We show that IL-23 is required for the long term containment of Mtb growth in mouse lungs and that it is critical for the CXCL13-dependent development of B cell follicles in infected lung tissue. We have previously reported that the lack of CXCL13 compromises immunity to tuberculosis and that this is linked to poor lymphoid follicle formation and increased accumulation of T cells around the vessels (15
). In the model reported here we show that IL-23 is critical for CXCL13 expression in lymphoid follicles and that there is increased T cell cuffing in the absence of IL-23 as the disease becomes chronic. We hypothesize therefore that the increased susceptibility to bacterial growth that occurs later in disease in the absence of IL-23 is a result of poor CXCL13 expression and the subsequent reduced ability of T cells to migrate from the vessels to the infected phagocytes and activate them efficiently.
We screened a large number of genes for differences in expression between the Mtb-infected B6 and IL-23a deficient lung and found that the strongest differences noted was the loss of the type 17 inflammatory cytokines IL-17A and IL-22. The absence of these genes either individually or in concert did not however recapitulate the deficiency in bacterial control seen in the absence of IL-23. Deficiency of IL-17 signaling or IL-22 did however modestly impact the development of the B cell follicles but this was not a sustained effect. Our observation that these cytokines were able to induce production of CXCL13 from lung stromal cells suggests that while IL-23 is critical for the CXCL13 expression in the lymphoid follicles, IL-23 may act through induction of IL-17 and IL-22 to mediate its effect. The fact that absence of either cytokine only modestly affects the development of follicles suggests that they compensate for each other in vivo.
We know that IL-23 can compensate for the absence of IL-12 in the IFN-γ-response during mouse tuberculosis (7
) and we see here a minor decrease in the IFN-γ producing cells in the chronically infected Il23a−/−
mice. Important recent data has shown that during chronic inflammatory conditions such as examined here, the development of antigen-specific cells from an IL-17-producing phenotype to an IFN-γ producing phenotype is dependent upon the IL-23a subunit (20
). Together with our data, this suggests that in the absence of IL-23 there may be a chronic, if small, inability to develop an IFN-γ-producing phenotype over time and this may also contribute to the increase in bacterial growth over the long term.
IL-17 has been implicated in germinal center formation (21
) and promotion of IgG2a and IgG3 isotypes (22
) as well as being associated with lymphoid neogenesis in graft rejection (23
). In a model of neonatal pulmonary inflammation wherein LPS treatment results in tertiary lymphoid tissues in the lung a clear dependence on IL-17-producing CXCR5-expressing TFH
cells was demonstrated (24
). We propose that as we see an early, albeit low, level of IL-23a independent IL-17A and delayed initiation of B cell follicle formation in the IL-17RA deficient mice that a similar process of IL-17 induction of CXCL13 production by lung stromal cells occurs during the early response to Mtb. In contrast to the data reported here however, the size of tertiary lymphoid tissues in the neonatal inflammation model was not impacted by the absence of IL-23a, although there were fewer lymphoid accumulations seen. Together these two different models suggest that while IL-17 and IL-23 play significant roles in the development of tertiary lymphoid structures, their relative roles may vary depending upon the nature of the stimulus. A critical issue may be the length of time and nature of the stimulus and the actual nature of the induced lymphoid follicle. We further show that IL-22 can augment the induction of CXCL13 in lung stromal cells and as infection progresses, it may be that there is a requirement for IL-22 in driving and maintaining CXCL13 production and this is why we see a requirement for IL-22 in B cell follicle formation in Mtb-infected lungs by day 80 of our study. As disease progresses however, B cell follicle growth in the Il17ra−/−
mice becomes equivalent with the B6 mice and it is the absence of IL-23 that has the greatest effect on the maintenance of the B cell follicle. Recent data show that IL-23 is critical for the expansion of infection-associated lymphoid tissue inducer (LTi) cells (25
) and that IL-22 is a critical product of these cells (26
). While these cells are associated with the development of lymphoid tissues they are also able to mediate protection against bacterial infection in the gut (25
) however preliminary studies do not show any appreciable differences in the LTi populations between infected control and Il23a−/−
mice (not shown).
While we propose that IL-23 is required to drive CXCL13 production and focus T cells to the granuloma, the possibility that IL-17 and IL-22 may be mediating direct effects should be considered. In more acute models of bacterial infection (27
) and specifically in high dose intratracheal BCG infection, IL-17 activity has been shown to be required for the early inflammatory response as well as protection; these activities are dependent upon IL-17 derived from γδ T cells (28
). We do not see a requirement for IL-17RA in protection in the low dose aerosol model suggesting that rapid expression of IL-17 activity within the lung is a requirement that depends upon dose and acuteness of challenge. Others have also reported little role for IL-22 in protection against Mtb infection (30
). We have not directly investigated the impact of the loss of IL-23 on the B cell function here but others have shown that blockade of CXCL13 does not impact B cell activation in tertiary lymphoid follicles (31
Our data support the need to investigate the role of IL-23 in the containment of tuberculosis. Humans lacking the IL-12p40 subunit that contributes to both IL-12p70 and IL-23 cytokine (32
) are particularly susceptible to mycobacterial diseases and this deficiency may reflect a role of both IL-12 and IL-23 in control of infection. In contrast to a protective role for IL-23, we recently published that excess IL-23 is associated with increased pathologic consequences (8
) and this highlights that, for a chronic disease such as tuberculosis, a balance in the level of this cytokine is essential. Too much and there is pathology, too little and there is a loss of protection.