IL-17 protein in BAL fluid and mRNA expression in lung homogenate from C3H/HeN (LPS-sensitive) and C3H/HeJ (LPS-insensitive) mice were determined at 0, 4, and 16 h after i.t. infection (). IL-17 in BAL fluid was at the lower limit of detection by ELISA until 16 h. At this time point, C3H/HeJ mice demonstrated significantly lower IL-17 levels compared with C3H/HeN mice. We have previously observed that IL-17 in BAL fluid is reflective of IL-17 levels in lung homogenates (10
). The lower IL-17 induction in C3H/HeJ mice was also confirmed by real-time RT-PCR for IL-17 transcripts. There was a highly statistically significant induction of IL-17 mRNA in C3H/HeN mice at 4 and 16 h compared with that of C3H/HeJ mice. Because IL-23 has been shown to induce IL-17 production in vitro (15
), we investigated the time course of IL-23 p19 mRNA induction in this model. Compared with the low levels of transcripts present 0 h after infection in both strains, there was significant and earlier induction of p19 in C3H/HeN compared with C3H/HeJ mice as soon as 4 h after bacterial challenge (). By 16 h, there was a delayed yet significant rise in p19 mRNA in the C3H/HeJ mice, although C3H/HeN p19 transcripts remained greater at this time point as well.
FIGURE 1 LPS-insensitive C3H/HeJ mice display markedly reduced IL-17 production and delayed p19 mRNA expression in response to i.t. challenge with K. pneumoniae. A, BAL fluid IL-17 protein content at the time of bacterial inoculation and 4 and 16 h later. B, Copy (more ...)
The putative cellular source of IL-17 is CD4+
T cells (2
). Stimulated CD8+
T cells from human preparations have also been demonstrated to generate significant IL-17 mRNA (17
). We next depleted CD4+
, or both T cell subsets in C57BL/6 mice with Ab GK1.5 or 2.43, respectively, which deplete over 97% of lung and splenic CD4+
T cells (18
depletion resulted in 45% reduction in measurable IL-17 in BAL fluid 24 h after K. pneumoniae
challenge (). However, the combination of CD4+
T cell depletion resulted in ~ 90% reduction in IL-17 following K. pneumoniae
challenge, suggesting that CD8+
T cells play a significant role in either mediating or directly producing IL-17 in this in vivo model of pneumonia.
FIGURE 2 The presence of both CD4+ and CD8+ T cells is necessary for normal IL-17 production in response to K. pneumoniae. Ab depletion of CD4+ or CD8+ T cells reduces the IL-17 content in BAL fluid from C57/BL6 mice 24 h after i.t. challenge. Combined neutralization (more ...)
As TLR4 is expressed on both macrophages and DC (20
), we designed in vitro experiments to determine whether TLR4 expression on DC was required for IL-17 elaboration by T cells and whether this stimulation required cell contact. DC obtained from C3H/HeN or C3H/HeJ mice had similar levels of class II MHC, CD80, and CD86 expression by FACS (data not shown). Moreover, DC from both mice strains produced comparable IL-12 (data not shown) upon stimulation with 1 μ
g/ml soluble CD40 ligand (a gift from Dr. W. Fanslow (Immunex, Seattle, WA)). However, C3H/HeN DC pulsed with K. pneumoniae
were capable of inducing much higher levels of IL-17 by total splenic T cells (CD90+
) compared with C3H/HeJ DC (). There was no IL-17 induction in T cells incubated with unpulsed DC or T cells incubated directly with bacteria but not DC (data not shown).
FIGURE 3 In vitro coculture of total spleen T cells (CD90+) with K. pneumoniae-pulsed DC. A, Twenty-four-hour cell culture supernatant analysis confirms that C3H/HeJ-derived cells exhibit diminished IL-17 expression compared with that of C3H/HeN cells. B, IL-17 (more ...)
Physical contact between DC and CD4+ or CD8+ T cells is not required for IL-17 production, because experiments using the Transwell membrane coculture system to separate DC and T cells showed no significant difference in IL-17 mRNA or protein expression compared with a nonseparated group (). In this system, CD8+ T cells from C3H/HeN mice were capable of expressing greater amounts of IL-17 mRNA transcripts and secreted protein after 24 h of culture compared with an equal number of CD4+ T cells. Similar IL-17 induction in CD4+ and CD8+ T cells was observed with cell-free conditioned medium from DC pulsed with K. pneumoniae. The same conditioned medium, when boiled, did not stimulate IL-17 (data not shown).
FIGURE 4 In vitro DC/T cell physical contact is not required for IL-17 production in response to K. pneumoniae. CD8+ T cells produce more IL-17 protein and mRNA transcripts compared with those of CD4+ T cells in response to bacteria-pulsed DC. A, IL-17 in cell (more ...)
As TLR4 signaling appears to be critical for the timely expression of IL-23 p19 induction in vivo, we investigated whether IL-23 elaboration by DC is critical for IL-17 induction by T cells. Neutralization experiments were performed in vitro on bacteria-pulsed C57BL/6 DC-conditioned medium using an anti-IL-12 p40 Ab which blocks IL-23 signaling (15
). We observed a significant reduction in IL-17 protein and mRNA in both CD4+
T cells exposed to the p40 Ab-treated medium but not isotype control (). Gene knockout experiments, using DC from mice on a C57 background, suggest that this activity is not due to IL-12 neutralization, because DC from p35−/−
mice (capable of making IL-23 but not IL-12) pulsed with K. pneumoniae
actually induced higher levels of IL-17 protein and mRNA. This finding is consistent with published data that rIL-12 can, in fact, inhibit IL-17 production in a dose-dependent manner (15
). Conversely, DC from p40−/−
mice (capable of making neither IL-23 nor IL-12) showed a significantly lower level of IL-17 induction compared with wild-type or p35−/−
mice, supporting the conclusion that IL-23, not IL-12, is responsible for T cell IL-17 expression in this in vitro system. We observed a greater percentage of inhibition in CD8+
T cell IL-17 production using anti-p40 Ab or supernatant from p40−/−
mice compared with that of CD4+
T cells. These data suggest that IL-23 is critical for both CD4+
T cell IL-17 production in response to TLR4 signaling, although other soluble factors may be operative in CD4+
T cell elaboration of IL-17. Toward this end, we performed in vitro Ab neutralization of TNF-α
, IL-6, and IL-15. All of these manipulations failed to inhibit IL-17 elaboration by CD4+
T cells (data not shown). Given the proven cause and effect of IL-23 on in vitro IL-17 elaboration combined with the earlier rise in p19 mRNA transcripts in our in vivo pneumonia model, we speculate that IL-23 is likely a key proximal mediator of the early, physiologically important induction of IL-17 in Gram-negative pneumonia. The previous finding that only APCs concomitantly express p40 and p19 (12
) and, hence, are the only cells believed capable of producing functional IL-23 underscores the importance of intact antigenic signaling via cognate receptors on APC (21
). Indeed, other studies have already proven the vital role of TLR2 on IL-23 expression (22
). The combination of our neutralization experiments and the time course of p19 induction in vivo supports the hypothesis that IL-23 in response to K. pneumoniae
is a key proximal event leading to IL-17 production in vivo. Future experiments using p19 knockout animals will enable us to answer this question more directly.
FIGURE 5 CD4+ and CD8+ T cell IL-17 response to conditioned medium from K. pneumoniae-pulsed DC requires biologically active IL-23. A, Conditioned medium from p35−/− (knockout (K/O)) DC (devoid of IL-12) elicits an augmented IL-17 response from (more ...)
These results significantly extend those recently obtained by Aggarwal et al. (15
). Our data demonstrate that TLR4 signaling is the principal mechanism to ultimately induce IL-17 in response to K. pneumoniae
infection both in vitro and in vivo. We also show CD8+
T cells are a significant source of IL-17, through an IL-23-dependent mechanism. C3H/HeN-derived CD8+
T cells were capable of greater in vitro IL-production compared with that of CD4+
T cells, whereas C57/BL6-derived CD8+
populations showed equivalent expression, suggesting strain differences may exist in the ability of T cell subsets to elaborate IL-17. Also, whereas the work of Aggarwal et al. focused on memory T cells that were activated and cultured for 3 days (hence representing a substantially different culture system than ours), we specifically focused on early (≤24 h) IL-17 elaboration in primary T cells, because IL-17 is induced rapidly in vivo in the lung after K. pneumoniae
In this model of Gram-negative pneumonia, a critical early host response is brisk neutrophil recruitment to quickly phagocytize invading pathogens before they begin geometric multiplication. Animals unable to mount this neutrophilic response to pulmonary K. pneumoniae
infection display greater lung bacterial burden, bacteremia, and death (23
). Earlier work has shown a critical role of IL-17 in neutrophil chemoattraction via its up-regulation of CXC chemokines and G-CSF (3
). Given the ability of IL-17 to induce neutrophil chemokine induction as well as granulopoiesis, it stands to reason that this cytokine likely functions as an important signal from T lymphocytes in orchestrating an augmented innate immune response to enhance pathogen clearance.
Our work proves that both CD4+
T cells can produce IL-17 in response to Gram-negative bacteria via TLR4 signaling, presumably by APC in the lung. It will be important to further define subpopulations of CD4+
T cells responsible for in vivo IL-17 production. Because IL-17 expression is not clearly dichotomized into Th1- or Th2-like expression profiles (2
), it is quite possible that IL-17-producing T cells do not represent either entity of this paradigm of acquired immunity.
Despite their lacking TLR4 signaling ability, the C3H/HeJ mice nevertheless demonstrate an increase, albeit attenuated and delayed, in both IL-17 and p19 mRNA. Based on our microarray data, this finding is consistent with a delayed expression of other proinflammatory genes, including MIP-2 and IL-6, in the C3H/HeJ strain. These later time points of gene induction may be due to LPS/TLR4-independent mechanisms, such as lipopeptide (TLR2) or unmethylated CpG motif (TLR9) signaling, which remain intact in C3H/HeJ mice. In addition, by 16 h, C3H/HeJ mice suffer a greater bacterial burden in the lung compared with that of controls. This differential inflammatory stimulus may invoke other, less potent signaling pathways to become operant in the expression of IL-17. As stated, this C3H/HeJ catch-up phenomenon is seen in many other proinflammatory signals, but likely represents too little too late, given the aforementioned increase in bacterial load and earlier occurrence of death in the C3H/HeJ strain compared with the C3H/HeN.
Our data show that intact TLR4 signaling represents the early and physiologically requisite signaling pathway in the timely expression of IL-23 and IL-17 in this Gram-negative pneumonia model. Further characterization of IL-23 and other downstream T cell mediators will greatly enlighten our understanding of the early communication between innate and acquired immunity in response to bacterial challenge.