Alveolar epithelial STAT3 was required early (24 h) for maximal neutrophil recruitment and bacterial killing during E. coli pneumonia. Later in the infection (48 h), STAT3 prevented alveolar edema and lung injury. In addition, our in vitro findings suggest that IL-6 family cytokines, particularly LIF, in alveolar lining fluid are necessary for STAT3 activation. We conclude from these data that STAT3 activity in alveolar epithelial cells is downstream of IL-6 family cytokine expression and functions to both promote innate host defense and limit inflammatory injury during pneumonia.
The phenotype of Stat3Δ/Δ
mice at 24 hours of infection was similar to that of IL-6–deficient mice with E. coli
pneumonia, including decreased neutrophil recruitment and bacterial clearance (15
). IL-6 deficiency transiently decreased total lung pSTAT3 content (15
), and the present data demonstrate a transient role for IL-6 in activation of STAT3 in BALF-stimulated MLE-15 cells. Together, these studies implicate alveolar epithelial STAT3 as one avenue through which IL-6 promotes innate immunity. It is likely, however, that IL-6 signals through other cell types as well, such that deletion of STAT3 in only alveolar epithelial cells understates the full contribution of IL-6 to innate immunity in the lungs. The mechanisms, however, through which STAT3 signaling promotes inflammation at this stage of infection remain unknown. STAT3 has been shown to influence the expression of multiple inflammatory mediators (7
), yet our current results indicate no contribution of STAT3 on the expression of CXC chemokines, early response cytokines, or S100 proteins during E. coli
pneumonia. Future research will be necessary to elucidate the factors that link IL-6 family-induced STAT3 activity to alveolar neutrophil emigration.
At 48 hours, the deficiency of STAT3 in alveolar epithelial cells resulted in exacerbated lung injury as measured by alveolar flooding and lung liquid accumulation. Although bacterial burdens were increased in Stat3Δ/Δ mice compared with controls at this later time point, the CFU values observed at 48 hours represent less than 2% of the original E. coli inoculum. Therefore, exaggerated lung injury at 48 hours likely resulted from dysregulated inflammation or epithelial integrity rather than overwhelming infection. The results at 48 hours also indicate the possibility that STAT3 influences host defense independent of its early contribution to neutrophil recruitment (at 24 h), since bacterial burdens were higher in Stat3Δ/Δ mice despite normal or perhaps increased emigrated neutrophils. While neutrophil recruitment is a critical determinant of bacterial clearance in the lower respiratory tract, it is also possible that other bactericidal factors are downstream of STAT3 activation in the epithelium.
STAT3 activity has diverse biological consequences, but perhaps the most consistent role is tissue protection (10
). Many mechanisms are possible, including but by no means limited to regulation of apoptosis (46
), surfactant production (47
), VEGF expression (40
), and heme oxygenase-1 expression (48
). STAT3 in alveolar epithelial cells helps prevent lung injury after hyperoxia or adenovirus administration via mechanisms that include the expression of surfactant protein-B and possibly the anti-apoptotic protein Bcl-xL
). The overexpression of a constitutively active form of STAT3 in the airway epithelium protects mice from hyperoxic lung injury, in part due to decreased expression of matrix metalloproteinases 9 and 12 (11
). VEGF is also expressed in response to STAT3 activation (40
) and can be cytoprotective in the lungs (41
), but our current data suggest that this growth factor was not responsible for the phenotype observed in Stat3Δ/Δ
mice. STAT3 target genes important during pneumonia may include those identified above as well as others. The net effects of STAT3 activity in the alveolar epithelium of infected lungs are to increase bacterial clearance and limit lung injury.
It was recently shown that dominant-negative mutations in the Stat3
gene in humans result in the hyper-IgE syndrome (24
). Recurrent pneumonias are a hallmark of this disease, and lung infection is either directly or indirectly the cause of death in these patients (23
). The present results establish a causal link between STAT3 and host responses to bacteria in the lungs. Our current data indicate that STAT3 deficiency in the alveolar epithelial cells reduces both innate immune responsiveness and tissue protection during pneumonia, which suggests that defective functions in these cells may contribute to the pathogenesis of hyper-IgE syndrome.
Because alveolar epithelial STAT3 has such important functions during pneumonia, we sought to determine upstream factors contributing to its activation. Multiple cytokines and growth factors stimulate STAT3 signaling. We focused on IL-6 family members due to the importance of IL-6 during pneumonia (15
) and the shared requisite use of the gp130/STAT signaling pathway by this family of cytokines (49
). Alveolar epithelial cells express gp130 and are responsive to IL-6 family cytokines (26
). Little is known about IL-6 family members other than IL-6 during pneumonia, but OSM and LIF are increased in serum and BALF of patients with pneumonia and acute lung injury (51
). We observed increased lung mRNA for IL-6, OSM, LIF, and IL-11 after intratracheal E. coli
. The combined blockade of both IL-6 and LIF completely inhibited STAT3 phosphorylation induced by BALF from mice infected 24 hours previously, suggesting these two cytokines as essential to activating alveolar epithelial STAT3 early during pneumonia. Despite its comparatively lower level of mRNA induction, LIF was the most essential of the IL-6 family cytokines in BALF for activating STAT3 phosphorylation in MLE-15 cells, supporting a role for LIF in particular during pneumonia.
As with STAT3, LIF has protective effects during inflammation. Hyperoxia-induced lung injury, which is exacerbated in the absence of alveolar epithelial STAT3 (10
), is ameliorated when LIF is overexpressed in the lungs (53
). Similarly, intratracheal administration of recombinant LIF protects rats from hyperoxia-induced lung injury (54
) and inhibits LPS-induced pulmonary inflammation (55
). The roles of endogenous LIF are less clear. However, increased susceptibility to endotoxemia-induced shock in LIF-deficient mice again suggests a protective effect of this IL-6 family member during inflammation (56
). The regulation and function of LIF in pneumonic lungs is an important direction for future research.
Together these results indicate a critical role for STAT3 in alveolar epithelial cells during pneumonia. Early during infection, epithelial STAT3 contributes to neutrophil recruitment, whereas later it serves a role in protection from lung injury. During E. coli pneumonia, both IL-6 and LIF activate STAT3 phosphorylation in alveolar epithelial cells. Variation among patients in the LIF/IL-6:STAT3 signaling pathway may influence both infection and lung injury during pneumonia.