Limited data concerning the innate immune response against microsporidians are available. The number of reports of microsporidian infections not only in immunocompromised individuals but also in immunocompetent individuals is increasing, indicating that the incidence of infection may be higher than the incidence that is typically reported (1
). Histological studies of microsporidiosis show that infiltrates are often composed of several cell types, including monocytes, granulocytes, and lymphocytes (1
); however, infected macrophages have been observed in disseminated disease (30
) and are thought to be the vehicle for multiorgan infections.
The production of chemokines to recruit various immune cells during parasitic infections is important in innate immunity (24
). Our data indicate that MDM infected with Encephalitozoon
spp. induce a functional chemokine gradient that supports the recruitment of monocytes to sites of infection. In our study, E. cuniculi-
and E. intestinalis
-infected primary human macrophages induced an approximately threefold increase in migration of naïve monocytes after 24 h of incubation in a chemotaxis system. The delay in migration may reflect parasitic evasion of the immune response, providing time to invade and replicate (Fig. ).
Focused microarray analyses of MDM infected with E. cuniculi
revealed increases in expression of several critical chemokines and receptors needed to recruit a variety of effector cells in order to mount a successful immune response. In a case study, Boldorini et al. found that in a patient with AIDS, an Encephalitozoon
sp. infection of kidney epithelium was surrounded by lymphocytes, plasma cells, and macrophages (1
), suggesting that these cells have a role in clearing the infection. We found that several genes involved in neutrophil recruitment (namely, the genes encoding C-X-C chemokines) were strongly induced, as were genes involved in the chemotaxis of dendritic and TH1 cells (namely, the genes encoding CCL1 and CCL5) (24
). Khan and Moretto reported that in a murine model a TH1 response against microsporidian infections was critical (18
), and later Khan et al. further defined a role for CD8+
and γδ T cells in resolving these infections in mice (20
). CCL5 is a potent chemoattractant for memory T cells, interleukin-2-activated T cells, and eosinophils (24
). Among the upregulated genes that are important in monocyte recruitment (24
) are the genes encoding CCL2, CCL3, CCL4, and CCL4L1. These four chemokines have been identified as agonists for the key monocyte receptors CCR1 and CCR5 (27
), both of which exhibited increased expression in our model of infection. A similar response has also been demonstrated for Candida albicans
infection of human monocytes, where increased expression of CCL2, CCL3, and CCL4 chemokine genes and CCR1 and CCR5 receptor genes has been observed (21
). Interestingly, as observed in our model, C. albicans
induces CCL4 expression that is many-fold greater than expression of either CCL2 or CCL3 at 6 h postinfection (21
To determine which of the genes give rise to protein products, a focused protein analysis was performed using proteome profiler arrays. Data obtained from the limited array revealed that there were >15-fold increases for the chemokines CCL3, CCL4, and CCL5 (Table ). Both CCL3 and CCL4 are known to be strong inducers of movement of monocytes to areas of infection and are also ligands for CCR5, an HIV coreceptor, whereas CCL5 predominately recruits T cells. In addition, the levels of expression CCL2, which is another potent monocyte chemoattractant, were moderate but significant.
The secretion kinetics of the monocyte chemokines most likely to participate in the spread of infection were further analyzed by ELISA to determine whether their expression coincided with the delayed recruitment observed. The increases in levels began around 6 h, and the levels peaked between 12 and 24 h. These times corresponded to the observed chemotactic response. It has yet to be determined whether microsporidians can dampen immediate immune responses during host uptake until they have established the meront stage within the macrophage. Delayed responses to other fungal pathogens, including Aspergillus fumigatus
, have been attributed to altered host responses based on the recognition of conidia verses hyphal forms or to C. albicans
yeast and hyphal forms which are thought to contribute to the pathology observed in opportunistic infections (35
To determine the individual roles of these chemokines in the recruitment of potential new hosts for microsporidians, neutralizing antibodies were employed in the coculture system. This analysis revealed that CCL2 and CCL3 contributed to the migration of monocytes, but inhibiting CCL4 in the cultures resulted in the most dramatic reduction (Fig. ). Neutralization of all three major chemokines reduced the levels of migrating monocytes to levels near the level of uninfected MDM, suggesting that these three chemokines were the chemoattractants responsible for the monocytic infiltration and, therefore, are potential targets of chemotherapeutic agents for controlling microsporidiosis (34
). In comparison, using a murine model, Huffnagle et al. showed that Cryptococcus neoformans
, a yeast-forming fungus known to cause disseminated disease, could evoke a monocyte chemoattractant protein 1 and macrophage inflammatory protein 1α response; in neutralization studies, the cryptococcal burden in the lungs of mice increased, while a decrease in macrophage and CD4+
T-cell recruitment was observed, resulting in inhibited clearance of the infection (16
While the immune response generated against Encephalitozoon spp. can result in recruitment of monocytes, it also has the potential to mediate adaptive immunity. However, in individuals with impairment of the adaptive arm of the immune system, such as AIDS patients, organ transplant recipients, the young, and the very old, the same recruited monocytes could amplify the infection. Understanding how macrophages function in propagating disease and how the life cycle of microsporidians can influence their responses is critical in developing antimicrosporidial compounds or anti-inflammatory intervention strategies. Studies to define host recognition of the parasite, signaling pathways, and subsequent cytokine profiles are under way.