Intestinal inflammation Salmonella style
It has been long known that intestinal inflammation is central for the pathology that follows infection with non-typhoidal Salmonellae such as Salmonella Typhimurium. However, in recent years work carried out in the laboratories of Wolf-Dietrich Hardt and Andreas Baumler have established that the inflammatory response is also required for Salmonella Typhimurium to compete with the resident intestinal microbiota and to secure essential nutrients. Unlike most other tissues, where the mere presence of bacterial products capable of stimulating innate immune receptors can trigger inflammation, the intestinal tract presents a challenge to those pathogens that need inflammation to sustain their livelihood. Indeed, the presence in the intestinal tract of an abundance of microbial products with the potential to stimulate innate immune receptors demands for the intestinal epithelium to be subject to negative regulatory mechanisms that can prevent the pathology that could result from the indiscriminate firing of these receptors. In fact, misregulation of those mechanisms can result in chronic inflammatory conditions such as inflammatory bowel disease. Consequently, to initiate an inflammatory response in the gut, S. Typhimurium cannot relay on the stimulation of innate immune receptors by the standard “pathogen-associated molecular patterns” (e. g LPS, peptidoglycan, flagellin) that, like many other bacteria, posses in abundance. Therefore, the mechanisms by which Salmonella trigger intestinal inflammation have been a long-standing question in the field and have been the subject of some controversy. We believe that a paper that we recently published in Nature Microbiology has finally clarified this important issue.
The paper in Nature Microbiology is here: go.nature.com/2xyNGxJ
Before getting into the nitty-gritty, let’s review some of the context for these studies. More than two decades ago 1,2, our laboratory made the observation that S.Typhimurium can stimulate MAP kinases and NF-kB signaling in cultured epithelial cells, and that this signaling results in the production of pro-inflammatory cytokines. More importantly, we showed at that time that stimulation of these responses required the activity of the type III secretion system (T3SS) encoded within its pathogenicity island 1 (SPI-1). Although these observations were made before innate immunity receptors and innate immunity as field of research have come into the forefront, these findings already suggested something unique about the mechanisms utilized by S.Typhimurium to trigger inflammation. However, our own work later showed that, intriguingly, the transcriptional responses stimulated by S.Typhimurium in cultured epithelia cells resemble those stimulated by innate immune receptors 3. The requirement of a functional SPI-1 T3SS presumably eliminated the possibility that the pro-inflammatory responses stimulated by S. Typhimurium in epithelial cells were triggered by conserved agonists of innate immune receptors (i. e. LPS, peptidoglycan, flagellin, etc) abundantly present in Salmonella. Therefore these observations resulted in a conundrum: how could S.Typhimurium trigger “innate immune-like” signaling presumably without engaging innate immune receptors? A major clue to this puzzle emerged from our discovery that the pro-inflammatory responses triggered by S. Typhimurium were dependent on the redundant activities of three of its SPI-1 T3SS effector proteins, SopE, SopE2, and SopB, which our laboratory had previously shown were able to activate Rho-family GTPases, including Rac1 and Cdc42. We went on to show that Cdc42, but not Rac1, was required for S.Typhimurium to stimulate pro-inflammatory signaling 3,4. Although these results clarified the picture significantly, the fact remained that these observations could not explain the similarity between the responses induced by Salmonellawith those induced by innate immune receptors as no connection between Cdc42 and canonical innate immune signaling mechanisms had been reported. Furthermore, studies have suggested that the activation of Rac1 by the S. Typhimurium effectors per se through unknown mechanisms was sensed as a “danger associated molecular pattern” by the innate immune receptor NOD1 leading to NF-kB activation and pro-inflammatory transcriptional responses 5. However, these findings were not consistent with our own previous studies showing that removal of Cdc42 abolished S. Typhimurium stimulation of inflammatory signaling, even though the absence of Cdc42 did not affect the ability of S. Typhimurium to activate Rac1. Furthermore, we had also shown that in Rip2-defficient mice, which are impaired in NOD1 signaling, S. Typhimurium stimulates intestinal inflammation in a manner that is indistinguishable from wild type mice 3. The clarification of these issues demanded the identification of the mechanisms by which activation of Cdc42 by Salmonella leads to “innate-immune-like” signaling. The description of these mechanisms is the topic of our paper just published in Nature Microbiology.
We found that stimulation of Cdc42 by the S.Typhimurium T3SS effector proteins leads to the activation of PAK1 and the subsequent formation of a previously unreported signaling complex composed of PAK1/TRAF6/TAK1. The key experimental breakthrough was to use endogenous levels of properly tagged relevant signaling molecules (i. e. Cdc42 and PAK1) to identify relevant interacting proteins exactly at the time that these molecules were being engaged by the Salmonellaeffectors as no interactions were detected in uninfected cells. Removal of PAK1, TRAF6, or TAK1 from various cell lines using CRISPR/Cas9-mediated genome editing abrogated the ability of S. Typhimurium to stimulate inflammatory signaling, thus confirming their involvement in the pathogen-induced responses. We also demonstrated the relevance of this signaling pathway in a mouse model of infection. PAK1-defficiency in mice resulted in only a partial inflammation phenotype after S. Typhimurium infection, which is expected given its redundancy with the related kinase PAK2 (a double knockout out mouse is not viable). However, oral administration of a highly specific inhibitor of group I PAKs (PAK1, PAK2, and PAK3) drastically reduced the inflammatory response and the replication of S. Typhimurium in the intestinal tract. Interestingly, oral administration of the inhibitor resulted in a significant increase in the number of S.Typhimurium in systemic tissues, indicating that while the inflammatory response is critically important for the replication of S. Typhimurium within the intestine, this response is also central for the host to anatomically restrict the pathogen and prevent its access to deeper tissues. These findings are very exciting because given the known roles of TRAF6 and TAK1 in signal transduction pathways emanating from innate immune receptors, these findings provide an explanation for the strong similarities observed between the pro-inflammatory transcriptional responses resulting from S.Typhimurium infection and those resulting from the stimulation of innate immune receptors. By engaging innate immune signaling pathways downstream from the actual receptors Salmonellais able to stimulate a response that shares great similarity with the responses stimulated by the activation of canonical innate immune receptors, while avoiding the negative regulatory mechanisms that prevent the activation of these receptors in the intestinal tract.
Our findings are an example of the unique balance that emerges from the host/pathogen co-evolution in that pathogen-initiated responses that help pathogen replication are also important to prevent pathogen spread to deeper tissues. Furthermore, the mechanisms describe here could help develop anti-pathogen therapeutic strategies by targeting specific host-signaling pathways.
1 Chen, L. M., Hobbie, S. & Galan, J. E. Requirement of CDC42 for Salmonella-induced cytoskeletal and nuclear responses. Science274, 2115-2118 (1996).
2 Hobbie, S., Chen, L. M., Davis, R. & Galán, J. E. Involvement of the mitogen-activated protein kinase pathways in the nuclear responses and cytokine production induced by Salmonella typhimuriumin cultured intestinal cells. J. Immunol.159, 5550-5559 (1997).
3 Bruno, V. M.et al.Salmonella Typhimurium type III secretion effectors stimulate innate immune responses in cultured epithelial cells. PLoS Pathog5, e1000538, doi:10.1371/journal.ppat.1000538 (2009).
4 Patel, J. C. & Galan, J. E. Differential activation and function of Rho GTPases during Salmonella-host cell interactions. J Cell Biol175, 453-463, doi:10.1083/jcb.200605144 (2006).
5 Keestra, A.et al.Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1. Nature496, 233-237 (2013).
Jorge E. Galán
Department of Microbial Pathogenesis
Yale University School of Medicine