application. Upon purification of chlamydial HSP60 with polymyxin B-agarose columns, its ability to induce TNF secretion in vitro is much reduced. However, purified chlamydial HSP60 causes increased serum levels of the CXC chemokines KC and MIP2 in vivo, as well as a strong accumulation of polymorphonuclear neutrophils (PMN) in the peritoneal cavity upon intraperitoneal challenge. With respect to PMN accumulation, chlamydial HSP60 is more potent than endotoxin or the CpG oligonucleotide 1668. The responses observed are completely abolished in Toll-like receptor (TLR)2/4-double-deficient mice, while single-deficient mice respond almost normally. Furthermore, KC induction and PMN accumulation are largely dependent on MyD88. In conclusion, HSP60 from C. pneumoniae triggers inflammatory responses in vivo that differ from responses induced by endotoxin or CpG oligonucleotides and are dependent on TLR2 and 4.     

Serological responses of patients with ectopic pregnancy to epitopes of the Chlamydia trachomatis 60 kDa heat shock protein

Clinical and histopathological correlations of immunoreactivity to Chlamydia trachomatis and to epitopes of the C. trachomatis 60 kDa heat shock protein (hsp60) among women with ectopic pregnancy were evaluated in a case-control study. Serological responses to 13 synthetic peptides corresponding to major epitopes of the chlamydial hsp60 were determined in 67 women treated for ectopic pregnancy and 45 women with uncomplicated pregnancy in utero. Plasma cell salpingitis was detected in 29 (43.3%) of the ectopic patients. Its presence correlated with antibodies to two hsp60 epitopes, encompassing amino acids 260-271 and 411-422 (P = 0.02). Antibodies to these two epitopes, along with five other epitopes, also correlated with peritubal adhesion formation in ectopic pregnant patients (P < 0.01). Antibodies to epitopes 260-271 and 188-199 also correlated with a history of pelvic inflammatory disease (PID; P = 0.05). Patients with ectopic pregnancy were also more likely than their intrauterine pregnant controls to have present anti- chlamydial immunoglobulin G (P < 0.005). Women positive for both C. trachomatis and hsp60 epitope antibodies had an increased prevalence over controls of salpingitis, pelvic adhesions or history of PID (P < 0.05). In contrast, patients who were positive for only C. trachomatis antibodies or only hsp60 epitope antibodies did not differ from antibody-negative patients in each of these categories.      

Humoral immune response to membrane components of Chlamydia trachomatis and expression of human 60 kDa heat shock protein in follicular fluid of in-vitro fertilization patients

Recent evidence suggests that Chlamydia trachomatis can persist in the female upper genital tract in an unculturable state. Since unsuspected C. trachomatis infection has been associated with adverse in-vitro fertilization (IVF) outcome we sought to detect further evidence of C. trachomatis in the genital tracts of women undergoing IVF. The prevalence and distribution of antibodies to the major structural proteins of C. trachomatis in paired follicular fluid and sera of women undergoing IVF were examined. Sera and follicular fluid samples from 149 women were assayed for immunoglobulin (Ig)G and IgA antibodies to two C. trachomatis antigens, the major outer membrane protein (MOMP) and a recombinant lipopolysaccharide (rLPS) fragment. Additionally, the expression of human 60 kDa heat shock protein (hsp 60) in follicular fluid was determined. All cervical and follicular fluid samples were negative for C. trachomatis by polymerase chain reaction, ligase chain reaction and DNA probe. Sera from 60% of the subjects were positive for antichlamydial rLPS IgG; 36% were positive for anti-MOMP IgG. Similarly, rLPS-directed and MOMP-directed IgA were detected in sera of 34 and 14% of the subjects respectively. IgG antibodies to MOMP and rLPS were detected in 42 and 41% of the follicular fluid examined respectively. Anti-MOMP IgA was identified in 8.7% of the follicular fluid while 27.5% were positive for anti-rLPS IgA. Human hsp 60 expression was documented in 11.6% of the follicular fluid tested. IgA antibodies to both MOMP (P = 0.03) and rLPS (P = 0.02) in follicular fluid were associated with a failure to become pregnant after embryo transfer. IgG antibodies in sera and follicular fluid and IgA antibodies in sera were unrelated to IVF outcome. Similarly only anti- MOMP IgA (P = 0.02) and anti-rLPS IgA (P = 0.04) in follicular fluid were correlated with human hsp 60 expression in follicular fluid. The unique association between IgA antibodies to two chlamydial antigens in follicular fluid and both hsp 60 expression and IVF failure provides further support for the possibility that a persistent upper genital tract chlamydial infection contributes to IVF failure in some women.      

Helicobacter species in the atherosclerotic plaques of patients with coronary artery disease

IntroductionSeveral epidemiological studies have proposed an association between Helicobacter pylori infection and coronary artery disease. In the current study, we aimed to evaluate the prevalence and relevance of H. pylori infection, using polymerase chain reaction (PCR) methods, in the coronary arterial wall of Iranian patients who have already undergone coronary bypass grafting (CABG).MethodsA total of 105 consecutive patients who underwent CABG at the Department of Cardiovascular Surgery of Baqiyatallah University of Medical Sciences were included in the study, and biopsy specimens from their coronary plaques were taken and analyzed using the PCR methods for detecting Helicobacter species (H Spp.). Fifty-three specimens from biopsies of the left internal mamillary artery in the same patients were also collected and tested.ResultsH. Spp. PCR test result was positive for 31 (29.5%) specimens from coronary artery atherosclerotic plaques. Serologic test results also showed 25 (23.8%) positive cases for H. pylrori immunoglobulin A (IgA) and 56 (53.3%) positive for anti-H. pylori immunoglobulin G. None of the specimens from the mamillary artery were positive for H Spp. genome when it was evaluated using PCR (P<.0001). Patients with positive test result for H. pylori IgA were significantly more likely to have higher total cholesterol and low-density lipoprotein (LDL) levels than IgA-negative patients.ConclusionH Spp. infection replication in the coronary arterial wall is associated with atherosclerotic plaque formation. Seropositivity for H. pylori IgA may also enhance blood values of total cholesterol and LDL in these patients.     

Levels of intra- and extracellular heat shock protein 60 in Kawasaki disease patients treated with intravenous immunoglobulin

Immune reactivity to autologous heat shock protein 60 (HSP60) has been reported to be associated with a favorable prognosis in autoimmune diseases. To provide a clue for the possible role of HSP60 in Kawasaki disease (KD), we investigated the levels of intra- and extracellular HSP60 in the course of KD. In KD patients, autologous HSP60 was abundantly expressed in CD11c(+) cells during the acute phase and subsequently decreased during the subacute phase. Most of HSP60-expressing CD11c(+) cells observed in the acute phase was composed of CD11c(low) cells instead of CD11c(high) cells, which were dominant in the subacute phase. In contrast, circulating HSP60 levels were higher in the subacute phase than those in the acute phase, reflecting higher level of HSP60 exposure to the immune system of patients during recovery. These changes in the levels of intra- and extracellular HSP60 were not observed in patients with other febrile diseases. The observed features of HSP60 expression in patients with KD are in favor of a role for autologous HSP60 as a regulator for control of inflammation, rather than a proinflammatory mediator in KD.     

Prevalence of Chlamydophila pneumoniae is higher in aorta and coronary artery than in carotid artery of coronary artery disease patients

Jha HC, Srivastava P, Divya A, Prasad J, Mittal A. Prevalence of Chlamydophila pneumoniae is higher in aorta and coronary artery than in carotid artery of coronary artery disease patients. APMIS 2009; 117: 905–11. Coronary artery disease (CAD) is a public health problem accounting for an estimated one‐third of deaths overall. A potential link between infectious agents and atherosclerosis has been suggested. Data obtained from several seroepidemiological studies have suggested that infection with Chlamydiophila pneumoniae, Helicobacter pylori, cytomegalovirus and herpes simplex virus‐1 can initiate or maintain the atherosclerotic process. However, there is no single study in which multiple infectious agents have been detected together in different vascular locations in the same population. This would help in determining if there is any leading pathogen in atheromatous plaques of CAD patients. Hence, we screened for C. pneumoniae, H. pylori, CMV and HSV‐1 in different vascular locations of CAD patients using quantitative real‐time (RT) PCR. We performed multiplex RT‐PCR for detecting pathogens, viz. C. pneumoniae, H. pylori, CMV and HSV‐1 in different vascular locations of CAD patients. Percent positivity scores for C. pneumoniae, H. pylori, CMV and HSV‐1 in different vascular locations were as follows: aorta (64.7, 35.3, 11.7 and 11.7 respectively); carotid (27.2, 27.2, 9 and 0 respectively); coronary artery (58.3, 33.3, 16.6 and 8.3 respectively). Combined positivity for C. pneumoniae (C. pneumoniae IgA and RT‐PCR for C. pneumoniae) was the highest compared with all other groups. Aorta and coronary artery were more susceptible to these pathogens as compared with carotid artery. Moreover, CAD patients’ characteristics were associated with C. pneumoniae positivity (C. pneumoniae IgA and RT‐PCR), suggesting thereby that C. pneumoniae may have caused chronic persistent infection in CAD.     

Effect of clarithromycin treatment on Chlamydia pneumoniae in vascular tissue of patients with coronary artery disease: a randomized, double-blind, placebo-controlled trial

Several small clinical trials have indicated that antibiotic treatment of Chlamydia pneumoniae infection is associated with a better outcome in patients with coronary artery disease (CAD). It has not been demonstrated whether antibiotic treatment eradicates C. pneumoniae from vascular tissue. The aim of the present study was to assess the effect of clarithromycin on the presence of C. pneumoniae in the vascular tissue of patients with CAD. Patients who had CAD and who were waiting for coronary artery bypass graft surgery were enrolled in a randomized, double-blind, placebo-controlled trial. Patients were treated with clarithromycin at 500 mg or placebo once daily from the day of inclusion in the study until surgery. Several vascular tissue specimens were obtained during surgery. The presence of C. pneumoniae in vascular tissue specimens was examined by immunohistochemical staining (IHC) and two PCR assays. Chlamydia immunoglobulin G (IgG) titers were determined by an enzyme-linked immunosorbent assay at the time of inclusion in the study and 8 weeks after surgery. A total of 76 patients were included, and 180 vascular tissue specimens were obtained (80 specimens from the group treated with clarithromycin and 100 specimens from the group treated with placebo). Thirty-five patients received clarithromycin (mean duration, 27 days; standard deviation [SD], 12.2 days), and 41 patients received placebo (mean duration, 27 days; SD, 13.9 days). IHC detected the C. pneumoniae major outer membrane protein antigen in 73.8% of the specimens from the group treated with clarithromycin and 77.0% of the specimens from the group treated with placebo (P was not significant). Chlamydia lipopolysaccharide antigen was found in only one specimen from the group that received placebo. C. pneumoniae DNA was not detected in any specimen. Baseline Chlamydia-specific IgG titers were equally distributed in both groups and were not significantly different after treatment. There was no indication of an active C. pneumoniae infection in vascular tissue. Chlamydia-specific IgG titers remained unchanged throughout the study in both the antibiotic- and the placebo-treated patients.     

Poor correlation between microimmunofluorescence serology and polymerase chain reaction for detection of vascular Chlamydia pneumoniae infection in coronary artery disease patients

Chlamydia pneumoniae has been associated to coronary artery disease by various methods including recovery of viable bacteria from plaques. The pathogenetical relevance of this is unclear but investigation of antichlamydial therapy in coronary arteriosclerosis is already in progress. The microimmunofluorescence test (MIF), the only species-specific serological assay available, might be considered useful in identifying patients with vascular chlamydial infection. However, this has never been systematically examined. We compared levels of C. pneumoniae antibodies in sera using MIF with direct detection of C. pneumoniae in coronary artery segments from 158 patients undergoing myocardial revascularization. A polymerase chain reaction (PCR) protocol, recently evaluated for use with vascular materials, detected C. pneumoniae infection in 34 patients. Correlation of serology and PCR was poor: in relation to PCR, MIF-IgG analysis had 21% sensitivity, 90% specificity, 37% positive predictive value, and 81% negative predictive value for detection of chlamydial presence. Thus, the MIF test currently appears not suitable to predict individual vascular C. pneumoniae infection. Received: 19 June 1998