Protection against Chlamydia trachomatis infection in vitro and modulation of inflammatory response in vivo by membrane-bound glycosaminoglycans

Glycosaminoglycans (GAG) efficiently inhibit adherence of several strains of Chlamydia trachomatis to cell lines in vitro, but none of the GAG have been able to inhibit infections in vivo. One possible cause for failure of GAG inhibition in vivo is the inability to deliver a sustained concentration of GAG at the mucosal surface. We tested the possibility of enhancing cell protection by increasing the cell-surface concentration of GAG using membrane-anchored GAG (MAG), composed of phosphatidylethanolamine (PE)-linked GAG. These lipid conjugates were originally designed as extracellular phospholipase A2 (PLA2) inhibitors and exhibit a dual effect: the lipid moiety incorporates into the cell membrane, interfering with the action of PLA2 on cell membranes, and the anchored GAG protects the cell membrane from exogenous inflammatory mediators. We tested the ability of MAG to block chlamydia infection in vitro and in vivo. The MAG blocked infection of epithelial cells in vitro when added to the cells at the same time or before infection, but not if added after the bacteria had already invaded the host cells. One of the MAG led to the production of aberrant Chlamydia vacuoles, suggesting it may inhibit intracellular PLA2 associated with development of the vacuole. Although the MAG did not inhibit vaginal infection of mice, they decreased significantly the level of secretion of the inflammatory cytokines TNF-alpha and IFN-gamma but had no effect on secretion of the neutrophil chemokine, macrophage inflammatory protein-2 (MIP-2). Acute and chronic inflammatory cell infiltrates were not altered by MAG treatment. These findings suggest that lipid conjugation of GAG could be used as a novel approach for increasing cell-surface concentrations of GAG. The inconclusive in vivo results might be due to the physical properties of the tested MAG or an insufficient application protocol, and their improvement might provide the desired inhibitory effects.     

Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection

The roles of Toll-like receptor (TLR) 2 and TLR4 in the host inflammatory response to infection caused by Chlamydia trachomatis have not been elucidated. We examined production of TNF-alpha and IL-6 in wild-type TLR2 knockout (KO), and TLR4 KO murine peritoneal macrophages infected with the mouse pneumonitis strain of C. trachomatis. Furthermore, we compared the outcomes of genital tract infection in control, TLR2 KO, and TLR4 KO mice. Macrophages lacking TLR2 produced significantly less TNF-alpha and IL6 in response to active infection. In contrast, macrophages from TLR4 KO mice consistently produced higher TNF-alpha and IL-6 responses than those from normal mice on in vitro infection. Infected TLR2-deficient fibroblasts had less mRNA for IL-1, IL-6, and macrophage-inflammatory protein-2, but TLR4-deficient cells had increased mRNA levels for these cytokines compared with controls, suggesting that ligation of TLR4 by whole chlamydiae may down-modulate signaling by other TLRs. In TLR2 KO mice, although the course of genital tract infection was not different from that of controls, significantly lower levels of TNF-alpha and macrophage-inflammatory protein-2 were detected in genital tract secretions during the first week of infection, and there was a significant reduction in oviduct and mesosalpinx pathology at late time points. TLR4 KO mice responded to in vivo infection similarly to wild-type controls and developed similar pathology. TLR2 is an important mediator in the innate immune response to C. trachomatis infection and appears to play a role in both early production of inflammatory mediators and development of chronic inflammatory pathology.     

Role of proapoptotic BAX in propagation of Chlamydia muridarum (the mouse pneumonitis strain of Chlamydia trachomatis) and the host inflammatory response

The BCL-2 family member BAX plays a critical role in regulating apoptosis. Surprisingly, bax-deficient mice display limited phenotypic abnormalities. Here we investigate the effect of BAX on infection by the sexually transmitted pathogen, Chlamydia muridarum (the mouse pneumonitis strain of Chlamydia trachomatis). Bax(-/-) cells are relatively resistant to Chlamydia-induced apoptosis, and fewer bacteria are recovered after two infection cycles from Bax(-/-) cells than from wild-type cells. These results suggest that BAX-dependent apoptosis may be used to initiate a new round of infection, most likely by releasing Chlamydia-containing apoptotic bodies from infected cells that could be internalized by neighboring uninfected cells. Nonetheless, infected Bax(-/-) cells die through necrosis, which is normally associated with inflammation, more often than infected wild-type cells. These studies were confirmed in mice infected intravaginally with C. muridarum; since the infection disappears more quickly from Bax(-/-) mice than from wild-type mice, secretion of proinflammatory cytokines is increased in Bax(-/-) mice, and large granulomas are present in the genital tract of Bax(-/-) mice. Taken together, these data suggest that chlamydia-induced apoptosis via BAX contributes to bacterial propagation and decreases inflammation. Bax deficiency results in lower infection and an increased inflammatory cytokine response associated with more severe pathology.     

Cell death and inflammation during infection with the obligate intracellular pathogen, Chlamydia

Infections by Chlamydia are followed by a strong inflammatory response, which is necessary to eliminate the infection, but at the same time is responsible for the pathology of infection. Resistance of infected cells against apoptosis induced by external ligands, together with the effects of IFNgamma secreted during infection, would be expected to contribute to persistence of infection. Secretion of TNFalpha plays an important role during clearance of the chlamydiae, but also triggers apoptosis of uninfected cells in infected tissues. Apoptosis of infected host-cells towards the end of the infection cycle is thought to participate in the release of chlamydiae from infected cells and propagation of the infection. Dysregulation of the apoptotic program during infection leads to a less efficient infection, but paradoxically, results in a higher inflammatory response and more severe pathology.     

Evidences on sodium ions compartmentalization in biological systems due to pathological states. A noninvasive NMR study

Noninvasive nuclear magnetic resonance was used to measure the relaxation decay curves of naturally occurring 23Na ions in several biological systems. Experimental results showed an increase of membrane bound population for pathologic samples as compared with control. The bound sodium population was put in evidence using singular value decomposition method. Thus, the singular values that are obtained without any a priori from the fitting the relaxation decay curves are a new parameter in characterizing the cellular state. In the presence of artificial biological membranes, 23Na bound strongly to membranes containing phosphatidylcholine (PC) and phosphatidylserine (PS), but not to membranes consisting of only PC. A large bound population also appeared in the presence of apoptotic epithelial cells, which are known to translocate PS to the cell surface. A role for PS was confirmed by showing that sodium binds to the surface of epithelial cells infected with Chlamydia psittaci, and the amplitude of the bound population increases with a time-course similar to the appearance of PS on the surface of dying cells. Finally, this approach could distinguish between normal perfused liver and liver undergoing ischemia, due most likely to the exposure of surface PS on apoptotic and necrotic cells in the damaged tissue. Taken together, these studies demonstrate that the analysis of 23Na relaxation decay curves could reveal the presence of cells undergoing apoptosis and/or necrosis in living tissues. Noninvasive 23Na NMR measurements could thus be envisioned for controlling the quality of organs before transplantation, for the detection of asymptomatic infections that result in death of the host cell or inflammation of the tissue, and for characterizing the efficiency of novel apoptosis-inducing drugs to treat cancer.     

'Infectious web'

A comprehensive list of all known bacterial pathogens of humans is now available at various web-sites on the internet. The sites contain hyperlinks to original scientific literature, along with general information on laboratory testing, antibiotic resistance and clinical treatment. More specific sites highlight the fungus Pneumocystic carinii, arguably the main cause of pneumonia in immunosuppressed individuals.     

Fluorescent labelling of intracellular bacteria in living host cells

The fluorescent reagent, CellTracker, labels metabolically-active cells and was used here to label Chlamydia in vivo during their exponential phase of growth in infected cells. HeLa cells infected with C. psittaci were labelled with the CellTracker reagents between 15 and 48 h post-infection. The fluorescent label accumulated in the host-cell membrane compartment (inclusion) within which Chlamydia reside and replicate, and was also incorporated by the bacteria. Labelling with the CellTracker affected neither the growth nor the differentiation of the chlamydiae, and labelled chlamydiae isolated from infected cells were infectious. Our results demonstrate that the CellTracker could become a valuable tool for in vivo labelling of obligate intracellular parasites for which no genetic tools exist.