Induction of Host Chemotactic Response byEncephalitozoonspp

Abstract

Microsporidians are a group of emerging pathogens typically associated with chronic diarrhea in immunocompromised individuals. The number of reports of infections with these organisms and the disseminated pathology is growing as diagnostic tools become more readily available. However, little is known about the innate immune response induced by and generated against these parasites. Using a coculture chemotaxis system, primary human macrophages were infected with Encephalitozoon cuniculi or Encephalitozoon intestinalis, and the recruitment of naïve monocytes was monitored. Encephalitozoon spp. induced an average threefold increase in migration of naïve cells 48 h postinfection, which corresponded to optimal infection of monocytederived-macrophages. A limited microarray analysis of infected macrophages revealed several chemokines involved in the inflammatory responses whose expression was upregulated, including CCL1, CCL2, CCL3, CCL4, CCL7, CCL15, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8. The levels of 6 of 11 chemokines also present in the microarray were confirmed to be elevated by protein profiling. Kinetic studies confirmed that secreted CCL2, CCL3, and CCL4 were expressed as early as 6 h postinfection, with peak expression at 12 to 24 h and expression remaining until 48 h postinfection. Neutralization of these chemokines, specifically CCL4, significantly reduced the number of migrating cells in vitro, indicating their role in the induction of monocyte migration. This mechanism of recruitment not only supports the evidence that in vivo cellular infiltration occurs but also provides new hosts for the parasites, which escape macrophages by rupturing the host cell. To our knowledge, this is the first documentation that chemokine production is induced by microsporidian infections in human macrophages.

Toll-Like Receptor 2 Recognition of the MicrosporidiaEncephalitozoonspp. Induces Nuclear Translocation of NF-κB and Subsequent Inflammatory Responses

“…Pathology and epidemiological reports have identified immune effector cells at sites of infection (32) and detected increased levels of serum antibodies for microsporidia in humans (17,39). Recent in vitro studies have shown that infections lead to the production of numerous cytokines and chemokines, including TNF-␣, CCL2, CCL3, and CCL4 (12,14). Although these responses have been well documented, to date, no known receptor involved in microsporidian recognition and signaling has been identified.…”

“…Spores were washed once in PBS containing 0.2% Tween 20, resuspended in supplemented DMEM, and counted with a hemacytometer (9). Spores were used at a 5:1 spore-to-MDM ratio (12). Some spores were heat inactivated in a 95°C water bath for 30 min.…”

“…Peripheral blood mononuclear cells were isolated from buffy coats of healthy donors (Our Lady of the Lake Regional Blood Bank, Baton Rouge, LA) by gradient centrifugation on lymphocyte separation medium according to guidelines established by the Internal Review Board (Louisiana State University, Baton Rouge, LA). Monocyte-derived macrophages (MDM) were obtained by adherence assays, as previously reported (12). Briefly, monocytes were plated onto 6-well (2 ϫ 10 6 cells/well), 24-well (5 ϫ 10 5 cells/well), and 96-well (1 ϫ 10 5 cells/well) culture plates and cultured for 3 h in supplemented DMEM containing 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 0.1 g/ml gentamicin at 37°C in 5% CO 2 .…”

“…6A). After a 12-h challenge with the parasites, cell culture supernatants were collected and measured for the levels of TNF-␣ and IL-8 mediators as well as two NF-B-driven chemokines, CCL3 and CCL4, previously described as being produced in response to Encephalitozoon challenge (12). The knockdown of TLR2 resulted in an approximately twofold decline in the production of TNF-␣ (Fig.…”

“…However, studies involving microsporidian infection have provided some evidence that suggests the involvement of NF-B. Analyses of culture supernatants from primary human macrophages challenged with Encephalitozoon spores revealed increases in the levels of the cytokine TNF-␣ (14) and the chemokines CCL3, CCL4, and CCL2 in response to infection (12). Taken together, these reports of inflammatory mediator production and the previously described biological similarities between microsporidia and pathogens that are known to activate TLR suggest that microsporidia may also be recognized by and elicit a TLR response.…”