This study shows that different immune responses are induced by immunization with viable versus dead C. trachomatis MoPn EBs. Intraperitoneal or intranasal immunization with viable chlamydiae elicited IFN-γ dominant responses associated with strong DTH and high levels of EB-specific IgG2a and IgA antibodies. The IFN-γ/IL-10 and IgG2a/IgG1 ratios among viable-EB-immunized mice were significantly higher than the ratios among mice immunized with dead EBs. Inactivated organisms together with IFA induced IL-10-dominant responses, with lower levels of IgG1 and IgG2a antibodies. Interestingly, immunization with viable organisms either given intranasally or intraperitoneally induced high levels of serum IgA antibodies, while immunization with dead organisms with adjuvant induced significantly lower levels of IgA antibodies.
Although we made our observations with C. trachomatis
in a single mouse strain (BALB/c), the phenomenon that viable and dead organisms induce different immune responses has been noted with other bacterial pathogens in multiple mouse strains and appears to be especially important for intracellular bacteria such as salmonellae and mycobacteria (5
). For instance, one informative study by Thatte et al. (37
) demonstrated that intraperitoneal immunization with live Salmonella typhimurium
induced an IFN-γ-dominant response associated with strong DTH and IgG2a antibody production, while heat-killed S. typhimurium
elicited an IL-4-dominant response with lower DTH and higher levels of IgG1 production.
Why should the immune responses induced by live and dead organisms be so different? Possible explanations include the density of antigenic peptides presented by APCs; also, the types of APC used to prime naive T cells could be different under the two immunization conditions. Since Th1
cells can be induced by identical antigenic peptides, T-cell receptor ligand density on the APCs has been considered a determining factor for differential induction of Th1
- or Th2
-like responses (6
). Such experiments show that a high density of MHC-antigen peptide complexes on the surface of APCs tends to stimulate Th1
cell responses, while low ligand density tends to stimulate Th2
responses. Therefore, it may be that infection of APCs by live organism results in higher ligand density, thus inducing Th1
-like responses. In the case of chlamydial infection, however, this mechanism may not be valid because recent observations suggest that APCs such as DCs do not readily support the replication of C. trachomatis
). Nonetheless, we still consider that ligand density may be relevant to the observations we have made and that further experiments are indicated. The ex vivo pulsing of DCs with killed or viable chlamydiae as reported by Su et al. (36
) offers an opportunity to evaluate the quantitative importance of this parameter.
Another and possibly more critical factor in determining the differences in immune responses induced by live and dead MoPn EBs is the difference in activation and differentiation of APCs. When viable and dead organisms are introduced into a host, the early innate response that they elicit may be critical in canalizing the subsequent adaptive immune responses. We observed that high levels of GM-CSF and IL-12 were produced in the peritoneal cavity of mice immunized with viable but not dead C. trachomatis
. GM-CSF was abundant at the early stages of inflammation (day 3 to day 7) and then declined in amount by day 21 (data not shown). Analysis of peritoneal cell composition demonstrated the enrichment for DC-like cells occurring during the time interval when GM-CSF and IL-12 levels were high (from day 3 to day 7) (Table and Fig. ). The correlation between GM-CSF production and DC-like cell enrichment suggests that GM-CSF production may be critical for development of DC-like cells following viable EB immunization. C. trachomatis
replication in epithelial cells is known to stimulate production of proinflammatory cytokines including GM-CSF (33
), and since chlamydiae are able to grow in mesothelial cells (39
), it may be that infection of these cells following intraperitoneal immunization with live organism induces the GM-CSF cytokine production that we observed. Since submucosal spaces are known to have rich populations of DCs (24
), we speculate that such effects may also occur at epithelial sites of infection. Further studies that more directly test the causal relationships among in vivo C. trachomatis
growth, GM-CSF production, DC recruitment, and enhanced immunity are needed.
Matured DCs are the important source of IL-12 and other cytokines that help T-cell activation (18
). Since IL-12 is essential to the differentiation of naive T cells into Th1
effector cells, the high levels of IL-12 observed after immunization with viable MoPn EBs may be a mechanism by which viable EBs preferentially induce Th1
immune responses. This speculation is supported by the observation that GM-CSF and IL-12 appeared at only low levels in the peritoneal cavities of mice immunized with inactivated chlamydiae and that immunization with nonviable EBs elicited only marginal levels of CD11c+
DC-like cells (Fig. ). We speculate that immunization with nonviable organisms may fail to recruit and activate DCs and thus fail to induce strong cellular immune responses.
An interesting finding in this study is the preferential induction of high levels of IgA by immunization with viable MoPn EBs. This observation is not unprecedented since immunization with live but not killed Aro− S. typhimurium
also induced strong IgA class switching (10
). Based on the Th1
paradigm, IgA responses are traditionally thought to be associated with Th2
cytokines such as IL-4, IL-5, and IL-6 which have been shown to facilitate IgA class switching. We assayed for IL-4 and IL-5 in the supernatants of antigen-stimulated spleen cells from mice immunized with viable or dead organisms, but neither of these cytokines was readily detected (data not shown). Since we found that mice immunized with viable EBs mounted IFN-γ-dominant Th1
responses and also had high levels of IgA production, this created an apparent paradox. The paradox may be explained by the recently reported observation that DC-like cells regulate IgA isotype switching of CD40-activated human B cells (9
). Fayette et al. (9
) reported that in these experiments, DCs in the presence of IL-10 and tumor necrosis factor alpha potentiate IgA class switching by CD40-activated naive B cells. Thus, the enrichment for DC-like cells in the peritoneal exudate together with antigen-specific IL-10 secretion could account for the strong IgA responses that were elicited under Th1
-dominant circumstances following viable EB immunization.
The finding of difference in protective immunity induced by viable and dead MoPn EBs and its association with proinflammatory cytokine (GM-CSF and IL-12) production and DC-like cell differentiation is helpful for the rational design of a chlamydial vaccine. It is self-evident that a protective vaccine should mimic viable organisms in terms of activating DCs and thus inducing strong protective immunity. Since antigen presentation by DCs can activate both protective Th1
-like responses (DTH and IFN-γ production) and IgA responses, it may be possible to develop a chlamydial vaccine which induces both CMI and IgA, the two important lines of defense against chlamydial infection by specifically targetting in vivo vaccine delivery to DCs. The recent observations of Su et al. (36
) demonstrating the remarkable immunogenicity of ex vivo chlamydia-laden DCs in producing protective immunity to vaginal chlamydial infection and oviductal pathology are extremely encouraging for this line of research.