STAT4 is a critical mediator of early innate immune responses against pulmonary Klebsiella infection

Abstract

Bacterial pneumonia is a leading cause of morbidity and mortality in the U.S. An effective innate immune response is critical for the clearance of bacteria from the lungs. IL-12, a key T1 cytokine in innate immunity, signals through STAT4. Thus, understanding how STAT4 mediates pulmonary immune responses against bacterial pathogens will have important implications for the development of rational immunotherapy targeted at augmenting innate immunity. We intratracheally administered Klebsiella pneumoniae to wild-type BALB/c and STAT4 knockout (STAT4-/-) mice. Compared with wild-type controls, STAT4-/- mice had decreased survival following intratracheal Klebsiella administration, which was associated with a higher lung and blood bacterial burden. STAT4-/- animals also displayed impaired pulmonary IFN-gamma production and decreased levels of proinflammatory cytokines, including the ELR- CXC chemokines IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma. Although total lung leukocyte populations were similar between STAT4-/- and wild-type animals following infection, alveolar macrophages isolated from infected STAT4-/- mice had decreased production of proinflammatory cytokines, including IFN-gamma, compared with infected wild-type mice. The intrapulmonary overexpression of IFN-gamma concomitant with the systemic administration of IFN-gamma partially reversed the immune deficits observed in STAT4-/- mice, resulting in improved bacterial clearance from the blood. Collectively, these studies demonstrate that STAT4 is required for the generation of an effective innate host defense against bacterial pathogens of the lung.

FIGURE 1

Survival in wt BALB/c and STAT4^−/− animals following i.t. Klebsiella infection. The wt and STAT4^−/− mice were i.t. inoculated with K. pneumoniae (2.5 × 10² CFU/mouse) on day 0, and followed for survival. *, p < 0.01 by two-tailed Mantel-Haenszel test (10 animals in each group; representative of 2 separate experiments).

FIGURE 2

a, Lung homogenate CFU following i.t. Klebsiella administration. The wt BALB/c and STAT4^−/− mice were i.t. inoculated with K. pneumoniae on day 0 (6 × 10² CFU/mouse). Total lung bacterial burden was assessed on days 2 and 3 postinfection. *, p < 0.05, wt vs STAT4^−/− lung CFU, day 3, by unpaired t test (n = 5 per group; data are representative of 3 separate experiments). b, Blood CFU following i.t. Klebsiella administration. K. pneumoniae (4–6 × 10² CFU/animal) was i.t. administered to wt BALB/c mice and STAT4^−/− mice on day 0, and serum bacterial burden was assessed on days 2 and 3 postinfection. *, p < 0.05, wt vs STAT4^−/− blood CFU, day 3, by unpaired t test (data were combined from 2 independent experiments with a total of 10 animals per group). c, H&E staining of lung tissue (×400) at baseline (top panels) and 3 days after i.t. administration of 2.5 × 10² CFU K. pneumoniae (bottom panels). Arrows indicate the presence of bacterial organisms in the alveolar spaces of infected STAT4^−/− mice.

FIGURE 3

a, Levels of whole lung IFN-γ mRNA in wt and STAT4^−/− mice following i.t. Klebsiella administration (n = 5 per group). b, IFN-γ levels in whole lung homogenates, day 3 following i.t. Klebsiella. *, p < 0.05, by Mann-Whitney U test (n = 5 per group; data are representative of 2 separate experiments).

FIGURE 4

Cytokine levels in BAL fluid. The wt BALB/c and STAT4^−/− mice were i.t. inoculated with K. pneumoniae on day 0, and then lavaged on days 1 and 2 postinfection. *, p < 0.05; **, p < 0.01 for Klebsiella-infected wt vs Klebsiella-infected STAT4^−/− groups as determined by unpaired t test (n = 5 per group; data are representative of 2 separate experiments).

FIGURE 5

Cell counts and differentials from BAL fluid. The wt and STAT4^−/− animals underwent bronchoalveolar lavage at baseline, 1 and 2 days after i.t. Klebsiella administration. n = 3–5 per group; data are representative of 2 separate experiments.

FIGURE 6

Number of IFN-γ⁺ cells in lung tissue. Lungs were harvested from wt and STAT4^−/− mice on day 2 after Klebsiella i.t. infection for total lung leukocyte isolation. Cells were stained for CD45 (leukocyte marker), CD4, CD8, DX5 (NK cell marker), and γδTCR (γδ T cell marker). Cells then were stained for IFN-γ after undergoing ex vivo stimulation with PMA and ionomycin. Analysis was performed after gating for CD45⁺ lymphocyte-sized cell populations. n = 2 per group; data are representative of 2 separate experiments.

FIGURE 7

Lung and blood bacterial burden in STAT4^−/− animals following IFN-γ therapy. All animals were given i.t. Klebsiella (2.7 × 10² CFU) on day 0. STAT4^−/− animals were administered either i.t. Adeno-IFN-γ (5 × 10⁸ PFU on day 0)/s.c. rmIFN-γ (500 ng on days 0, 1, and 2), or i.t. control adenovirus (5 × 10⁸ PFU on day 0)/s.c. saline (days 0, 1, and 2). On day 3, animals were sacrificed, and lung (a) and blood (b) CFU determined. *, p < 0.01 by unpaired t test (n = 6–8 per group).