In the present study, we evaluated the initial cellular responses of human intestinal epithelial cells to T. gondii
infection. This type of study is critical for identifying the very early innate immune responses to parasitic infection of the intestinal mucosa. Using an in vitro
model, where an isolated cell type is directly exposed to the infectious agent offers the advantage over mixed cell populations in identifying the response of a specific cell type. Most studies on the immune response to T. gondii
infection have used a peritoneal challenge model for this orally acquired pathogen. More recently, the importance of studying the natural route of infection has revealed the importance of epithelial cell response in influencing the outcome of the local and systemic immune response (40
). By examining the response of the cells most likely to first encounter the pathogen, we can begin to uncover the early responses that may limit, or induce, the spread of T. gondii
to other tissues such as muscle and brain, where a persistent infection results. Therefore, it is highly relevant to study the response of these cells during T. gondii
infection, which occurs both locally in the intestine and systemically.
Very little is known about the human intestinal response to T. gondii
infection; therefore, a major finding of this study is that human small intestinal epithelial cells directly respond to T. gondii
within minutes to activate signaling cascades. The neutrophil chemoattractant IL-8 is upregulated both at the protein and mRNA level within hours. Several additional cytokines and chemokines are also upregulated at the mRNA level within 4 hours. During mouse infections with T. gondii
, neutrophils are critical for host defense and are one of the first cells recruited to the site of infection. They play a key role in the recruitment and activation of macrophages and DCs (27
). Therefore, our findings that the human small intestine epithelial cell line, Henle 407, directly responds to T. gondii
infection suggests that in vivo
epithelial response would modulate the local inflammatory environment to initiate host defense against infection.
This series of studies also elucidates the molecular mechanism for epithelial response to T. gondii
infection. We show that, similar to macrophages and DCs, epithelial cells activate the MAPK pathway. The inability of MyD88 deficient epithelial cells to secrete IL-8 and activate ERK1/2 during infection suggests that TLRs play a critical role in the initiation of mucosal inflammatory process. Human TLR2 responds to live T. gondii
infection in our heterologous reconstitution assay. However, knocking down TLR2 with short hairpin RNAs in intestinal epithelial cells only partially reduced the activation of ERK1/2. So while TLR2 contributes to epithelial response to T. gondii
infection, there is likely an interaction with additional TLRs or other receptors that we could not detect in our assay. In fact, preliminary examination of the dependence of several cytokines and chemokines on MyD88 and TLR2 using shRNA knockdown revealed a complex pattern. While several genes were TLR2 dependent (IL-8, CCL10, CCL15), a few were TLR2 independent, MyD88 dependent (CCL5 (RANTES) and CCL11). IL-18 and CCL20 did not depend on either MyD88 or TLR2 (data not shown). TLR4 and TLR9 are candidates to work in concert with TLR2 for the production of cytokines and chemokines since mice deficient in these TLRs have reduced intestinal pathology during oral T. gondii
infection. Furthermore, TLR9 in either hematopoietic or nonhematopoietic compartments is important for efficient T cell responses to oral infection (21
). However, in our in vitro
system, knocking down TLR9 in Henle 407 cells or reconstituting TLR9 in HEK293 cells does not affect the MAPK or elicit NF-κB response to T. gondii
. Commensal bacteria are present in the intestine and are capable of activating TLR9. Therefore, it is likely that the TLR9 dependent pathology induced during oral infection with T. gondii
is secondary to epithelial damage and recognition by TLR9 of bacteria that penetrate the epithelial barrier.
Ligands for TLR2 include lipopeptides, lipoproteins and GPIs. GPI anchored proteins are abundant on the surface of T. gondii
tachyzoites, and GPIs from Trypanosoma cruzi, Plasmodium falciparum
activate TLR2 (43
). T. gondii
GPIs also stimulate cytokine production in macrophages through TLR2 and TLR4 (45
). Direct studies on the role of TLR2 in human intestinal epithelial cells during T. gondii
infection are lacking. Our data suggest that activation of TLR2 requires live parasites, or at least components not present in STAg, or damaged or lost during the preparation of STAg. The molecular component of T. gondii
that intestinal epithelial cells recognize via TLR2 remains unknown.
The molecular mechanisms by which T. gondii
activates of epithelial cells and macrophages are different. In macrophages, T. gondii
exploits Gi-protein mediated signaling to activate PI-3 kinase that leads to Akt and ERK1/2 activation, a process that is independent of MyD88 signaling (36
). We show that inhibition of PI-3 kinase had no effect on the ability of epithelial cells to respond to infection. T. gondii
infection of macrophages fails to induce NF-κB nuclear translocation and in fact inhibits activation in response to TLR ligands such as LPS (47
). In epithelial cells, NF-κB translocation to the nucleus was not impaired. Similar results in infected murine embryonic fibroblasts (MEFs) have been reported (49
). Both ERK1/2 and p38 kinases were activated by T. gondii
infection of epithelial cells; however, activation was almost entirely dependent on MyD88 but only partially dependent on TLR2. Additional innate immune receptors or other recognition mechanisms present in the intestinal epithelial cells may cooperate to regulate the immune response of T. gondii
infection. These other receptors are unlikely to be TLRs since attempts to co-express other TLRs with TLR2 did not enhance the response in our HEK293 based stimulation assay.
There are three clonal lineages of T. gondii
that differ ability to induce cytokines and in virulence. Low virulence type II parasites show higher induction level of IL-12p40, IL-10, IL-1β, and IL-6, where as high virulence type I parasites attract more neutrophils during infection (51
). We predicted that ability to activate TLR2 might correlate with lower virulence due to an increased activation of the immune response especially since NF-κB activation and cytokine secretion in immune cells correlates with genotype (53
). However, all types elicited ERK1/2 and p38 MAPK activation through TLR2. Although the strains varied in the level of activation, there was no correlation with genotype. Virulence and cytokine induction differences among strains are not due to ability to activate epithelial cells via TLR2.
In summary, this study demonstrates that human intestinal epithelial cells directly respond to T. gondii infection via MyD88 and TLR2 driven ERK1/2 kinase and NF-κB signaling pathways. An interesting question for future studies is how epithelial cells cross talk and influence immune cells during infection. Our preliminary data point to a complex pattern of cytokine regulation, in which TLR2 is important but not the whole story. Understanding the local immune response against pathogens in the intestine will provide insight into the development of intestinal disorders, mechanisms for enhancing immune response to infection, or targets for vaccine development.