IL-23/Th17 pathway is critical for protective immunity against extracellular bacterial infections (Happel et al., 2005
; Ye et al., 2001
). However, the IL-23/Th17 axis is thought to be dispensable for protection against intracellular pathogens (Khader et al., 2005
; Schulz et al., 2008
; Aujla et al., 2008
). We show that absence of IL-23 and IL-17 but not IL-22 results in increased susceptibility to pulmonary LVS
infection. We also show that IL-17 can induce IL-12 and IFNγ from DCs and mediate Th1 responses, as well as induce IL-12 and IFNγ from macrophages and mediate bacterial clearance. Thus, we demonstrate a novel biological function for IL-17 in regulating IL-12/Th1 immunity in protection against an intracellular pathogen.
Our data shows that LVS
effectively induces the production of IL-12p40, IL-12 and IL-23 in the lungs of infected mice. Similar to other pulmonary infection models (Happel et al., 2005
), early induction of IL-23p19 and the more sustained production of IL-12p35 in the LVS
-infected lungs suggests that IL-23 functions during the early immune response. Induction of IL-23 and IL-17 between days 2-4 may contribute to the sustained IL-12 mRNA levels detected in infected B6 lungs. Both Th1 and Th17 responses are induced in the lungs of LVS
-infected mice, and are dependent on IL-12p40 cytokines. Specifically, IL-12 is required for the generation of Th1 responses while IL-23 is required for the generation of Th17 responses. Further, IL-12 and IFNγ negatively regulate Th17 responses in vivo during LVS
infection. However, the failure of high levels of IL-17 or its inducible chemokines to protect in the absence of IL-12/Th1 axis suggests that Th17 pathway alone cannot confer protection against pulmonary tularemia.
Our data shows that the IL-17 response is antigen-driven (Figure S3A
) and a majority of the IL-17-producers were TCRB+ CD3+ and CD4+ T cells, with a smaller population of CD8 cells (). A proportion of IL-17-producing cells were detected in wells that did not receive antigen (Figure S3A
) and are produced by γδ T cells in LVS
infected lungs (). Furthermore, that γδ−/− mice have reduced levels of IL-17 and are susceptible to pulmonary tularemia suggests that IL-17 produced by innate cells may serve as a defense mechanism until the adaptive immune cells are recruited to control the infection.
Although depletion of neutrophils resulted in reduced protection against systemic Francisella
infection (Sjostedt et al., 1994
can evade neutrophil-killing (Allen and McCaffrey, 2007
). Therefore, although decreased recruitment of neutrophils in the lungs of infected IL-17/IL-17R−/− mice may impact protective immunity against LVS
, there is nonetheless significant recruitment of neutrophils in absence of the IL-17/IL-17R pathway. This suggests that the increased susceptibility seen in mice lacking IL-17/IL-17R is likely due to the markedly reduced Th1 responses. We show that IL-17, but not IL-17F or IL-22 can enhance the ability of LVS
-stimulated DCs to produce IL-12. This extends the recent finding that IL-17 can induce the IL-12 production in peritoneal macrophages (Ishigame et al., 2009
), to include BMDCs, BMDMs, lung alveolar macrophages and lung CD11C+ cells. Importantly, since IL-17 can induce the production of IL-12 and IFN-γ within one hour of treatment, we conclude that IL-17 dependent induction of IL-12 in APCs is a rapid event that can impact downstream T cell events. This pathway may provide the basis for the plasticity of conversion of Th17 to Th1 responses seen in vivo (Lee et al., 2009
), where it is possible that IL-17 produced by Th17 cells can impact the induction of IL-12 in APCs and induce the conversion of Th17 to Th1 cells.
Until recently it was thought that the primary responses to IL-17 occurred in non-hematopoietic cells such as fibroblast and epithelial cells (Shen and Gaffen, 2008
). However, our data, as well as data using peritoneal macrophages (Ishigame et al., 2009
), suggest that myeloid cells such as DCs and macrophages express significant levels of IL-17RA mRNA and respond to IL-17 by production of cytokines and chemokines. That this response is mediated primarily through IL-17RA but not IL-17RC, is consistent with studies that show that murine IL-17RC can only bind to murine IL-17F but not IL-17, whereas human IL-17RC can bind to both human IL-17 and IL-17F (Kuestner et al., 2007
). IL-17 is known to activate NF-κβ and our studies show that IL-17 treatment of BMDCs results in activation of NF-κβ. However, co-treatment with an NF-κβ inhibitor results in loss of some IL-17-induced cytokines such as IFNγ and IL-6, but not others such as IL-12. That NF-κβ inhibitor PTDC did not completely repress TLR driven IL-12 production in DCs (Bohnenkamp et al., 2007
) suggests that other pathways such as mitogen activated protein kinases (MAPK) are involved. Additional experiments using pathway specific inhibitors will delineate the signaling pathways involved in IL-17 mediated responses in APCs.
An important facet of our findings is that IL-17 can induce IL-12 and IFNγ production and enhance LVS
bacterial clearance in macrophages. In a Bordetella pertussis
model, it was shown that IL-17 treatment of macrophages enhanced bacterial clearance (Higgins et al., 2006
) and was thought to be mediated by direct macrophage activation. Our data suggest that IL-17-dependent activation of macrophages and bacterial killing is mediated through induction of IFNγ, since IFNγ−/− BMDMs could not mediate IL-17-dependent control of bacteria. This suggests that IL-17 can modulate the innate responses and contribute to immunity and bacterial clearance until the arrival of adaptive immune cells to the site of infection.
Given that IL-17 can induce IL-12 and IFNγ production from APCs in the absence of pathogen stimulation suggests that IL-17 acts downstream of the initial pathogen-APC interaction. Although it is the Th1 response that controls intracellular bacteria, the IL-17 pathway provides critical “help” for induction of the Th1 pathway. This is evident from the reduced IFNγ levels in the lungs of infected IL-17−/− and IL-17R−/− mice and the increased IFNγ responses in infected IL-23p19−/− and IL-17−/− mice treated with exogenous IL-17. Further proof for IL-17 in generating Th1 responses is reflected by the decreased frequencies of IFNγ-producing cells and decreased induction of IL-12p35 mRNA in CD11c+ cells and neutrophils in IL-17R−/− infected lungs. That IFNγ production in CD8 T cells and NK 1.1 cells is also decreased in IL-17R−/− infected lungs, suggests an even broader role for IL-17 in maintenance of Th1 immune responses in the lung. Recent work shows that IL-17 inhibits T-bet expression and Th1 differentiation in anti-CD3 anti-CD28 stimulated Th1 cultures (O’Connor et al., 2009
). However, our data shows that Th cultures generated in the presence of APC and IL-17 can induce Th1 responses via induction of IL-12, suggesting that differential outcomes can be expected depending on whether the Th1 differentiation is mediated in the presence or absence of DCs.
Absence of the IL-23/Th17 axis did not impact IFNγ responses during M.tuberculosis
(Khader et al., 2005
) or L.monocytogenes
(Aujla et al., 2008
) infections, but resulted in reduced IFNγ responses following M.bovis BCG
infection (Umemura et al., 2007
). The unique requirement for the IL-23/Th17 pathway in induction of Th1 responses during LVS
and M. bovis BCG
, but not other intracellular infections is intriguing. Induction of IL-12 by LVS
infection is dependent on TLR-2 signaling (Hong et al., 2007
), while induction of IL-12p40 by a heat shock protein of Francisella
is dependent on TLR-4 (Ashtekar et al., 2008
). M.bovis BCG
lipomannans induce IL-12p40 through TLR2 (Quesniaux et al., 2004
). In contrast, IL-12 production by DCs following M.tuberculosis
infection can take place in the absence of either TLR-2 and TLR-4 (Jang et al., 2004
) and requires signals from both TLR2 and TLR9 (Bafica et al., 2005
).This suggests that different intracellular bacteria may stimulate differential TLRs on APCs and produce distinct polarizing cytokines that impact host immune response to infection. It is likely that some intracellular bacterial infections can effectively induce IL-12/IFNγ responses in the host, while other pathogens require the IL-23/IL-17 pathway for effective induction of host IL-12/IFNγ responses for pathogen control.
In summary, we show that IL-23 dependent IL-17 is induced during LVS infection and is required for induction of IL-12, optimal induction of Th1 responses and host resistance to infection. Further studies to understand the unique requirement for IL-17 in protection against LVS will advance our understanding of the fundamental requirements for protective immunity to intracellular pathogens.