Anthony A. Azenabor

Effective macrophage redox defense against Chlamydia pneumoniae depends on L-type Ca2+ channel activation.

Abstract. Macrophage immune capability depends on their efficient redox potential expressed in the effective release of reactive oxygen species (ROS) and nitric oxide. In this study the effect of the activation of a specialized Ca2+ channel on macrophage redox function during Chlamydia pneumoniae infection was explored. C. pneumoniae exhibited a profound and sustained Ca2+ influx capacity, with evidence of activity attributable to their lipopolysaccharide (cLPS) content. Also the organism showed an additional Ca2+ influx signal in macrophages exposed to thapsigargin, and there was evidence for the operation of a single ion channel of the L type as demonstrated by the effect of L-type channel antagonists (methoxyverapamil and nimodipine) despite exposure to Ca2+-rich medium. C. pneumoniae or cLPS induced intracellular ROS and NO generation in a manner consistent with dependence on intracellular calcium. L-type Ca2+ channel blocking significantly prompted C. pneumoniae inclusion formation. These findings suggest that Ca2+ influx signal and redox function in C. pneumoniae-infected macrophages depend on L-type Ca2+ channel activation.

Free intracellular Ca2+ regulates bacterial lipopolysaccharide induction of iNOS in human macrophages

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 Ca(2+) levels ([Ca(2+)]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 [Ca(2+)]i on LPS-mediated generation of NO and iNOS expression in these cell types. The role of free [Ca(2+)]i oscillation and the probable input of extracellular Ca(2+) influx on Ca(2+)-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-gamma. 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 [Ca(2+)]i affected iNOS expression, inhibition of extracellular Ca(2+) influx resulted in a steady increase in iNOS expression. The implications of these findings are: moderate free [Ca(2+)]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.

Elicitation of reactive oxygen species in Chlamydia pneumoniae-stimulated macrophages: a Ca2+-dependent process involving simultaneous activation of NADPH oxidase and cytochrome oxidase genes

Chlamydia pneumoniae, a respiratory pathogen implicated in the development and progress of atherosclerosis, is known to infect and survive in macrophages, despite macrophage producing reactive oxygen species (ROS). To gain insight into ROS generation in macrophages infected with C. pneumoniae and to explore factors accounting for their final levels and effect, we investigated the role of NADPH oxidase and cytochrome oxidase pathways in the production and modulation of ROS. We also determined the operational role of Ca2+ signaling in the process. Macrophages stimulated with C. pneumoniae exhibit early release of ROS via up-regulation of NADPH oxidase and cytochrome c oxidase activities. Increasing the dose of C. pneumoniae led to an increase in the expression of these enzymes’ gene production, which was accompanied by a significant up-regulation of their gene products, implying a probable activation of transcriptional and translational processes, respectively. The change in levels of free Ca2+, influx across plasma membrane and efflux from intracellular store into cytosol all exhibited a significant regulatory role on the ROS generation pathways in macrophages. The observed events were shown to be dependent on binding of C. pneumoniae to CD14 receptors of macrophages. The data reported here imply that macrophages infected with C. pneumoniae produce ROS through membrane-associated NADPH oxidase with oxidative phosphorylation levels depending on Ca2+ influx signals.

Chlamydia pneumoniae infected macrophages exhibit enhanced plasma membrane fluidity and show increased adherence to endothelial cells.

Chlamydia pneumoniae, an intracellular prokaryote, is known to have requirement for some lipids which it is incapable of synthesizing, and these lipids have important fluidizing roles in plasma membrane. We decided to examine if the trafficking of these lipids to C. pneumoniae alters the physicochemical properties of macrophage plasma membrane, affects the expression of genes and proteins of enzymes associated with metabolism of some of these lipids and assess if Ca2+ signaling usually induced in macrophages infected with C. pneumoniae modulates the genes of these selected enzymes. Chlamydia pneumoniae induced the depletion of macrophage membrane cholesterol, phosphatidylinositol and cardiolipin but caused an increase in phosphotidylcholine resulting in a relative increase in total phospholipids. There was increased membrane fluidity, enhanced macrophage fragility and heightened adherence of macrophages to endothelial cells despite the application of inhibitor of adhesion molecules. Also, there was impairment of macrophage 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene and protein expression independent of Ca2+ signaling, while phospholipase C gene and protein were up-regulated in a manner minimally dependent on Ca2+ signaling. The implications of these findings are that macrophages infected with C. pneumoniae have altered membrane physicochemical characteristics which may render them atherogenic. (Mol Cell Biochem 269: 69–84, 2005)

Induction of lipoprotein lipase gene expression in Chlamydia pneumoniae-infected macrophages is dependent on Ca2+ signaling events.

Abstract Unregulated uptake of low density lipoprotein (LDL) in macrophages is the hallmark of early atherogenic lesions, and Chlamydia pneumoniae infection of macrophages induces this process by an unknown mechanism. It was therefore aimed in this study to investigate (i) the role of C. pneumoniae in macrophage expression of the lipoprotein lipase (LpL) gene, (ii) the probable role of Ca[2+] influx signals and (iii) the effect of the process on LDL uptake. Lipoprotein lipase mRNA expression and LpL activity in infected RAW-264.7 cells were significantly upregulated. A biphasic Ca[2+] influx signal was observed in infected cells with a moderate influx (303 nM Ca[2+]) favoring optimal LpL gene expression. Also, the antagonists of Ltype Ca[2+] channel in macrophages significantly downregulated LpL gene expression and the biomolecular content of C. pneumoniae responsible for the observed events was in part found to be Chlamydia lipopolysaccharide (cLPS). Investigations aimed at determining the specific relevance of Ca[2+]dependent lipoprotein lipase gene expression in C. pneumoniae-infected macrophages showed that the condition caused enhanced uptake of LDL which was abrogated by Calphostin-C-mediated downregulation of LpL. This discovery of a specialized Ca[2+] influx signal-mediated LpL upregulation in C. pneumoniae-infected macrophages provides a mechanistic insight into early events involving C. pneumoniae in macrophage foam cell formation resulting from LDL uptake.

Chlamydia trachomatis induces anti-inflammatory effect in human macrophages by attenuation of immune mediators in Jurkat T-cells

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 3 h, 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.

Chlamydia trachomatis evokes a relative anti-inflammatory response in a free Ca2+ dependent manner in human macrophages.

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 Ca(2+) 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 Ca(2+)-poor medium, while there was significant increase in IL-10 in a Ca(2+)-rich medium. Also, when macrophages were infected after treatment with drugs that either facilitated Ca(2+) 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 Ca(2+) 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 Ca(2+) levels, may evoke a net anti-inflammatory response that presumably favors chronic chlamydia infections.

The roles of unfolded protein response pathways in Chlamydia pathogenesis.

Chlamydia is an obligate intracellular bacterium that relies on host cells for essential nutrients and adenosine triphosphate (ATP) for a productive infection. Although the unfolded protein response (UPR) plays a major role in certain microbial infectivity, its role in chlamydial pathogenesis is unknown. We hypothesized that Chlamydia induces UPR and exploits it to upregulate host cell uptake and metabolism of glucose, production of ATP, phospholipids, and other molecules required for its replicative development and host survival. Using a combination of biochemical and pathway inhibition assays, we showed that the 3 UPR pathway transducers-protein kinase RNA-activated (PKR)-like ER kinase (PERK), inositol-requiring enzyme-1α (IRE1α), and activating transcription factor-6α (ATF6α)-were activated during Chlamydia infection. The kinase activity of PERK and ribonuclease (RNase) of IRE1α mediated the upregulation of hexokinase II and production of ATP via substrate-level phosphorylation. In addition, the activation of PERK and IRE1α promoted autophagy formation and apoptosis resistance for host survival. Moreover, the activation of IRE1α resulted in the generation of spliced X-box binding protein 1 (sXBP1) and upregulation of lipid production. The vital role of UPR pathways in Chlamydia development and pathogenesis could lead to the identification of potential molecular targets for therapeutics against Chlamydia.

Plasmodium falciparum Treated with Artemisinin-based Combined Therapy Exhibits Enhanced Mutation, Heightened Cortisol and TNF-α Induction

The artemisinin-based combined therapy (ACT) post-treatment illness in Plasmodium falciparum-endemic areas is characterized by vague malaria-like symptoms. The roles of treatment modality, persistence of parasites and host proinflammatory response in disease course are unknown. We investigated the hypothesis that ACT post-treatment syndrome is driven by parasite genetic polymorphisms and proinflammatory response to persisting mutant parasites. Patients were categorized as treated, untreated and malaria-negative. Malaria positive samples were analyzed for Pfcrt, Pfmdr1, K13 kelch gene polymorphisms, while all samples were evaluated for cytokines (TNF-α, IL-12p70, IL-10, TGF-β, IFN-γ) and corticosteroids (cortisol and dexamethasone) levels. The treated patients exhibited higher levels of parasitemia, TNF-α, and cortisol, increased incidence of parasite genetic mutations, and greater number of mutant alleles per patient. In addition, corticosteroid levels declined with increasing number of mutant alleles. TGF-β levels were negatively correlated with parasitemia, while IL-10 and TGF-β were negatively correlated with increasing number of mutant alleles. However, IL-12 displayed slight positive correlation and TNF-α exhibited moderate positive correlation with increasing number of mutant alleles. Since post-treatment management ultimately results in patient recovery, the high parasite gene polymorphism may act in concert with induced cortisol and TNF-α to account for ACT post-treatment syndrome.