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Abstract: The chronic course of Chlamydia trachomatis infection is subtle with no obvious unusual inflammatory change. The reason for this is not clear. The data reported here explain how macrophage usual inflammatory response switches to anti-inflammatory response during C. trachomatis infection of mixed culture of macrophages and Jurkat T-cells. We assessed the establishment of productive infection in individual or mixed cell culture models, determined the status of C. trachomatis in the cells by monitoring HSP-60:MOMP or the proportions of the estimated IFUs that shed HSP-60 or MOMP. Also, the specific time-course expression of IL-12, IL-10 and IFN-γ or IL-12R, IL-10R, and IFN-γ-R during infection of cell models was assessed. Finally, the early events in cytokine elaboration in circumstances of varying intracellular Ca2+ levels were determined. There was evidence of productive infection in all individual and mixed cell culture models. The shedding of HSP-60 was highest in THP-1/Jurkat mixed cell culture model. The proportions of IFU that shed HSP-60 was heightened in infected THP-1/Jurkat mixed culture model, while the proportion of IFU that shed MOMP was higher in infected macrophage/Jurkat mixed culture and infected macrophages only. There was profound early elaboration of IL-10, varying significantly from IL-12 and IFN-γ in all infected individual or mixed cell culture models except in the case of Jurkat; where all cytokine elaboration was downregulated. The receptor to IL-10 was upregulated in infected macrophage/Jurkat cells and THP-1/Jurkat cells compared with other models in which IL-12 and IFN-γ receptors were more expressed. There was no observed significant change in cytokine in any model following the impairment of intracellular Ca2+ except in the case of macrophage/Jurkat cell model in which IL-12 and IL-10 were upregulated in 1h or 3h, respectively. The implication of these findings is that C. trachomatis mediates a switch from inflammatory to anti-inflammatory function in macrophages due to downregulation of the regulatory cytokine, IFN-γ in Jurkat cells, culminating in C. trachomatis chronic course. [Copyright &y& Elsevier]

Abstract: Chlamydia trachomatis infections manifest as unique, chronic inflammatory diseases, indicating a relative compromise in the capacity of early immune responders such as macrophages to resolve the infection. We decided to investigate whether or not the chronic inflammatory manifestations are influenced by a disturbance in the pattern of inflammatory:anti-inflammatory cytokine elaboration early in the infection cycle in macrophages and assess the possible modulatory role of Ca2+ signals in the process. Although the basal and functional levels of IL-12 and IL-10 are not identical in concentration, chlamydia initiated a significant decline in IL-12. This led to a difference in the ratio of time-course decline in IL-12 compared with IL-10 in a Ca2+-poor medium, while there was significant increase in IL-10 in a Ca2+-rich medium. Also, when macrophages were infected after treatment with drugs that either facilitated Ca2+ influx into cells or inhibited efflux from intracellular stores into cytosol, there was a significant enhancement of the elaboration of IL-10 compared with IL-12. The immobilization of cytosolic Ca2+ by BAPTA-AM resulted in the decline of macrophage IL-12 and IL-10 in both infected and uninfected cases. There was evidence that infectivity and status of chlamydial elementary bodies harvested from macrophages during these experiments were consistent with chronic forms as assessed by HSP-60:MOMP ratio. The implication of these findings is that chlamydia infection of macrophages, together with its capacity to moderate macrophage intracellular Ca2+ levels, may evoke a net anti-inflammatory response that presumably favors chronic chlamydia infections. [ABSTRACT FROM AUTHOR]

Abstract: Inducible nitric oxide synthase (iNOS) reaction is of enormous significance in innate immune protective response of host to microbial challenges. We speculate that microbial challenges, by virtue of their lipopolysaccharide (LPS) content, exert an effect on intracellular free Ca2+ levels ([Ca2+]i), which evokes cellular signaling in a manner that depends on LPS type. In this study, we investigated the effect of LPS from heterogeneous sources (Chlamydia pneumoniae [cLPS], Pseudomonas aeruginosa [pLPS], and Salmonella typhimurium [sLPS]) on nitric oxide (NO) release and iNOS expression in THP-1- and U937-derived macrophages, and determined the role of free [Ca2+]i on LPS-mediated generation of NO and iNOS expression in these cell types. The role of free [Ca2+]i oscillation and the probable input of extracellular Ca2+ influx on Ca2+-signals was monitored in relation to iNOS expression in LPS-stimulated macrophages. There was a significant difference in the time course release of NO in both macrophage types when stimulated with cLPS, or pLPS, or sLPS. In all instances, LPS induced a significant release of NO compared with interferon-γ. There was a difference in the pattern of basal iNOS reaction leading to NO release compared to iNOS expression observed at 24h post-stimulation. While a biphasic pattern was observed in the manner in which free [Ca2+]i affected iNOS expression, inhibition of extracellular Ca2+ influx resulted in a steady increase in iNOS expression. The implications of these findings are: moderate free [Ca2+]i affects macrophage release of NO and iNOS expression during LPS stimulation; also, LPS of heterogeneous sources differentially evokes macrophage iNOS reactions irrespective of macrophage types. [Copyright &y& Elsevier]

Abstract: Latency, chronicity and recurrent nature are the features of Chlamydia pneumoniae biology which play a central role in the course and outcome of C. pneumoniae–host interaction. Since redox status is directly an indicator of inflammatory response via molecular signaling mechanisms, we decided to study the regulatory role of macrophage cellular redox balance on the molecular indices of C. pneumoniae chronicity. We examined GSH–GSSG status, the activities of antioxidant enzymes (SOD, GPx and γ-GCS), along with their protein and gene expression, the MOMP and cHSP-60 protein and gene expression, and the consequence of redox balance on the establishment of productive infection in macrophages. Results showed that C. pneumoniae caused changes in GSH–GSSG levels, antioxidant enzymes activity, mRNA gene and protein expression in macrophages. The relevance of this to the state and status of C. pneumoniae in macrophages was assessed by inhibitor induced attenuation of antioxidant enzymes and there was evidence that, while SOD attenuation did not significantly affect MOMP and cHSP-60 gene and protein expression, γ-GCS attenuation increased cHSP-60 gene and protein expression. The increase in molecular evidence of chronic forms of C. pneumoniae (cHSP-60) was consistent with decrease in normal forms of C. pneumoniae. These findings reflect the importance of redox balance modulation on the outcome of C. pneumoniae infection in macrophages, a significant process in the pathogenesis of chlamydial diseases. [Copyright &y& Elsevier]

Summary: Recent data have shown a unique relationship between Ca2+ signaling in macrophages through L-type channels and the outcome of C. pneumoniae infection of such cells. The present investigation seeks to provide insights into the manner in which macrophage L-type Ca2+ channel operation affects major outer membrane protein (MOMP) and heat shock protein-60 (HSP-60) mRNA gene expression (factors associated with Chlamydia chronicity), and the possible effect of this on antibiotic susceptibility. Intracellular calcium ([Ca2+]i) chelation using varying doses of 1,2-bis (o-aminophenoxy) ethane-N,N,N′N′ – tetra acetic acid (acetoxymethyl) ester (BAPTA-AM) induced an increase in MOMP and a decrease in HSP-60 mRNA gene expression. L-type Ca2+ channel antagonists produced an identical but enhanced effect. Since these findings associate specialized Ca2+ channels to Chlamydia chronicity, it was important to determine Ca2+ channel effect on the usual antibiotic refractory form of C. pneumoniae in macrophages. Inhibition of macrophage L-type Ca2+ channel operation improved C. pneumoniae antibiotic susceptibility assessed by decreased inclusion counts or down-regulated MOMP and HSP-60 mRNA gene expression. These findings provide molecular insights into how specialized Ca2+ channels influence Chlamydia chronic course in macrophages and demonstrates a role for L-type Ca2+ channel inhibitors in enhanced C. pneumoniae susceptibility to antibiotic therapy. [Copyright &y& Elsevier]