TNF-alpha from inflammatory dendritic cells (DCs) regulates lung IL-17A/IL-5 levels and neutrophilia versus eosinophilia during persistent fungal infection.

Aspergillus fumigatus is commonly associated with allergic bronchopulmonary aspergillosis in patients with severe asthma in which chronic airway neutrophilia predicts a poor outcome. We were able to recapitulate fungus-induced neutrophilic airway inflammation in a mouse model in our efforts to understand the underlying mechanisms. However, neutrophilia occurred in a mouse strain-selective fashion, providing us with an opportunity to perform a comparative study to elucidate the mechanisms involved. Here we show that TNF-α, largely produced by Ly6c(+)CD11b(+) dendritic cells (DCs), plays a central role in promoting IL-17A from CD4(+) T cells and collaborating with it to induce airway neutrophilia. Compared with C57BL/6 mice, BALB/c mice displayed significantly more TNF-α-producing DCs and macrophages in the lung. Lung TNF-α levels were drastically reduced in CD11c-DTR BALB/c mice depleted of CD11c+ cells, and TNF-α-producing Ly6c(+)CD11b(+) cells were abolished in Dectin-1(-/-) and MyD88(-/-) BALB/c mice. TNF-α deficiency itself blunted accumulation of inflammatory Ly6c(+)CD11b(+) DCs. Also, lack of TNF-α decreased IL-17A but promoted IL-5 levels, switching inflammation from a neutrophil to eosinophil bias resembling that in C57BL/6 mice. The TNF-α(low) DCs in C57BL/6 mice contained more NF-κB p50 homodimers, which are strong repressors of TNF-α transcription. Functionally, collaboration between TNF-α and IL-17A triggered significantly higher levels of the neutrophil chemoattractants keratinocyte cytokine and macrophage inflammatory protein 2 in BALB/c mice. Our study identifies TNF-α as a molecular switch that orchestrates a sequence of events in DCs and CD4 T cells that promote neutrophilic airway inflammation.

Inhibition and role of let-7d in idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation. OBJECTIVES: To determine changes in expression and role of microRNAs in IPF. METHODS: RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry. MEASUREMENTS AND MAIN RESULTS: Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells. CONCLUSIONS: Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).

Early infection with respiratory syncytial virus impairs regulatory T cell function and increases susceptibility to allergic asthma.

Immune tolerance is instituted early in life, during which time regulatory T (T(reg)) cells have an important role. Recurrent infections with respiratory syncytial virus (RSV) in early life increase the risk for asthma in adult life. Repeated infection of infant mice tolerized to ovalbumin (OVA) through their mother's milk with RSV induced allergic airway disease in response to OVA sensitization and challenge, including airway inflammation, hyper-reactivity and higher OVA-specific IgE, as compared to uninfected tolerized control mice. Virus infection induced GATA-3 expression and T helper type 2 (T(H)2) cytokine production in forkhead box P3 (FOXP3)(+) T(reg) cells and compromised the suppressive function of pulmonary T(reg) cells in a manner that was dependent on interleukin-4 receptor α (IL-4Rα) expression in the host. Thus, by promoting a T(H)2-type inflammatory response in the lung, RSV induced a T(H)2-like effector phenotype in T(reg) cells and attenuated tolerance to an unrelated antigen (allergen). Our findings highlight a mechanism by which viral infection targets a host-protective mechanism in early life and increases susceptibility to allergic disease.

Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance of alternatively activated phenotype.

The ubiquitous fungus Aspergillus fumigatus is associated with chronic diseases such as invasive pulmonary aspergillosis in immunosuppressed patients and allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis or severe asthma. Because of constant exposure to this fungus, it is critical for the host to exercise an immediate and decisive immune response to clear fungal spores to ward off disease. In this study, we observed that rapidly after infection by A. fumigatus, alveolar macrophages predominantly express Arginase 1 (Arg1), a key marker of alternatively activated macrophages (AAMs). The macrophages were also found to express Ym1 and CD206 that are also expressed by AAMs but not NOS2, which is expressed by classically activated macrophages. The expression of Arg1 was reduced in the absence of the known signaling axis, IL-4Rα/STAT6, for AAM development. While both Dectin-1 and TLR expressed on the cell surface have been shown to sense A. fumigatus, fungus-induced Arg1 expression in CD11c(+) alveolar macrophages was not dependent on either Dectin-1 or the adaptor MyD88 that mediates intracellular signaling by most TLRs. Alveolar macrophages from WT mice efficiently phagocytosed fungal conidia, but those from mice deficient in Dectin-1 showed impaired fungal uptake. Depletion of macrophages with clodronate-filled liposomes increased fungal burden in infected mice. Collectively, our studies suggest that alveolar macrophages, which predominantly acquire an AAM phenotype following A. fumigatus infection, have a protective role in defense against this fungus.

Activation of c-Kit in dendritic cells regulates T helper cell differentiation and allergic asthma.

Dendritic cells (DCs) are integral to the differentiation of T helper cells into T helper type 1 T(H)1, T(H)2 and T(H)17 subsets. Interleukin-6 (IL-6) plays an important part in regulating these three arms of the immune response by limiting the T(H)1 response and promoting the T(H)2 and T(H)17 responses. In this study, we investigated pathways in DCs that promote IL-6 production. We show that the allergen house dust mite (HDM) or the mucosal adjuvant cholera toxin promotes cell surface expression of c-Kit and its ligand, stem cell factor (SCF), on DCs. This dual upregulation of c-Kit and SCF results in sustained signaling downstream of c-Kit, promoting IL-6 secretion. Intranasal administration of antigen into c-Kit-mutant mice or neutralization of IL-6 in cultures established from the lung-draining lymph nodes of immunized wild-type mice blunted the T(H)2 and T(H)17 responses. DCs lacking functional c-Kit or those unable to express membrane-bound SCF secreted lower amounts of IL-6 in response to HDM or cholera toxin. DCs expressing nonfunctional c-Kit were unable to induce a robust T(H)2 or T(H)17 response and elicited diminished allergic airway inflammation when adoptively transferred into mice. Expression of the Notch ligand Jagged-2, which has been associated with T(H)2 differentiation, was blunted in DCs from c-Kit-mutant mice. c-Kit upregulation was specifically induced by T(H)2- and T(H)17-skewing stimuli, as the T(H)1-inducing adjuvant, CpG oligodeoxynucleotide, did not promote either c-Kit or Jagged-2 expression. DCs generated from mice expressing a catalytically inactive form of the p110delta subunit of phosphatidylinositol-3 (PI3) kinase (p110(D910A)) secreted lower amounts of IL-6 upon stimulation with cholera toxin. Collectively, these results highlight the importance of the c-Kit-PI3 kinase-IL-6 signaling axis in DCs in regulating T cell responses.

Distinct responses of lung and spleen dendritic cells to the TLR9 agonist CpG oligodeoxynucleotide.

Dendritic cells (DCs) sense various components of invading pathogens via pattern recognition receptors such as TLRs. CpG oligodeoxynucleotides (ODNs), which mimic bacterial DNA, inhibit allergic airways disease and promote responses in the spleen to bacterial components. Because many TLR agonists are currently being tested for potential therapeutic effects, it is important to characterize the expression and function of TLRs in different tissues. We show that both myeloid and plasmacytoid DCs in the spleen express TLR9, the receptor for CpG ODNs, but lung DCs show no detectable expression in either subset. TLR4 expression in contrast was detected on both lung and spleen DCs. LPS was superior to CpG ODN in increasing the allostimulatory potential of lung DCs and their expression of CD40. However, both agonists efficiently stimulated spleen DCs. CpG ODNs administered to mice efficiently inhibited Th2 cytokine production both in the lung draining lymph node and in the spleen. Surprisingly, inhibition of Th2 cytokine production was evident despite high levels of expression of GATA-3 and additional transcription factors that regulate Th2 responses. Although in the spleen CpG ODNs induced IL-6, a key cytokine induced via TLR9-MyD88 signaling, no IL-6 was detectable in lung LN cells. These studies show for the first time that lung DCs lack TLR9 expression, but, despite this deficiency, CpG ODNs induce potent inhibitory effects on Th2 cytokine production in the lung without inducing expression of the proinflammatory cytokine, IL-6, which has been linked to chronic diseases in the lung and the gut.

Inducible expression of keratinocyte growth factor (KGF) in mice inhibits lung epithelial cell death induced by hyperoxia.

Oxidant-induced injury to the lung is associated with extensive damage to the lung epithelium. Instillation of keratinocyte growth factor (KGF) in the lungs of animals protects animals from oxidant-induced injury but the mechanism of protection is not well understood. An inherent problem in studying KGF function in vivo has been that constitutive overexpression of KGF in the lung causes embryonic lethality with extensive pulmonary malformation. Here we report the development of a stringently regulated, tetracycline-inducible, lung-specific transgenic system that allows regulated expression of KGF in the lung without causing developmental abnormalities from leaky KGF expression. By using this system, we show that exposure of KGF-expressing mice to hyperoxia protects the lung epithelium but not the endothelium from cell death in accordance with the selective expression of KGF receptor on epithelial and not on endothelial cells. Investigations of KGF-induced cell survival pathways revealed KGF-induced activation of the multifunctional pro-survival Akt signaling axis both in vitro and in vivo. Inhibition of KGF-induced Akt activation by a dominant-negative mutant of Akt blocked the KGF-mediated protection of epithelial cells exposed to hyperoxia. KGF-induced Akt activation may play an important role in inhibiting lung alveolar cell death thereby preserving the lung architecture and function during oxidative stress.