Our current studies show that IL-23 is required for the in vivo pulmonary IL-17 and IL-17F response to K. pneumoniae
infection. These results are novel in the light of previous work suggesting other cytokine signals, namely IL-15, are responsible for the pulmonary IL-17 response to lipopolysaccharide (30
). Although IL-15 appears to play a role in IL-17 induction in other models of inflammation (31
), our current data demonstrate a strict requirement for IL-23 in pulmonary IL-17/17F induction in response to K. pneumoniae
challenge. Our current work also identifies two potential sources of IL-23 in the lung—AMs and mDCs—and suggests IL-23 functions very early in lung response to pathogen compared with IL-12. As early as 4 h after in vitro exposure to bacteria, media from AM culture stimulates splenocyte IL-17 production, indicating a rapid induction of bioactive IL-23 (unpublished data). In contrast, IL-12 remained undetectable in AM culture supernatants even 24 h after in vitro challenge, and these supernatants did not induce splenocyte IFN-γ expression. These findings are consistent with prior work showing that AMs produce little or no IL-12 p70 in response to isolated challenge with K. pneumoniae
or LPS (5
). Hence, AM may be more important in initiating the early “ThIL-17
” response to this pathogen rather than directing T cells into Th1 polarization (34
In vivo, up-regulation of IL-23 p19 in BAL cells is seen as early as 2 h after K. pneumoniae infection. Cells obtained by BAL at this time are still more than 95% AMs, implicating these cells as the likely source of early IL-23 expression in the alveolar compartment, because they express p40 mRNA even before infection. Hence, induction of p19 transcription likely regulates the onset of IL-23 production in air spaces. The early increase in BAL cell p19 mRNA is followed by greater expression 16–24 h after infection, a pattern observed in both BALF cells and lung homogenate. Whether this finding is due to the alveolar recruitment of additional cell types expressing IL-23 p19 or increased AM gene expression is unknown.
Interestingly, we observed greater splenocyte IL-17 induction from bacteria-pulsed IL-12 p35−/−
AM conditioned media compared with WT AM. A regulatory role for IL-12 in IL-23–mediated signaling has been previously demonstrated (17
), because IL-12 and IL-23 share a common p40 subunit and both require IL-12Rβ1 binding to signal. However, we were unable to measure IL-12 p70 in bacteria-stimulated AM supernatants, regardless of genotype. One hypothesis is that AM production of bioactive IL-23 is greater in p35−/−
AMs, because more intracellular p40 is available to combine with p19. In support of this is, it has been shown that the elaboration of IL-12 p70 in AMs is under posttranscriptional control, and a second stimulus (such as IFN-γ) (5
) is required for AM release of IL-12 p70 heterodimer in response to LPS. Of note, higher IL-17 levels in IL-12 p35−/−
mice was not observed in vivo. However, deficient STAT1 signaling results in augmented IL-23 and IL-17 expression in the context of respiratory syncytial virus infection (35
Our prior work has shown that TLR4 signaling is required for early IL-23 p19 and IL-17 mRNA expression in the lung challenged with K. pneumoniae
). The greater up-regulation in p19 mRNA seen in mDCs compared with pDCs also supports a TLR4-dependent mechanism for IL-23 expression in this model, because granulocyte macrophage CSF–treated, bone marrow–derived mDCs are reported to express greater amounts of TLR4 and are more responsive to LPS than Flt3 ligand-generated pDCs (28
). Our data lead us to speculate that mDCs play an important role in the IL-17 recall response to bacterial challenge as mDCs readily migrate to draining lymph nodes upon antigen capture, a function not readily shared by AMs (36
). The subsequent T cell expansion, IL-17 expression, and augmented neutrophil recruitment as a result of the IL-23/IL-17 axis may represent a novel “cross-talk” loop between innate and adaptive pulmonary immunity, which enables the infected lung to more rapidly contain infection.
Although IL-12 p35 was not required for the pulmonary IL-17 response to K. pneumoniae,
it was requisite for IFN-γ expression in this infection. This finding is consistent with the well-studied stimulatory effect of IL-12 on IFN-γ expression (37
) as well as prior work that demonstrates the requirement of intact IL-12 for the pulmonary IFN-γ response to infection (38
). The inability of IL-23 to induce pulmonary IFN-γ expression in the absence of IL-12 is consistent with previous work showing the failure of recombinant IL-23 to induce splenocyte IFN-γ expression, despite up-regulation of IL-17 by this cytokine (17
). These observations are likely the result of differential receptor affinity and intracellular signaling events induced by IL-12 and IL-23. IL-12 binding to the IL-12Rβ1/Rβ2 complex predominantly activates STAT4. In contrast, IL-23 binds to the IL-23R/IL-12Rβ1 complex and induces STAT3, STAT1, and possibly STAT3/STAT4 heterodimer nuclear translocation, while only weakly activating STAT4 (39
Our finding of decreased survival following pulmonary K. pneumoniae infection in both p35−/− and p40−/− mice is consistent with other reports of the importance of intact IL-12 signaling in this infection model. The early and universal mortality observed in the p40−/− group compared with other strains suggests roles for both IL-12 and IL-23 in host defense. That a normally sublethal pathogen dose imparts 60% mortality in IL-23 p19−/− animals confirms the critical requirement for this cytokine in surviving pulmonary K. pneumoniae infection. Bacterial clearance could be significantly enhanced in IL-23 p19−/− mice by administration of recombinant IL-17 at 12 h into the infection, and this treatment restored G-CSF and LIX production without correcting IL-6 expression. These data suggest that the absence of IL-17 signaling in p19−/− mice mediates the observed phenotype and that IL-17–induced IL-6 signaling is not a critical component of host defense in this infection model. Despite the absence of IL-12 and markedly diminished lung IFN-γ induction in IL-12/23 p40−/− mice, IL-17 treatment still reduced significantly the high bacterial burden observed in these mice, further suggesting IL-17 plays a significant role in host defenses in this model independent of IL-12/IFN-γ signaling.
We recognize that the current study has important limitations. Namely, we have not identified the specific effector immune functions defective in the absence of IL-23 signaling. Because IL-17 and IL-17F elicit neutrophil recruitment in the lung (40
), defects in the number or function of these cells may also underlie the observed phenotype. Impaired antimicrobial peptide production may also contribute to enhanced mortality, as IL-17 signaling has also been shown to induce airway epithelial cell expression of mucin and β defensin 2 proteins, molecules important in bacterial clearance (41
Our data support a critical role for IL-23 and IL-17 in early host resistance to K. pneumoniae
independent of IL-12 and IFN-γ. It is possible that IL-23 and the subsequent IL-17 pathway have evolved to handle extracellular gram-negative bacterial challenges, because IL-17 is not required for host resistance against intracellular organisms such as Listeria
monocytogenes or Mycobacterium tuberculosis
(Kolls et al., unpublished observations), whereas IL-12 and IFN-γ have been shown to be critical for host resistance against these pathogens (44
). Moreover, because IL-23 is critical for autoimmune diseases such as arthritis and multiple sclerosis (47
), our data suggest that targeting IL-23 p19 would be less immunosuppressive than IL-12/23 p40.