Elucidation of a molecular mechanism controlling inflammation during bacterial infection

Recently we described a mechanism of gap junction-mediated communication between infected and uninfected epithelial cells that potentiates innate immunity during infection by the enteroinvasive bacterium Shigella flexneri. We showed that although S. flexneri secretes multiple effector proteins that downregulate inflammation in infected epithelial cells, NF-κB and the MAP kinases p38, JNK and ERK are activated in uninfected cells surrounding the sites of infection. The propagation of these proinflammatory signals leads to massive secretion of proinflammatory cytokines such as interleukin-8 (IL-8) by uninfected bystander cells. A genome wide RNAi-screen on Shigella-induced bystander activation confirmed the roles of the proteins TAK1 and NF-κB. Besides this, new candidates for bystander activation were found, including Na+/K+-ATPase (ATP1A1), the TRAF-interacting protein with a FHA domain (TIFA) and the TNF receptor-associated factor 6 (TRAF6). These proteins together with NOD1 and RIPK2, members of the NOD1 signaling pathway, which is induced by invasive Shigella, were studied in more detail. To our surprise we found that signals underlying cell-cell communication are produced independently of the receptor NOD1 and the downstream signaling proteins RIPK2, TAK1 and NF-κB as well as independent of TIFA and TRAF6. Unexpectedly, in bystander cells NOD1 and RIPK2 contribute to the proinflammatory response, whereas TAK1, NF-κB, TIFA and TRAF6 are indispensable for the production of cytokines. Furthermore, TIFA and TRAF6 are upstream of TAK1 and are required for TAK1 activation. In addition, selective stimulation of TIFA or TRAF6 depleted cells with the NOD1 ligand iE-DAP unraveled that TIFA and TRAF6 contribute to NOD1 signaling in bystander cells of S. flexneri infection. And finally, we propose a link between intercellular calcium signaling triggered by invasive S. flexneri and bystander IL-8 expression, since inhibition of calcium signals via a calcium chelator or inhibition of the IP3-receptor or phospholipase C (PLC) lead to a decreased bystander IL-8 response.