Induction of Host Chemotactic Response by <em>Encephalitozoon</em> spp.<sup><a href="#fn1" rid="fn1" class=" fn">▿</a></sup>

Jeffrey Fischer

Jeffrey West

Nnenaya Agochukwu

Colby Suire

Hollie Hale-Donze

Louisiana State University, 202 Life Sciences Building, Baton Rouge, Louisiana 70803

^{Corresponding author. Mailing address: Department of Biological Sciences, Louisiana State University, 202 Life Sciences Building, Baton Rouge, LA 70803. Phone: (225) 578-8411. Fax: (225) 578-2597. E-mail:}ude.usl@1delahh.

Received 2006 Sep 22; Revised 2006 Oct 30; Accepted 2006 Dec 5.

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 monocyte-derived-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.

Abstract

Microsporidiosis is a disease that is caused by an obligate intracellular eukaryotic parasite and has gained recognition as an opportunistic infection in AIDS patients, commonly causing chronic diarrhea leading to malabsorption of nutrients and wasting (23, 32). The true extent of microsporidiosis is difficult to determine, because it is often underreported due to a lack of proper facilities to diagnose this disease (13). Epidemiological reports indicate that anywhere from 5 to 50% (19) of patients with chronic diarrhea are positive for microsporidians, most notably Enterocytozoon bieneusi and Encephalitozoon spp. (7). Based on projections that in the course of human immunodeficiency virus (HIV) AIDS about 93% of individuals have at least one bout of chronic diarrhea (10), these pathogens could conservatively account for chronic diarrhea in approximately 1.3 to 13.5 million people suffering from a life-threatening loss of nutrients and liquids in the sub-Saharan region alone. (The number of cases of microsporidiosis was estimated using previously published reports [19] of the incidence [5 to 50%] of infection in individuals who have chronic diarrhea and are HIV positive. An estimated 93% of AIDS patients develop chronic diarrhea [10] in regions, such as sub-Saharan Africa, where water treatment and highly active antiretroviral therapy are limited. According to the 2005 UNAIDS/WHO AIDS report, 29 million individuals are infected with HIV in this region.)

More recently, cases of microsporidiosis in patients who received immunosuppressive therapy (2, 12, 14) or in immunocompetent individuals (26) who presented with symptoms of chronic diarrhea have been described. Although the most common symptoms of the disease are enteric, there have been many reports of more severe disseminated diseases, such as keratoconjunctivitis, sinusitis, tracheobronchitis, encephalitis, interstitial nephritis, hepatitis, cholecystitis, osteomyelitis, and myositis (7, 22).

Microsporidians are ubiquitous in nature and are known to infect a variety of vertebrate and invertebrate organisms (4, 6, 38, 40). Although these organisms were once classified as protozoans, new evidence based on phylogenetic analysis suggests that they are more closely related to fungi (4, 11, 39). Of the more than 1,200 species of microsporidians, only 14 have been reported to infect humans (4). Microsporidian infections are believed to occur when a spore in contaminated water or food is ingested (4). Infections of epithelial and endothelial cells and macrophages are common (19). Classically, cellular invasion occurs when a spore encounters a host cell and everts a polar tube, which penetrates the cell membrane of the host cell and injects the sporoplasm. Alternatively, spores can be internalized through phagocytosis or endocytosis (8). Further proliferation and spore production occur through merogony and sporogony within a parasitophorous vacuole, followed by lysis of the host cell and release of mature spores (9).

While most studies of microsporidians have focused on their genomes and life cycles (7), there are limited data on the host responses to these opportunistic pathogens and especially their roles in human macrophage infection and disseminated disease (8, 30). Some reports indicate that macrophages/monocytes are the source of the disseminated pathogen (33). In individuals with multifocal organ involvement, infiltrates of infected macrophages are evident in lesions, microabscesses, and granulomas (37). In animal models, injection of infectious spores results in a peritoneal infiltrate with predominately monocytes/macrophages, which is followed by disappearance of these cells and presumably spread to the lymph nodes and other tissues (28, 36). Determining which chemokines are present is critical in the development of therapeutic strategies that prevent dissemination of the pathogen and the resulting disease. Based on previous reports, we investigated two species of microsporidians that are known to cause disseminated diseases, Encephalitozoon cuniculi and Encephalitozoon intestinalis, and defined the production of a chemotactic gradient that is induced by infection with these intracellular parasites. This paper provides the first description of the innate immune response in disseminating infections with regard to chemokines and provides a foundation for describing the initial host reaction to these pathogens involving human macrophages.

Acknowledgments

This study was supported by NIH grant RR020159-01, by Louisiana Board of Regents grant LEQSF (2004-7)-RD-A-10, and by LSU FRG.

We thank Elizabeth Didier and Lisa Bowers for donating the E. cuniculi III strain and for their assistance, Andrew Whitehead and Jen Roach for their technical expertise, Earl Weidner for his knowledge and continuous support, and the staff at Our Lady of the Lake Blood Center for providing buffy coats. We also thank Dominique Homberger, James Moroney, and Karin Peterson for their critical review of the manuscript. We acknowledge the contributions of Nicole Hazard and our undergraduate researchers.

Acknowledgments

Notes

Editor: W. A. Petri, Jr.

Notes

Editor: W. A. Petri, Jr.

Footnotes

^{Published ahead of print on 18 December 2006.}

Footnotes

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