Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells.

Chlamydia pneumoniae has recently been associated with atherosclerotic lesions in coronary arteries. To investigate the biological basis for the dissemination and proliferation of this organism in such lesions, the in vitro growth of C. pneumoniae was studied in two macrophage cell lines, peripheral blood monocyte-derived macrophages, human bronchoalveolar lavage macrophages, several endothelial cell lines, and aortic smooth muscle cells. Five strains of C. pneumoniae were capable of three passages in human U937 macrophages and in murine RAW 246.7 macrophages. Titers were suppressed in both macrophage types with each passage, as compared with growth titers in HEp-2 cells. Both human bronchoalveolar lavage macrophages and peripheral blood monocyte-derived macrophages were able to inhibit C. pneumoniae after 96 h of growth. Eleven C. pneumoniae strains were capable of replicating in normal human aortic artery-derived endothelial cells, umbilical vein-derived endothelial cells, and pulmonary artery endothelial cells. Infection in human aortic artery smooth muscle cells was also established for 13 strains of C. pneumoniae. The in vitro ability of C. pneumoniae to maintain infections in macrophages, endothelial cells, and aortic smooth muscle cells may provide support for the hypothesis that C. pneumoniae can infect such cells and, when infection is followed by an immune response, may contribute to atheroma formation in vivo. More studies are needed to investigate the complex relationship between lytic infection and persistence and the potential for C. pneumoniae to influence the generation of atheromatous lesions.     

Differences in innate immune responses (in vitro) to HeLa cells infected with nondisseminating serovar E and disseminating serovar L2 of Chlamydia trachomatis

The inflammatory response associated with Chlamydia trachomatis genital infections is thought to be initiated by the release of proinflammatory cytokines from infected epithelial cells. This study focuses on the interactions between C. trachomatis-infected HeLa cells and immune cells involved in the early stages of infection, i.e., neutrophils and macrophages. First, we showed that the expression of interleukin-11 (IL-11), an anti-inflammatory cytokine mainly active on macrophages, was upregulated at the mRNA level in the genital tracts of infected mice. Second, incubation of differentiated THP-1 (dTHP-1) cells or monocyte-derived macrophages (MdM) with basal culture supernatants from C. trachomatis serovar E- or serovar L2-infected HeLa cells resulted in macrophage activation with a differential release of tumor necrosis factor alpha (TNF-alpha) and upregulation of indoleamine 2,3-deoxygenase (IDO) but not of Toll-like receptor 2 and 4 mRNA expression. Third, coculture of infected HeLa cells with dTHP-1 cells resulted in a reduction in chlamydial growth, which was more dramatic for serovar E than for L2 and which was partially reversed by the addition of anti-TNF-alpha antibodies for serovar E or exogenous tryptophan for both serovars but was not reversed by the addition of superoxide dismutase or anti-IL-8 or anti-IL-1beta antibodies. A gamma interferon-independent IDO mRNA upregulation was also detected in dTHP-1 cells from infected cocultures. Lastly, with a two-stage coculture system, we found that (i) supernatants from neutrophils added to the apical side of infected HeLa cell cultures were chlamydicidal and induced MdM to express antichlamydial activity and (ii) although polymorphonuclear leukocytes released more proinflammatory cytokines in response to serovar E- than in response to L2-infected cells, MdM were strongly activated by serovar L2 infection, indicating that the early inflammatory response generated with a nondisseminating or a disseminating strain is different.     

Temporal cytokine profiling of Francisella tularensis-infected human peripheral blood mononuclear cells

BACKGROUND AND PURPOSE: Francisella tularensis is an intracellular bacterium known to replicate in monocytes and macrophages and cause tularemia in humans. Because of its infectious nature, F. tularensis is considered a biowarfare agent. Early cytokine profiles of Francisella-infected human peripheral blood mononuclear cells were evaluated. METHODS: Populations of human peripheral blood mononuclear cells were infected in vitro with F. tularensis live vaccine strain at a very low multiplicity of infection of 1:10 (bacteria:cells). A multiplex bead kit which analyzes 30 cytokines, chemokines and growth factors was utilized to measure secreted cytokines in cell supernatants 1, 4, 8, 16, and 24 h post-infection. RESULTS: Compared with uninfected controls, infected cells showed no increase in cytokine secretion at 1 and 4 h, implying a threshold for activation of immune responses. Starting at 8 h post-infection and continuing through to 24 h, an array of cytokines and growth factors was secreted by the infected cells. Some cytokines not previously associated with Francisella infection in humans were detected at 8 h, including interleukin-17 and interleukin-1 receptor agonist and vascular endothelial growth factor. CONCLUSIONS: The cytokine profiles of F. tularensis-infected peripheral blood mononuclear cells indicate an intricate pattern of both pro- and anti-inflammatory responses, including early T-cell activation.     

Chlamydia pneumoniae Infection of Human Endothelial Cells Induces Proliferation of Smooth Muscle Cells via an Endothelial Cell-Derived Soluble Factor(s)

An association of Chlamydia pneumoniae with atherosclerosis and coronary heart disease has been determined epidemiologically and by the detection of C. pneumoniae organisms in atherosclerotic lesions in both humans and animal models of atherosclerosis. Previously, it has been shown that C. pneumoniae is capable of replicating in cell types found within atheromatous lesions, viz., endothelial cells, smooth muscle cells (SMC), and macrophages, yet the role of C. pneumoniae in the pathogenesis of atherosclerosis has not been determined. Since intimal thickening is a hallmark of atherosclerosis, we investigated whether C. pneumoniae infection of human umbilical vein endothelial cells (HUVEC) could induce the expression of a soluble factor(s) with mitogenic potential for SMC by using [3H]thymidine incorporation and direct cell counting. Conditioned medium harvested from HUVEC infected with C. pneumoniae stimulated SMC replication in a time- and dose-dependent fashion. Infection studies using various multiplicities of infection (MOIs) ranging from 0.001 to 1 demonstrated a dose-dependent production of the soluble factor(s). At an MOI of 1, SMC stimulation indices were 8.4 (P < 0.01) and 12.2 (P < 0.01) for conditioned media harvested at 24 and 48 h, respectively. To determine whether viable C. pneumoniae was required for production of the soluble factor(s), HUVEC were infected with heat-inactivated C. pneumoniae or with viable organisms in the presence of chloramphenicol. Both treatments produced stimulation indices similar to those for live C. pneumoniae in the absence of chloramphenicol (P > 0.05), indicating that the factor(s) was produced by HUVEC and not by C. pneumoniae and that signal transduction events following chlamydia endocytosis may be important in the production of a soluble factor(s). The ability of C. pneumoniae to elicit an endothelial cell-derived soluble factor(s) that stimulates SMC proliferation may be important in the pathogenesis of atherosclerosis.     

肺炎衣原体对血管内皮细胞的感染及其对ICAM-1表达的影响

目的:观察肺炎衣原体对人脐静脉内皮细胞(HUVECs)的感染及其对细胞分泌和表达细胞间粘附分子1(ICAM-1)的影响,探讨C.pneumoniae感染在动脉粥样硬化形成中的作用及其可能机制。方法:用人喉表皮癌(HEP-2)细胞培养C.pneumoniae,以C.pneumoniae感染HUVE细胞,经透射电镜及PCR检测有无感染。用流式细胞仪检测感染前后HUVE细胞表面ICAM-1蛋白的表达的变化,用荧光定量RT-PCR检测ICAM-1mRNA的变化。结果:C.pneumoniae能感染体外培养的HUVE细胞;感染后12h,细胞表面ICAM-1蛋白的表达即增加,其峰值约在感染后24h;荧光定量RT-PCR结果显示其增加在mRNA水平。结论:C.pneumoniae能感染体外培养的人脐静脉内皮细胞并增加ICAM-1的表达,提示C.pneumoniae感染可能是动脉粥样硬化的始动因子之一,其致动脉粥样硬化机制可能与感染后血管内皮细胞粘附分子表达的增加有关。     

Effect of Chlamydia pneumoniae on cellular ATP content in mouse macrophages: role of Toll-like receptor 2

Chlamydiae are obligate intracellular gram-negative bacteria and are dependent on the host cell for ATP. Thus, chlamydial infection may alter the intracellular levels of ATP and affect all energy-dependent processes within the cell. We have shown that both live C. pneumoniae and inactivated C. pneumoniae induce markers of cell death prior to completion of the bacterial growth cycle. As depletion of ATP could account for the observed increase in cell death, the effects of C. pneumoniae on ATP concentrations within mouse macrophages were investigated. Live, heat-killed, and UV-inactivated C. pneumoniae cultures (at multiplicities of infection [MOIs] of 0.01, 0.1, and 1.0) were incubated with mouse bone marrow macrophages isolated from C57BL/6J mice and mice deficient in Toll-like receptors. Treatment of the macrophages with both live and inactivated C. pneumoniae increased the ATP content of the cells. In cells infected with live C. pneumoniae, the increase was inversely proportional to the MOI. In cells treated with inactivated C. pneumoniae, the increase in ATP content was smaller than that induced by infection with live organisms and was proportional to the MOI. The increase in ATP content early in the developmental cycle was independent of the growth of C. pneumoniae, while sustained induction required live organisms. The capacity of C. pneumoniae to increase the ATP content was ablated in macrophages deficient in expression of either Toll-like receptor 2 or the Toll-like receptor accessory protein MyD88. In contrast, no effect was observed in macrophages lacking expression of Toll-like receptor 4.     

In vitro susceptibility of human vascular wall cells to infection with Chlamydia pneumoniae.

Chlamydia pneumoniae is a common respiratory pathogen. Recent studies have demonstrated the presence of C. pneumoniae in coronary and aortic atherosclerotic lesions. To study the role of C. pneumoniae in atherosclerosis, we investigated the susceptibilities of three different cells of the human vascular wall to infection with C. pneumoniae AR-39. These cell types were endothelial cells, smooth muscle cells, and macrophages derived from peripheral blood monocytes. Infection was assessed by using a direct fluorescent antibody to assess inclusion counts. Duplicate cell samples were harvested 3 days postinfection and were passed in HL cells, a susceptible human epithelial cell line, to determine if infectious organisms were produced. Endothelial cells, smooth muscle cells, and macrophages were capable of supporting C. pneumoniae growth in vitro. These results showed that three different cell types known to be important in atherogenesis are susceptible to infection with C. pneumoniae.     

Chlamydia pneumoniae infection induces differentiation of monocytes into macrophages

Migration and differentiation of monocytes to the intima of blood vessels may be a crucial first step in the development of atherosclerosis associated with Chlamydia (Chlamydophila) pneumoniae. However, the involvement of C. pneumoniae infection in such steps is not clear. In the present study, therefore, the differentiation-inducing activity of C. pneumoniae to monocytes was examined. Human THP-1 monocytic cell line cells were infected with C. pneumoniae, and the differentiation of monocytes to macrophages was assessed by cell morphology, phagocytic activity, and expression of a cell surface adhesion molecule. The monocytic cells infected with viable bacteria markedly differentiated into macrophages associated with diffused cell morphology, increased uptake of polystyrene beads and increased ICAM-1 (intercellular adhesion molecule 1) expression on the cell surfaces. Heat-killed bacteria did not induce any morphological changes or increase of phagocytosis, but they did induce an increase of cell surface ICAM-1 expressions in THP-1 monocytic cells. The antibiotic minocycline treatment of infected cells resulted in marked inhibition of the cell differentiation as well as C. pneumoniae growth in the cells, but not ICAM-1 expression. In addition, the experiments with human peripheral blood monocytes infected with C. pneumoniae also showed the differentiation of macrophages assessed by morphological change and phagocytic activity. These results indicate that C. pneumoniae infection may directly induce the differentiation of monocytes to macrophages. However, antigenic stimulation of monocytes with bacteria may not be sufficient for a full macrophage differentiation.     

Effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta and RANTES mRNA expression in guinea pig cells exposed to attenuated and virulent mycobacteria

The effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta (IL-1 beta) or regulated-upon-activation, normally T-cell-expressed and -secreted chemokine (RANTES) mRNA expression in guinea pig spleen cells stimulated with concanavalin A, lipopolysaccharide (LPS), phorbol myristate acetate (PMA) plus ionomycin, or purified protein derivative (PPD) was studied in vitro. Similarly, peritoneal exudate cell-derived macrophages from na?ve and BCG-vaccinated guinea pigs were infected with M. bovis BCG, Mycobacterium avium, the attenuated Mycobacterium tuberculosis H37Ra strain, or virulent strains H37Rv and Erdman of M. tuberculosis. Total RNA was subjected to Northern blot analysis using probes generated from guinea pig IL-1 beta or RANTES cDNA. Although IL-1 beta and RANTES mRNA could be detected in the spleen cells from na?ve animals stimulated with LPS or PMA plus ionomycin, the levels were significantly enhanced after BCG vaccination. mRNA expression was also elevated in macrophages infected with live mycobacteria after BCG vaccination. However, macrophages infected with the virulent H37Rv strain of M. tuberculosis showed 75 to 90% reductions in IL-1 beta expression and 25 to 60% reductions in RANTES mRNA expression compared with macrophages infected with the attenuated H37Ra strain. The IL-1 beta mRNA levels peaked as soon as 1 h after PPD stimulation and 4 h after M. tuberculosis H37Rv infection of macrophages. In contrast, RANTES mRNA expression was delayed until 48 h after infection. These results indicate that molecular mediators produced in response to various stimuli associated with protective immunity against mycobacteria are upregulated after BCG vaccination; however, a significantly weaker response was observed with virulent M. tuberculosis. These initial studies indicate that BCG vaccination has a positive effect on IL-1 beta and RANTES mRNA expression by host cells in a highly relevant animal tuberculosis model.     

Effects of CD14, TLR2, TLR4, LPB, and IL-6 Gene Polymorphisms on Chlamydia pneumoniae Growth in Human Macrophages In Vitro

Chlamydia pneumoniae is an obligate intracellular gram-negative bacterium, which causes respiratory infections in humans. It can infect various cell types, e.g. vascular endothelial cells, smooth muscle cells and monocyte-derived macrophages in vitro . The susceptibility of macrophages from healthy individuals to C. pneumoniae infection is highly variable. In this study, we evaluated the effects of innate immunity genes CD14 −260 C>T, TLR2 Arg753Gln, TLR4 Asp299Gly, LBP Phe436Leu and IL6 −174 G>C polymorphisms on C. pneumoniae growth in human macrophages in vitro. The growth of C. pneumoniae was highest in CD14 −260 C>T TT genotype cells and the difference to CC and CT genotypes was statistically significant ( P  = 0.032 and 0.022 respectively). The G-allele of the IL6 −174 G>C polymorphism had a positive influence on chlamydial growth; the difference was statistically significant only between CC and GC genotypes ( P  = 0.018). TLR2 Arg 753Gln, TLR4 Asp299Gly, LBP Phe436Leu polymorphisms showed no effect on chlamydial growth.     

脂氧素受体激动剂对巨细胞病毒感染巨噬细胞的免疫负调节

背景：脂氧素可以抑制炎症细胞、内皮细胞、小鼠脾脏树突状细胞等合成细胞因子,还可以抑制炎性细胞因子对细胞的生物效应。 目的：分析脂氧素受体激动剂BML-111对人巨细胞病毒感染THP-1源巨噬细胞的免疫调节作用。 方法：用人巨细胞病毒 AD169毒株感染THP-1源巨噬细胞(MOI=0.5),细胞培养后设立对照组、人巨细胞病毒感染组及人巨细胞病毒+BML-111组。在感染后0,1,2,4,12,24,48,72 h收集各组细胞培养上清液,以ELISA检测各组细胞因子的表达水平; RT-PCR检测各指标mRNA的表达强度;Western blot检测感染4 h的细胞核内p65亚基蛋白表达。 结果与结论：与对照组相比,其他两组各细胞因子蛋白及mRNA表达明显升高(P < 0.05)。与人巨细胞病毒感染组相比,人巨细胞病毒+BML-111组白细胞介素1β和肿瘤坏死因子α显著降低,转化生长因子β显著升高(P < 0.05),白细胞介素10差异无显著性意义(P > 0.05);人巨细胞病毒+BML-111组各细胞因子mRNA均明显降低(P < 0.05)。与对照组相比,其他两组细胞核内NF-κB p65亚基蛋白浓度明显升高(P < 0.05);人巨细胞病毒+BML-111组显著低于人巨细胞病毒感染组(P < 0.05)。说明BML-111可能通过抑制NF-κB的p65亚基核转位,减少白细胞介素1β、肿瘤坏死因子α的表达,促进转化生长因子β的表达,从而对人巨细胞病毒感染的THP-1源巨噬细胞发挥其免疫负调节作用。     

Effect of hypoxia on gene expression of bone marrow-derived mesenchymal stem cells and mononuclear cells

MSC have self-renewal and multilineage differentiation potential, including differentiation into endothelial cells and vascular smooth muscle cells. Although bone marrow-derived mononuclear cells (MNC) have been applied for therapeutic angiogenesis in ischemic tissue, little information is available regarding comparison of the molecular foundation between MNC and their MSC subpopulation, as well as their response to ischemic conditions. Thus, we investigated the gene expression profiles between MSC and MNC of rat bone marrow under normoxia and hypoxia using a microarray containing 31,099 genes. In normoxia, 2,232 (7.2%) and 2,193 genes (7.1%) were preferentially expressed more than threefold in MSC and MNC, respectively, and MSC expressed a number of genes involved in development, morphogenesis, cell adhesion, and proliferation, whereas various genes highly expressed in MNC were involved in inflammatory response and chemotaxis. Under hypoxia, 135 (0.44%) and 49 (0.16%) genes were upregulated (>threefold) in MSC and MNC, respectively, and a large number of those upregulated genes were involved in glycolysis and metabolism. Focusing on genes encoding secretory proteins, the upregulated genes in MSC under hypoxia included several molecules involved in cell proliferation and survival, such as vascular endothelial growth factor-D, placenta growth factor, pre-B-cell colony-enhancing factor 1, heparin-binding epidermal growth factor-like growth factor, and matrix metalloproteinase-9, whereas the upregulated genes in MNC under hypoxia included proinflammatory cytokines such as chemokine (C-X-C motif) ligand 2 and interleukin-1alpha. Our results may provide information on the differential molecular mechanisms regulating the properties of MSC and MNC under ischemic conditions. Disclosure of potential conflicts of interest is found at the end of this article.