The production of NO is an important antimicrobial defense mechanism, but when uncontrolled it can lead to pathological inflammatory states (28
). Indeed, elevated NO levels and increased expression of iNOS have been implicated in clinical problems such as rheumatoid arthritis and inflammatory bowel disease (1
). Elucidating the mechanisms that regulate iNOS expression is thus relevant to understanding both innate immunity, and the pathogenesis and treatment of chronic inflammatory conditions.
Like several other microorganisms, Salmonella
has been noted to increase expression of iNOS in mammalian cells, although the mechanism of induction has not been worked out in detail. Two recent studies have shown that infection of intestinal epithelial cell lines by Salmonella
is associated with increased iNOS expression, and a correlation between invasiveness and iNOS induction was observed (33
). Our findings extend these studies to macrophages and also demonstrate the involvement of SipB, SipC, and SipD in the regulation of iNOS expression. These effectors have been previously shown to play an important role in invasion, thereby providing an explanation for the link between invasiveness and iNOS induction noted in the earlier studies (33
). However, our results show clearly that the two processes can be dissociated based on the effects of cytochalasin D treatment (Fig. and ). This uncoupling from invasion has been noted for other effects induced by Salmonella
, i.e., the activation of NF-κB, as well as transepithelial signaling for neutrophil recruitment (8
), and is consistent with the observation that Sips can be translocated into mammalian cells by extracellularly situated bacteria (4
One of the functions of Sips B, C, and D is the formation of a translocation apparatus for the delivery of certain effector proteins (AvrA, SopB, SopE, and SptP) into the mammalian cytosol (9
). The involvement of one of these effectors in iNOS induction seemed likely based on our observation that Sips B, C, and D were all required for the function. However, our initial series of experiments ruled out a role for AvrA, SopB, SopE, and SptP (Table ), suggesting that yet another Sip-dependent effector might be the ultimate mediator of iNOS induction. Indeed, we found that a strain deficient in SopE2, a recently identified homolog of SopE that is also likely to be dependent on Sips B, C, and D for translocation (2
), was clearly impaired in its ability to induce iNOS expression (Fig. ).
Our findings thus suggest a simple model in which SopE2, translocated into the mammalian cell via the SPI1 type III secretion system and the SipB, -C, and -D complex, initiates signals leading to increased iNOS expression. SopE2 is likely to have the same function as SopE in relation to the activation of Rho GTPases (14
), thus providing an obvious mechanism for the initiation of signaling. The close structural, and presumed functional, homology between the two molecules raises the possibility that SopE may also have the ability to induce iNOS expression. Although strains GG5 and SE1 (both deficient in SopE) behaved like the corresponding wild-type strains with respect to iNOS expression (Table ), the presence of SopE2 in these strains probably compensated for the lack of SopE. A strain expressing SopE but deficient in SopE2 will be required to test the involvement of the former effector in iNOS induction. Finally, it is worth mentioning here that since Rho GTPases have been implicated in the Salmonella
-activated signaling pathways leading to the expression of proinflammatory cytokines in epithelial cells (20
), SopE and SopE2 are likely to have a broad role in inducing inflammation that extends beyond the production of NO in macrophages.
Complementation of the SopE2-deficient strain SE2.1 with a plasmid-encoded SopE2 gene (2
) did not restore iNOS induction to wild-type levels (data not shown). Although this failure of rescue raises the possibility that some other gene might be involved in the regulation of iNOS expression, we think it unlikely since (i) the sopE2
mutation does not cause obvious effects on the expression or secretion of any other Salmonella
secretory protein (2
) and (ii) our analysis of the SipB-, SipC-, and SipD-deficient mutants and their transcomplemented derivatives clearly indicates a role for a Sip-dependent effector in iNOS induction. The failure of complementation may reflect a requirement for specifically regulated expression of SopE2 (expression at a specific time during invasion, or at a specific site within the macrophage) that cannot be achieved by the plasmid-encoded effector.
Although our findings have highlighted the involvement of Sips B, C, and D and SopE2 in iNOS induction, they do not exclude a role for LPS in this process. LPS is an important inducer of iNOS expression in macrophages, especially in conjunction with cytokines such as interferon gamma (42
). Furthermore, a mutant of Salmonella
with altered LPS structure has been shown to be impaired in inducing NO production, both in vitro and in vivo (26
). Sip-deficient, as well as SopE2-deficient, mutants of Salmonella
have residual iNOS inducing ability (Fig. and , for example), which could be attributable to the effects of LPS acting through the recently described Toll-like receptors (30
). It is likely that the final level of iNOS expression is determined by signals activated by both LPS and SopE2.
We have not directly addressed the in vivo significance of our observations. However, evidence from the published literature suggests that our findings are likely to be relevant to Salmonella
pathogenicity. B1, the mutant strain of serovar Dublin deficient in Sips A, B, C, and D, has been found to be markedly impaired in inducing intestinal fluid secretion and neutrophil transmigration in bovine ligated ileal loops (11
). Similar findings were obtained when serovar Typhimurium mutant strains deficient in SipB, SipC, or SipD were fed to calves (37
). Although the decreased pathogenicity of the Sip-deficient strains in these studies could be related to their poor invasiveness, at least some aspects of virulence (neutrophil transmigration, NF-κB activation) have been shown to be independent of cell invasion (8
). An alternative possibility, therefore, is that one of the effectors dependent on Sips B, C, and D for translocation might be involved in inducing intestinal pathology. The involvement of several such effectors has been examined in bovine models. Strains deficient in SopB, SipA, or SptP all behave like wild-type Salmonella
after being fed to calves (36
). In the bovine ligated ileal loop model, strains deficient in SptP or AvrA are unimpaired in enteropathogenesis, and strains deficient in SopB or SopE are only modestly impaired (11
). Thus, these in vivo observations have an excellent correlation with our in vitro findings: Sips B, C, and D are required for inducing both intestinal pathology and iNOS expression; conversely, SipA, SopB, SopE, AvrA, and SptP are not required for either of these functions. Together with the known involvement of NO in inflammatory conditions of the bowel (7
), our results suggest that the SopE2-dependent induction of iNOS in epithelial cells and tissue macrophages of the gut will have an important role in the intestinal pathology associated with Salmonella
infection. Experiments to address this possibility will be a fruitful avenue of further research.