These results demonstrate that NF-κB RelA in alveolar epithelial cells is essential for induction of select cytokines in the lung, including CXCL5 and CCL20. The effects of alveolar epithelial mutation of RelA on cytokine expression were more modest than anticipated. Many cytokines were expressed similarly regardless of the epithelial cell mutation, including IL-1β, CXCL1, and CXCL2, which contrasts with prior publications in which the NF-κB pathway was targeted by overexpression of dnIκBα (14
). This discrepancy may result from differences in the models, including distinct delivery systems for LPS or off-target effects of the SPC-driven dnIκBα transgene, or conceivably they may suggest possible roles for factors other than RelA being downstream of pathways inhibited by dnIκBα overexpression. Cytokines such as TNF-α and IL-1β more likely result from leukocytes than epithelial cells (47
), suggesting that decreases observed in the SPC-dnI κBα transgenic mice may reflect indirect effects rather than direct expression by alveolar epithelial cells. In contrast to other cytokines, two chemokines were strongly diminished by RelA mutation in alveolar epithelial cells during LPS-induced inflammation, CCL20 and CXCL5. CCL20 is induced in human AT2 cells with LPS stimulation (49
), but it can also be elaborated by other cells such as T lymphocytes (50
). CCL20 has direct antimicrobial activities against gram-negative bacteria (50
), and it signals to dendritic cells and T cells in adaptive immune responses (52
). CXCL5 has been suggested to be an AT2 cell-specific product based on immunohistochemistry (53
), and it influences neutrophil influx to the lung after LPS inhalation and during E.coli
). Our results from targeted RelA mutation identify alveolar epithelial cells as essential sources of select innate immunity mediators important to lung host defense.
CCL20 induction was consistently abrogated by mutation of RelA in the alveolar epithelial cells. The AT2-like MLE15 cells make much more CCL20 than do the AT1-like E10 cells, and this induction is consistently dependent upon RelA. The sorted cell studies revealed that CCL20 is induced in cells other than AT1 cells or leukocytes. Altogether, these 3 sets of results suggest that AT2 cells may be unique sources of CCL20 during acute pulmonary inflammation, and this chemokine expression is uniformly dependent upon RelA.
In our study, CXCL5 was induced by LPS and pneumococcus in T1α−
cells which include AT2 cells, consistent with a prior study demonstrating CXCL5 induction in this cell type following an intrapulmonary LPS challenge (53
). Excitingly, our in vivo
studies revealed AT1 cells as a novel source of CXCL5 during pneumococcal pneumonia. Previous studies have demonstrated that purified bacterial products are capable of inducing chemokines in primary AT1-like cells or cell lines under in vitro
culture conditions (55
), which we have here expanded and confirmed with studies of AT1-like E10 cultures. Due to their anatomic localizations and morphological characteristics, AT1 cells have been difficult to study using immunohistochemistry or in situ
hybridization. There is little or no information about in vivo
AT1 responses to bacterial infection. The T1α-based sorting method allows the study of primary AT1 cells collected from living lungs. The present studies reveal that AT1 cells respond in vivo
during a bacterial infection of the lung. To our knowledge, this is the first demonstration that AT1 cells induce innate immunity genes during pneumonia. The discovery that the cells constituting the majority of the alveolar surface respond to infection with the elaboration of innate immunity mediators broadens our understanding of lung cell biology and pulmonary immunity.
Our conclusion that AT1 cells have roles in innate immunity is further supported by the evidence that the pattern recognition receptors, TLR2 and STING, are induced in AT1 cells during pneumococcal pneumonia, although TLR2 it is more highly expressed by leukocytes. Interestingly, STING is most highly expressed in AT1 cells at baseline and induced to the highest levels during infection in AT1 cells. STING mediates recognition pathways for pneumococcal DNA and drives the expression of innate immunity mediators such as type I interferons (43
). Thus, our data suggest that AT1 cells during infection increase their expression of both membrane and cytosolic receptors capable of responding to pneumococcus.
The significance of AT1 cell induction of innate immunity genes (including but likely not limited to TLR2, STING, and CXCL5) is unclear. Because the vast majority of the lung surface is AT1 cells, the increased expression of pattern recognition receptors and neutrophil chemokines may limit the spread of microbes throughout alveoli or across epithelial barriers. The fact that we did not see defects in inflammation and host defense is certainly not evidence for a lack of a role; these particular challenges and the limitations of this transgenic mouse model (discussed above) may have been inadequate to reveal functionally significant effects of AT1-derived genes. It is tempting to speculate that AT1 responses may be especially important in settings where microbes subvert macrophage responses, since alveolar macrophages are typically sentinel cells alerting to lung infection (2
). The functional significance of innate immunity gene induction by AT1 cells now becomes an important focus for future research.
Not only do the results indicate that CXCL5, TLR2, and STING are induced in AT1 cells during infection, they also illuminate distinct transcriptional regulation in these cells. TLR2 induction in AT1 cells was entirely abrogated by RelA deletion, revealing that AT1 cells can use RelA to induce the expression of innate immunity mediators, similar to prior observations with other cells (12
). In contrast, the induction of CXCL5 and STING was unaffected by the mutation of RelA. This was unexpected and represents the first observations of truly NF-κB RelA-independent innate immunity gene induction in pneumonic lungs. These results highlight novel pathways for innate immunity gene expression yet to be discovered.
Most significantly, these results demonstrate that AT1 cells are under-appreciated players in lung immunity and antibacterial host defense. These cells constitute the vast majority of the surface with which bacteria can interact during lung infection. Which products they elaborate, by what recognition and signaling and transcriptional pathways, and to what effects on integrated immune and physiological processes now become critical next questions. An improved understanding of the mechanisms and the significance of AT1 cell responses to infection may suggest new susceptibility determinants and therapeutic approaches for acute bacterial pneumonia.