Pneumocystis jirovecii is an opportunistic fungal agent that causes Pneumocystis pneomonia (PCP) in immunocompromised individuals. Although the incidences of PCP have decreased in HIV seropositive individuals after the introduction of highly active anti-retroviral therapy (HAART), it remains one of the most common opportunistic infections in such patients. A higher morbidity and mortality has been observed due to PCP in HIV negative immunocompromised individuals, in comparison to HIV positive individuals. One possible reason for this difference among the two groups of patients could be the difference in ability to mount an inflammatory response. It has been shown that non HIV infected patients have a more robust inflammatory response against the organism in comparison to HIV infected individuals. This exuberant immune response towards the organism has been shown to be more harmful to the host than the organism itself.
A lot of research has been going on Pneumocystis. Today, I read an interesting article entitled, "Pneumocystis cell wall beta D glucan stimulates calcium dependent signaling of IL-8 secretion by human airway epithelial cells". This work has been performed in the lab of Dr. Andrew H. Limper, USA. Pneumocystis organisms are present within the alveolus in lungs in two different forms, i.e. trophic and cyst forms. The cyst forms have a thick cell wall which is rich in beta glucan. Recent studies have shown that this beta glucan is a major initiator of lung inflammation during Pneumocystis infection. However, the mechanisms by which beta glucans induce this exaggerated inflammatory response is not fully clear.
The authors of this paper have earlier demonstrated that beta glucan in the wall of Pneumocystis induce NFkappaB translocation and TNF-alpha production in macrophages. The authors have also demonstrated that Pneumocystis beta glucans (PCBG) stimulate rat airway epithelial cells to secrete macrophage inhibitory protein-2 (MIP-2) through NFkappaB dependent mechanisms. However, the events by which PCBG activates airway epithelial cells are unclear. It has been shown that several bacterial pathogens e.g. Pseudomonas and Salmonella activate airway epithelial cells by increasing intracellular concentration of calcium. Based on this knowledge, the authors have hypothesized that binding of PCBG stimulates airway epithelial cells. These cells induce changes in cytosolic calcium influx. The change in intracellular calcium subsequently activates signal transduction pathways. The activation of these pathways finally leads to cytokine secretion by airway epithelial cells.
Adhesion of the fungus to host cells is an important step to establish infection. Several receptors have been proposed to bind Pneumocystis particles. Lactosylceramide is one such receptor. This group has earlier observed that lactosylceramide is responsible for MIP-2 production. Thus, this study also evaluated the role of glycosphingolipids in cytokine signaling by airway epithelial cells activated with PCBG.
Major Results:
1. PCBG induces IL-8 secretion from airway epithelial cells: Human airway epithelial cells 1HAEo were cultured and challenged with PCBG or S. cerevisiae derived beta glucan or LPS and IL-8 secretions were measured by ELISA. SCBG and PCBG challenged cells secreted IL-8 in a dose dependent manner compared with LPS-challenged cells.
2. IL-8 secretion by airway epithelial cells stimulated with PCBG is calcium dependent- HAEo cells were loaded with Fura-2AM, a calcium binding dye, and incubated with either PCBG or a positive control peptide for indicated time and transient intracellular calcium release was measured. To further show the importance of intracellular calcium mobilization, HAEo cells were pretreated with various extracellular and intracellular Ca chelating agents before stimulating with PCBG. Then the secretion of IL-8 was measured. It was observed that in cells preincubated with intracellular chelating agents, a significant decrease in IL-8 production was observed. In contrast, cells pretreated with extracellular Ca chelating agents did not show any decrease.
3. IL-8 secretion by airway epithelial cells is mediated by NFkappaB and AP-1- IL-8 promoter contains a variety of transcrption factors binding sites (including those of NF and AP-1). To investigate the importance of NF and AP-1 in IL-8 production induced by PCBG, HAEo cells were transfected with IL-8 luciferase reporter construct (promoter) or with IL-8 luciferase reporter construct (promoter) that had targeted mutations in NF or AP-1 binding sites. These cells were than challenged with PCBG. After certain incubation the cells were harvested and luciferase activity was measured. The results showed that PCBG failed to activate IL-8 transcription in cells transfected with either the mutant NF or mutant AP-1 promoter. IL-8 transcription was normal in cells transfected with wild type promoter.
4. IL-8 secretion by PCBG stimulated airway epithelial cells is mediated by MAP kinases- HAEo cells were pretreated with a inhibitor of ERK (PD98059) and then stimulated with PCBG. A dose dependent decrease in IL-8 production was observed. To further understand the kinetics of MAPK/ERK activation, phosphorylation of ERK was determined by western blotting after stimulation of cells for different periods of time. Phosphorylation of ERK p44/42 was detected within five minutes of stimulation and remained slightly elevated as long as two hrs after the initial challenge.
Next, the authors tried to evaluate whether p38, an independent MAPKs pathway, participated in beta glucan mediated IL-8 secretion from airway epithelial cells in response to PCBG. For this, cells were treated with a p38 inhibitor, SB202190 and then stimulated with PCBG. A dose dependent decrease in IL-8 production was observed indicating the role of p38 in release of IL-8. Furthermore the kinetics of p38 was followed by western blot studies. Phosphorylation of p38 was detected at 15 minutes post stimulation and reached a peak at 30 minutes. After 1 hr, phosphorylation of p38 returned to a baseline level. These data show different kinetics of these two pathways (ERK p44/42 and p38). Similar experiments were repeated for JNK. A JNK inhibitor, was used prior to treatment with PCBG. No inhibition in production of IL-8 was observed. These results indicated that JNK does not participate in IL-8 response.
5. MAPK activation in PCGB stimulated HAEo cells stimulates downstream NF expression: To determine whether MAPK activation following beta glucan stimulation also results in downstream NF dependent activation in HAEo cells, HAEo cells transiently infected with NF promoter plasmid were pretreated with inhitor for ERK p44/42 or p38 or JNK and then stumulated with PCBG. Pretreatment of cells with ERK p44/42 and p38 inhibitors led to decrease in transcritional activity of NF. Treatment with JNK inhibitor showed no change.
6. Inhibition of glycophospholipids synthesis further impairs IL-8 secretion by airway epithelial cells stimulated with BCPG- IL-8 secretion in PCBG stimulated cells was assessed in the presence of a potent glycophospholipd synthesis inhibitor. Il-8 production was decreased.
Conclusions: Thus, the present study demonstrated that human airway epithelial cells secrete significant amounts of IL-8 in response to Pneumocystis beta D Glucan. Also, airway epithelial cells mobilize intracellular Ca within seconds after PCBG stimulation. This intra calcium flux initiates the activation of two major MAPk pathways, ERK p44/42 and p38 and subsequent activation of NF and AP-1, which results in release of IL-8.
Future Studies: Better knowledge of the molecular mechanisms regulating chemokine generation will be essential to understand the recruitment of inflammatory cells to the lung during Pneumocystis infection and to design therapeutic strategies for exaggerated lung inflammation.
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