Chen J, Wen F, Chen C
… +3 more, Wang Y, Sun J, Sun Y
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 42089312
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WNT ligands have been critically implicated in many aspects of lung cancer progression. This study explores the impacts of Wnt family member 10A (WNT10A) in progression and immune response in lung adenocarcinoma (LUAD) a...WNT ligands have been critically implicated in many aspects of lung cancer progression. This study explores the impacts of Wnt family member 10A (WNT10A) in progression and immune response in lung adenocarcinoma (LUAD) and investigates the underpinning mechanisms. Expression, prognosis, and immune correlations of WNT10A in LUAD were explored using TCGA-LUAD, GEO datasets, and tissue microarrays. In vitro, WNT10A was knocked down in LUAD cell lines (H441 and H1299), followed by RNA sequencing and functional assays. Tumor-bearing mouse models with WNT10A knockdown were used to assess tumor growth and immune response. The functions of WNT10A on CD8 + T cell cytotoxicity were further explored through co-culture experiments and flow cytometry. FRAT1 overexpression, Wnt activators (BML-284), and CXCL12 manipulations were employed to verify mechanistic links. High WNT10A expression showed a trend toward poor overall survival, reduced CD8 + T cell infiltration, and unfavorable prognosis in LUAD patients. WNT10A loss in LUAD cells reduced growth, mobility, and inhibited Wnt signaling. In vivo, WNT10A loss prolonged animal survival in immunocompetent mice, reduced tumorigenic activity of mouse 3LL cells, and enhanced CD8 + T cell activity and tumor cell apoptosis. Mechanistically, WNT10A promoted FRAT1-mediated suppression of GSK3β, facilitating CTNNB1 activation and CXCL12 expression, which restricted CD8 + T cell function. WNT10A silencing synergized with anti-PD-1, reducing tumor burden and boosting CD8 + T cell infiltration/function. Generally, WNT10A promotes LUAD progression and CD8 + T cell dysfunction through FRAT1-mediated Wnt signaling and CXCL12 activation. Focusing on WNT10A could offer a compelling approach to boosting T cell-driven anti-tumor responses for the treatment of LUAD.
Uddin MB, McKelvey M, Palani S
… +3 more, Shao S, Liang Y, Sun K
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 42089309
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Secondary bacterial pneumonia following influenza A virus (IAV) infection markedly exacerbates lung inflammation and contributes to acute respiratory distress syndrome (ARDS); however, the immunologic pathways that drive...Secondary bacterial pneumonia following influenza A virus (IAV) infection markedly exacerbates lung inflammation and contributes to acute respiratory distress syndrome (ARDS); however, the immunologic pathways that drive lung injury and determine protective versus pathogenic inflammation remain incompletely defined. Using a clinically relevant murine model of sublethal IAV infection followed by methicillin-resistant Staphylococcus aureus (MRSA) challenge under antibiotic therapy, this study investigated the dynamic role of type I interferon (IFN-I) signaling in disease progression. The findings demonstrate that IFN-I exerts dual and contrasting effects on the host inflammatory response: it enhances myeloid-derived TNF-α while indirectly suppressing T cell-derived IFN-γ. Reporter mouse models identified recruited monocytes and dendritic cells (DCs) as the primary IFN-I-targeted populations, whereas neutrophils, T cells, and alveolar macrophages exhibited limited direct responsiveness. Myeloid-specific deletion of IFNAR1 reduced TNF-α production, restrained inflammatory monocyte differentiation, and improved survival without disrupting IFN-γ and IL-10 balance. Temporal IFNAR1 blockade further revealed that early IFN-I signaling supports alveolar macrophage maintenance and primes monocytes/DCs for immune activation, whereas sustained signaling during bacterial superinfection drives persistent monocyte chemoattractant production, excessive monocyte activation, and delayed resolution of inflammation. Collectively, these findings position IFN-I as a temporal immune rheostat-protective during acute viral infection but pathogenic when prolonged-and define a therapeutic window in which selective IFNAR inhibition enhances host antibacterial defense, either alone or in combination with antibiotic therapy. These insights highlight a promising immunomodulatory strategy to improve outcomes in severe viral-bacterial pneumonia and ARDS.
Thomas L, Serizay J, Mani R
… +22 more, Couvet S, Papon JF, Tamalet A, Mitri R, Faucon C, Murris-Espin M, Blanchard E, Langeois M, Blesson S, Tissier S, Montantin G, Moal FD, Levergeois R, Louis B, Beydon N, Thouvenin G, Escudier E, Amselem S, Giurgea I, Zaragosi LE, Meunier A, Legendre M
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 42089308
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Fluid mobilization on epithelia is ensured by the motile cilia of differentiated multiciliated cells (MCCs). Key transcriptional regulators of motile ciliogenesis include CCNO, MCIDAS, RFXs, and the transcription factor...Fluid mobilization on epithelia is ensured by the motile cilia of differentiated multiciliated cells (MCCs). Key transcriptional regulators of motile ciliogenesis include CCNO, MCIDAS, RFXs, and the transcription factor FOXJ1, whose precise role in humans remains unclear. We show that, unlike CCNO and MCIDAS, FOXJ1 expression persists in well-differentiated human airway epithelial cells (hAECs), suggesting functions beyond cilia initiation. ChIP-seq in hAECs allowed us to refine the consensus target motifs of FOXJ1 and RFXs, as well as their close proximity, which strongly suggests functional cooperation within a transcriptional complex. By combining ChIP-seq in normal cells with RNAseq from patients with FOXJ1-related primary ciliary dyskinesia (PCD), we identified 683 direct FOXJ1 target genes. Among these, 89 MCC-enriched genes-particularly those encoding axonemal proteins such as microtubule-inner proteins (MIPs) and dynein arm docking components-were downregulated in FOXJ1-deficient cells. Collectively, these findings provide new insights into how FOXJ1 contributes to human motile ciliogenesis, and reveal a potential role for FOXJ1 in maintaining MCCs differentiation and ciliary function by sustaining the expression of ciliary proteins, particularly those in direct contact with axonemal tubulin.
Wang Z, Chen Z, Ye T
… +6 more, Fan J, Cai Y, Zhou H, Ou T, Lu D, Cai K
Am J Respir Cell Mol Biol
· 2026 May · PMID 42089307
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BACKGROUND: Single-lung ventilation (SLV), commonly used in thoracic surgery, carries a risk of acute lung injury and acute respiratory distress syndrome. Our previous studies suggest that pulmonary microbiota may play a...BACKGROUND: Single-lung ventilation (SLV), commonly used in thoracic surgery, carries a risk of acute lung injury and acute respiratory distress syndrome. Our previous studies suggest that pulmonary microbiota may play a role in SLV-induced lung injury, but the precise mechanisms remain unclear. Palmitoleic acid (PoA), a crucial metabolite, is associated with protective physiological processes. This study explores the variations in pulmonary microbiota and metabolites during SLV-induced lung injury and investigates the potential protective role of PoA. RESULTS: The development of SLV-induced lung injury correlated with the changes of pulmonary microbiota and alterations of certain microbial genera and species. Through metabolomics and correlation analysis of pulmonary metabolites and microbiota, several metabolites exhibited a negative association with SLV-induced lung injury, with PoA being particularly noteworthy. Changes in pulmonary microbiota appeared to contribute to the reduction of PoA levels, ultimately leading to lung injury. Supplementation with PoA significantly attenuated the severity of lung injury, both in vivo and in vitro, by upregulating PPARγ expression and its downstream signaling pathways. CONCLUSIONS: The dysregulation of pulmonary microbiota contributes to SLV-induced lung injury, with PoA reduction playing a role in this process. PoA supplementation offers a protective effect by activating the PPAR pathway, suggesting its therapeutic potential in mitigating SLV-induced lung injury.
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 42089305
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Alveolar type 2 (AT2) cells play numerous roles in the alveolus related to stem cell, immunoregulatory, and secretory functions. Primary human AT2 cells can now be isolated and studied as organoids consisting of self-org...Alveolar type 2 (AT2) cells play numerous roles in the alveolus related to stem cell, immunoregulatory, and secretory functions. Primary human AT2 cells can now be isolated and studied as organoids consisting of self-organizing epithelial tissues as pure populations without the need for stromal support cells. However, genetic manipulation of AT2 cells to investigate their biology has relied on expensive and time-consuming processes requiring the use of viral vectors or conducting gene editing with induced pluripotent stem cells (iPSCs)-derived AT2 cells. Here we describe a high-efficiency method of accomplishing highly effective gene editing in cultured primary human AT2 cells, which can be done rapidly and at significantly lower costs. Using an optimized CRISPR ribonucleoprotein (RNP) approach, we can achieve nearly complete genetic knockout while preserving AT2 identity and viability. Our results simplify the process of genetically manipulating human AT2 cells to better understand the role of the alveolar epithelium in human lung biology.
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 42089304
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Neutrophil elastase (NE) is a key contributor to the pathogenesis of chronic obstructive pulmonary disease (COPD), driving airway inflammation and tissue destruction. While the RIPK3/MLKL-mediated necroptosis has been im...Neutrophil elastase (NE) is a key contributor to the pathogenesis of chronic obstructive pulmonary disease (COPD), driving airway inflammation and tissue destruction. While the RIPK3/MLKL-mediated necroptosis has been implicated in cigarette smoke (CS)-induced COPD, its role in NE-mediated lung injury remains undefined. In this study, we demonstrate that NE does not induce necroptosis in lung epithelial cells in vitro. Although genetic deletion of RIPK3 or MLKL did not prevent NE-induced apoptosis, the absence of MLKL significantly reduced NE-induced cellular senescence. Mechanistically, we found that after NE is internalized into endosomes, MLKL facilitates its escape into the cytoplasm and subsequent translocation to the nucleus, where it induces DNA damage and senescence. In contrast, MLKL deficiency retains NE within endosomes, promoting its degradation via lysosomal trafficking. Furthermore, Mlkl knockout mice were protected from porcine pancreatic elastase (PPE)-induced emphysema, highlighting the pathological relevance of this mechanism. Collectively, our findings reveal a novel, necroptosis-independent function of MLKL in regulating intracellular NE trafficking, suggesting a new therapeutic target for protease-driven lung injury in COPD.
CD55 is an immune regulator that inhibits T cell activation and also binds to CD97, a molecule involved in immune cell migration and signaling. While CD55 expression is reduced in chronic beryllium disease (CBD), its fun...CD55 is an immune regulator that inhibits T cell activation and also binds to CD97, a molecule involved in immune cell migration and signaling. While CD55 expression is reduced in chronic beryllium disease (CBD), its functional role in disease pathogenesis remains unclear. We hypothesized that CD55 downregulation in peripheral blood mononuclear cells (PBMCs) contributes to heightened beryllium (Be)-specific immune responses in CBD. To test this, we characterized CD55 expression and function in PBMCs from individuals with CBD and beryllium sensitization (BeS), as well as in a human Be-specific T cell model. CD55, sCD55, and CD97 mRNA expression were quantified by qRT-PCR in PBMCs from CBD (n = 25), BeS (n = 36), and control (n = 7) subjects. BeSO4 stimulation was used to assess CD55, STAT1, JAK2, and TNF-α expression over time in CBD PBMCs (n = 8). Serum sCD55 was measured by ELISA in an independent cohort. Functional studies using anti-CD55 neutralizing antibodies and the JAK2 inhibitor TG101348 evaluated effects on TNF-α production and lymphocyte proliferation (BeLPT) in PBMCs, and IL-2 production in a Jurkat-Be cell model. CD55, sCD55, and CD97 were significantly downregulated in CBD PBMCs compared to BeS (P < .001). Serum sCD55 was also reduced in CBD (P < .05). BeSO4 stimulation further downregulated CD55 and upregulated STAT1. CD55 blockade increased TNF-α production and BeLPT responses in CBD (P < .001) and BeS (P < .05); these effects were reversed by JAK2 inhibition. In the Be-cell model, CD55 inhibition enhanced IL-2 production (P < .01), attenuated by JAK2 blockade. These findings suggest that CD55 downregulation amplifies Be-induced immune responses in CBD via JAK2/STAT1 signaling.
Cooley JC, Penick SK, Wilson JA
… +9 more, Javkhlan N, Foster DG, Edelman BL, Schott CA, Humphries SM, Lynch DA, Schwartz DA, Riches DWH, Redente EF
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 42089301
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RATIONALE: Progressive fibrosing interstitial lung diseases (PF-ILDs) have a large global impact and there is a pressing need to develop new therapies. OBJECTIVES: We investigate if nintedanib augments apoptosis of pro-f...RATIONALE: Progressive fibrosing interstitial lung diseases (PF-ILDs) have a large global impact and there is a pressing need to develop new therapies. OBJECTIVES: We investigate if nintedanib augments apoptosis of pro-fibrotic fibroblasts induced by BCL-2 inhibition with ABT-199, and if combination therapy reverses pulmonary fibrosis. METHODS: Healthy and PF-ILD fibroblasts were treated with nintedanib and BCL-2 family member expression was measured. Fibroblasts and precision cut lung slices (PCLS) were analyzed for apoptosis after ABT-199 and nintedanib treatment. Therapeutic intervention with ABT-199 and nintedanib in mice with repetitive bleomycin-induced progressive pulmonary fibrosis were assessed for pro-fibrotic fibroblast and aberrant transitional epithelial cell apoptosis, lung collagen content, longitudinal oxygen saturation and micro-CT disease burden. MEASUREMENTS AND MAIN RESULTS: Nintedanib treatment increased expression of pro-apoptotic BIM and anti-apoptotic BCL-2 in PF-ILD primary fibroblasts. There was significantly increased apoptosis of fibroblasts in vitro after ABT-199. This was augmented after nintedanib co-treatment both in PF-ILD fibroblasts and PCLS. ABT-199 significantly improved fibrosis in mice, which was further enhanced after nintedanib co-treatment, by targeted apoptosis of pathogenic fibroblasts and transitional epithelial cells. CONCLUSIONS: The pro-apoptotic effects of ABT-199 were increased with nintedanib co-treatment and reversed fibrosis in mice. Combination treatment induced pro-fibrotic fibroblast and aberrant transitional epithelial cell apoptosis. These studies highlight a new mechanistic strategy to treat PF-ILD.
Cooney AL, Lamer S, Yang P
… +10 more, Wegner DJ, White FV, Cole FS, Wohlford-Lenane C, Hennessey E, Bawa P, Kotton DN, Sinn PL, Wambach JA, Mccray PB
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 42089300
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The ATP-binding cassette subfamily A member 3 (ABCA3) protein in the limiting membrane of lamellar bodies in alveolar type 2 (AT2) cells transports phospholipids required for pulmonary surfactant assembly. ABCA3 deficien...The ATP-binding cassette subfamily A member 3 (ABCA3) protein in the limiting membrane of lamellar bodies in alveolar type 2 (AT2) cells transports phospholipids required for pulmonary surfactant assembly. ABCA3 deficiency results from biallelic pathogenic variants in ABCA3 and causes progressive neonatal respiratory failure or childhood interstitial lung disease (chILD). Palliative care or lung transplantation are the only current definitive treatments for progressive respiratory failure due to ABCA3 deficiency. Complementing dysfunctional ABCA3 by gene addition has therapeutic potential. Previous studies show that repairing or complementing ABCA3 in induced pluripotent stem cell (iPSC)-derived AT2 cells rescues lamellar body morphology and surfactant phospholipid composition. Pathogenic variants disrupt ABCA3 function through altered protein trafficking (type 1) or by impaired phospholipid transport (type 2) into lamellar bodies. Here we tested ABCA3 gene complementation using a human pulmonary epithelial cell line (A549) with a genomically silenced ABCA3 locus (ABCA3 KO). Using this line, we generated additional cell lines that stably express individual ABCA3 variant cDNA constructs from a single genomic locus: L101P (type 1), E292V (type 2), E690K (type 2), or wild-type (WT) ABCA3. Lentiviral-mediated delivery of WT ABCA3 to each cell line partially rescued localization to LAMP3 + vesicles, lamellar body-like structure morphology, and cell proliferation. A functional assay measuring NF-κB signaling suggested that ABCA3 complementation ameliorated aberrant inflammatory signaling in E292V or E690K (type 2) mutant lines, but not in L101P (type 1) or knockout lines. These studies highlight the therapeutic potential of gene complementation as well as differences between ABCA3 pathogenic variants that may influence genetic therapy outcomes.
Klymenko O, Mohr J, Danopoulos S
… +29 more, Mižík I, Kuiper-Makris C, Stephan S, Mahjoub MA, Alvira CM, Rheindorf D, Imhof T, Akgül S, Donato M, Ebert LK, Müller S, Vohlen C, Hirani D, Bartenhagen C, Bartkuhn M, Procida T, Schermer B, Khatri P, Bellusci S, Goth K, Alexopoulos I, Georgomanolis T, Pryhuber GS, Pullamsetti SS, Seeger W, Al Alam D, Dötsch J, Koch M, Alejandre Alcazar MA
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 42089296
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Alveolar growth and repair are central processes in development and chronic lung disease, such as bronchopulmonary dysplasia (BPD), a neonatal lung disease without curative therapy. Alveolar epithelial type 2 cells (AT2)...Alveolar growth and repair are central processes in development and chronic lung disease, such as bronchopulmonary dysplasia (BPD), a neonatal lung disease without curative therapy. Alveolar epithelial type 2 cells (AT2) are the endogenous progenitor pool giving rise to AT1 and promoting alveolar repair. Since netrin-1, a regulator of cell homeostasis and stemness, has been linked to lung diseases, we now investigated its signaling and function in AT2 in a hyperoxia-based model of BPD and in lungs of infants with BPD. First, we demonstrated that prolonged hyperoxia reduced both netrin-1 and its receptor Unc5b in neonatal mouse lungs and in primary AT2 cells. Second, ex vivo studies using precision-cut lung slices (PCLS) and transgenic netrin-1 null mice showed that netrin-1 regulates AT2 survival and the expression of Krüppel-like factor 4 (Klf4) through Unc5b, a transcription factor regulating cell survival. Third, single cell and bulk transcriptomic analysis as well as proximity-dependent biotin identification assay, showed Klf4 to be upregulated in AT2 during alveolarization, downstream of netrin-1, and to regulate AT2 survival. In vivo, Klf4 gene expression and protein abundance was significantly reduced in total lung homogenates and in AT2 cells of neonatal mice exposed to hyperoxia. Finally, KLF4 + cells, KLF4 + epithelial, and specifically KLF4 + AT2 cells were reduced in clinical BPD. In summary, our data identify a novel netrin1-Unc5b-Klf4 axis in AT2 that is disrupted in BPD and could offer a novel target for endogenous alveolar repair.
Sun D, Wang Y, Ma R
… +3 more, Dilixiati N, Song Y, Ye Q
Am J Respir Cell Mol Biol
· 2026 Jul · PMID 42089295
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Secreted phosphoprotein 1-positive (SPP1+) macrophages play a critical role in the progression of silicosis, but their regulatory mechanisms remain unclear. In this study, we integrated single-cell RNA sequencing and spa...Secreted phosphoprotein 1-positive (SPP1+) macrophages play a critical role in the progression of silicosis, but their regulatory mechanisms remain unclear. In this study, we integrated single-cell RNA sequencing and spatial transcriptomics to characterize crucial macrophage subpopulations and their associated regulatory pathways during silicosis. Our results demonstrate that silica exposure significantly promotes the accumulation of SPP1+ macrophages within silicotic nodules. These macrophages contribute to inflammation by releasing pro-inflammatory cytokines such as TNF-α, MCP-1, and IL-6. Concurrently, they exhibit elevated ACTA2 RNA transcription levels, which enhance TGF-β production and drive fibrotic progression. These functional transitions are regulated by the ERK1/2 and JNK signaling pathways. Inhibition of SPP1 markedly attenuated silica-induced lung inflammation and fibrosis. Moreover, plasma SPP1 levels in silicosis patients were strongly correlated with disease onset and severity. Collectively, our findings indicate that SPP1+ macrophages are involved throughout the entire course of silicosis development, under the regulation of ERK/JNK signaling. Targeting SPP1+ macrophages may thus represent a promising therapeutic strategy for silicosis.
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 42089294
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Optogenetics involves genetic insertion of light-sensitive ion channels into nerves, such as with cre-based recombination. The recent increase in cre-based mouse lines has expanded our ability to test the roles of periph...Optogenetics involves genetic insertion of light-sensitive ion channels into nerves, such as with cre-based recombination. The recent increase in cre-based mouse lines has expanded our ability to test the roles of peripheral nerves in homeostasis and in disease models. Here, we generated an optogenetic mouse line in which hyperpolarizing inhibitory halorhodopsin channels were inserted into vagal cholinergic (parasympathetic) nerves using choline acetyltransferase (ChAT)-cre, enabling targeted inhibition of parasympathetic nerves. Halorhodopsin expression in parasympathetic neurons was confirmed by RT-PCR and confocal microscopy. Nerve reflex responses were generated with aerosolized serotonin or methacholine, during which mice were exposed to hyperpolarizing 645 nm or control 454 nm and 570 nm light. Inhibition of parasympathetic nerves by halorhodopsin attenuated airway hyperreactivity in house dust mite challenged mice while inhibition of cardiac cholinergic nerves increased heart rate. Our findings demonstrate the utility of halorhodopsin as a tool for testing the roles of parasympathetic nerves in cardiopulmonary function.
Banday MM, Banday S, Rahman M
… +23 more, Harrison AO, Singh N, Moore RM, Khan MM, Shankar S, Goda Y, Coppolino A, Movval N, Stutts S, Woolley A, Dishaw L, Goldberg H, Mebratu Y, Polson SW, Patel KN, Gaggar A, Hayes D, Krishnamoorthy N, Gewurz BE, Washko G, Tesfaigzi Y, Rehman R, Sharma NS
Am J Respir Cell Mol Biol
· 2026 Mar · PMID 42089290
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BACKGROUND: The lung virome (LV) and its interactions with the host-immune system leading to allograft injury after lung transplantation are not well characterized. METHODS: Shotgun metagenomics and qPCR was performed on...BACKGROUND: The lung virome (LV) and its interactions with the host-immune system leading to allograft injury after lung transplantation are not well characterized. METHODS: Shotgun metagenomics and qPCR was performed on a multicenter BAL/serum cohort from lung transplant recipients (LTRs). Viral constructs from betatorquevirus clade and group 2 TTVs were transfected in primary bronchial epithelial cells (PBECs) or airways of C57BL/6 mice with and without exposure to Influenza A (IAV) or RSV. RESULTS: LV in LTRs was dominated by viruses from the family Anelloviridae. CLAD LV was characterized by the enrichment of betatorquevirus clade (BTV). Validation in an independent cohort confirmed BTV abundance in CLAD BAL, serum and lung tissues. BTV ORF1 protein (kV1) suppressed PBEC IFNα and IFNγ responses by preventing intranuclear STAT translocation. Co-culture of IAV or RSV in kV1 transfected PBECs significantly augmented replication of RSV and IAV and increased cellular injury. Likewise, in-vivo transfection of kV1 increased replication and lung injury associated with IAV. CONCLUSION: Our work illuminates a novel virus-associated dysregulation of host interferon responses that promote lung injury associated with respiratory viral infections and, in part, explain differential host responses to viral infections after lung transplantation.