Activation of Alveolar Epithelial ER Stress by ß-Coronavirus Infection Disrupt Surfactant Homeostasis in Mice: Implications for Covid-19 Respiratory Failure.

COVID-19 syndrome is characterized by acute lung injury, hypoxemic respiratory failure, and high mortality. Alveolar Type 2 (AT2) cells are essential for gas exchange, repair, and regeneration of distal lung epithelium. We have shown that the causative agent, SARS-CoV-2 and other ß-coronavirus genus members induce an ER stress response in vitro, however the consequences for host AT2 function in vivo are less understood. To study this, two murine models of coronavirus infection were employed- mouse hepatitis virus-1 (MHV-1) in A/J mice and a mouse adapted SARS-CoV-2 strain. MHV-1 infected mice exhibited dose-dependent weight loss with histological evidence of distal lung injury accompanied by elevated bronchoalveolar lavage fluid (BALF) cell counts and total protein. AT2 cells showed evidence of both viral infection and increased BIP/GRP78 expression, consistent with activation of the unfolded protein response (UPR). The AT2 UPR included increased IRE1α signaling and a biphasic response in PERK signaling accompanied marked reductions in AT2 and BALF surfactant protein (SP-B, SP-C) content, increases in surfactant surface tension, and emergence of a re-programmed epithelial cell population (Krt8+, Cldn4+). The loss of a homeostatic AT2 endophenotype was attenuated by treatment with the IRE1α inhibitor OPK711. As proof-of-concept, C57BL6 mice infected with mouse-adapted SARS-CoV-2 demonstrated similar lung injury and evidence of disrupted surfactant homeostasis. We conclude that lung injury from ß-coronavirus infection results from an aberrant host response activating multiple AT2 UPR pathways, altering surfactant metabolism/function, and changing AT2 endophenotypes offering a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and acute respiratory failure.

Impaired AMPK Control of Alveolar Epithelial Cell Metabolism Promotes Pulmonary Fibrosis.

Alveolar epithelial type II (AT2) cell dysfunction is implicated in the pathogenesis of familial and sporadic idiopathic pulmonary fibrosis (IPF). We previously described that expression of an AT2 cell exclusive disease-associated protein isoform (SP-CI73T) in murine and patient-specific induced pluripotent stem cell (iPSC)-derived AT2 cells leads to a block in late macroautophagy and promotes time-dependent mitochondrial impairments; however, how a metabolically dysfunctional AT2 cell results in fibrosis remains elusive. Here using murine and human iPSC-derived AT2 cell models expressing SP-CI73T, we characterize the molecular mechanisms governing alterations in AT2 cell metabolism that lead to increased glycolysis, decreased mitochondrial biogenesis, disrupted fatty acid oxidation, accumulation of impaired mitochondria, and diminished AT2 cell progenitor capacity manifesting as reduced AT2 self-renewal and accumulation of transitional epithelial cells. We identify deficient AMP-kinase signaling as a key upstream signaling hub driving disease in these dysfunctional AT2 cells and augment this pathway to restore alveolar epithelial metabolic function, thus successfully alleviating lung fibrosis in vivo.

PGF2α signaling drives fibrotic remodeling and fibroblast population dynamics in mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2α, and its cognate receptor FPr (Ptgfr) are implicated as a TGF-ß1-independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER-SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen-treated IER-SftpcI73T mice developed an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER-SftpcI73T mice crossed to a Ptgfr-null (FPr-/-) line showed attenuated weight loss and gene dosage-dependent rescue of mortality compared with FPr+/+ cohorts. IER-SftpcI73T/FPr-/- mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single-cell RNA-Seq, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts, which were reprogrammed to an "inflammatory/transitional" cell state in a PGF2α /FPr-dependent manner. Collectively, the findings provide evidence for a role for PGF2α signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.

Disruption of Prostaglandin F2α Receptor Signaling Attenuates Fibrotic Remodeling and Alters Fibroblast Population Dynamics in A Preclinical Murine Model of Idiopathic Pulmonary Fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2α, and its cognate receptor FPr (Ptfgr) are implicated as a TGFß1 independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER - SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen treated IER-SftpcI73T mice develop an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER-SftpcI73T mice crossed to a Ptgfr null (FPr-/-) line showed attenuated weight loss and gene dosage dependent rescue of mortality compared to FPr+/+ cohorts. IER-SftpcI73T/FPr-/- mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single cell RNA sequencing, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts which were reprogrammed to an "inflammatory/transitional" cell state in a PGF2α/FPr dependent manner. Collectively, the findings provide evidence for a role for PGF2α signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.

Chronic Expression of a Clinical SFTPC Mutation Causes Murine Lung Fibrosis with Idiopathic Pulmonary Fibrosis Features.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic interstitial lung disease. A barrier to developing more effective therapies for IPF is the dearth of preclinical models that recapitulate the early pathobiology of this disease. Intratracheal bleomycin, the conventional preclinical murine model of IPF, fails to reproduce the intrinsic dysfunction to the alveolar epithelial type 2 cell (AEC2) that is believed to be a proximal event in the pathogenesis of IPF. Murine fibrosis models based on SFTPC (Surfactant Protein C gene) mutations identified in patients with interstitial lung disease cause activation of the AEC2 unfolded protein response and endoplasmic reticulum stress-an AEC2 dysfunction phenotype observed in IPF. Although these models achieve spontaneous fibrosis, they do so with precedent lung injury and thus are challenged to phenocopy the general clinical course of patients with IPF-gradual progressive fibrosis and loss of lung function. Here, we report a refinement of a murine Sftpc mutation model to recapitulate the clinical course, physiological impairment, parenchymal cellular composition, and biomarkers associated with IPF. This platform provides the field with an innovative model to understand IPF pathogenesis and index preclinical therapeutic candidates.

Disruption of proteostasis causes IRE1 mediated reprogramming of alveolar epithelial cells.

Disruption of alveolar type 2 cell (AEC2) protein quality control has been implicated in chronic lung diseases, including pulmonary fibrosis (PF). We previously reported the in vivo modeling of a clinical surfactant protein C (SP-C) mutation that led to AEC2 endoplasmic reticulum (ER) stress and spontaneous lung fibrosis, providing proof of concept for disruption to proteostasis as a proximal driver of PF. Using two clinical SP-C mutation models, we have now discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop a reprogrammed cell state that heretofore has been seen only as a response to lung injury. Using single-cell RNA sequencing in vivo and organoid-based modeling, we show that this state arises de novo from intrinsic AEC2 dysfunction. The cell-autonomous AEC2 reprogramming can be attenuated through inhibition of inositol-requiring enzyme 1 (IRE1α) signaling as the use of an IRE1α inhibitor reduced the development of the reprogrammed cell state and also diminished AEC2-driven recruitment of granulocytes, alveolitis, and lung injury. These findings identify AEC2 proteostasis, and specifically IRE1α signaling through its major product XBP-1, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.

Immunophenotyping of Acute Inflammatory Exacerbations of Lung Injury Driven by Mutant Surfactant Protein-C: A Role for Inflammatory Eosinophils.

Acute inflammatory exacerbations (AIEs) represent immune-driven deteriorations of many chronic lung conditions, including COPD, asthma, and pulmonary fibrosis (PF). The first line of therapy is represented by broad-spectrum immunomodulation. Among the several inflammatory populations mobilizing during AIEs, eosinophils have been identified as promising indicators of an active inflammatory exacerbation. To better study the eosinophil-parenchymal crosstalk during AIE-PF, this work leverages a clinically relevant model of inflammatory exacerbations triggered by inducible expression of a mutation in the alveolar epithelial type 2 cell Surfactant Protein-C gene [SP-CI73T]. Unbiased single-cell sequencing analysis of controls and SP-CI73T mutants at a time coordinated with peak eosinophilia (14 days) defined heightened inflammatory activation, chemotaxis, and survival signaling (IL-6, IL-4/13, STAT3, Glucocorticoid Receptor, mTOR, and MYC) in eosinophils. To study the impact of eosinophils in inflammatory exacerbations, the SP-CI73T line was crossed with eosinophil lineage deficient mice (GATA1Δdbl) to produce the SP-CI73TGATA1KO line. Time course analysis (7-42 days) demonstrated improved lung histology, survival, and reduced inflammation in SP-CI73TGATA1KO cohorts. Spectral flow cytometry of tissue digests confirmed eosinophil depletion in GATA1KO mice and the absence of a compensatory shift in neutrophils and immature monocyte recruitment. Eosinophil deletion resulted in progressive monocyte-derived macrophage accumulation (14 days post-injury), combined with declines in CD3+CD4+ lymphocyte and B220+ B cell abundance. Histochemical analysis revealed atypical inflammatory cell activation in SP-CI73TGATA1KO mice, with reduced numbers of Arg-1+ and iNOS+ cells, but increases in tgfb1 mRNA expression in bronchoalveolar lavage cells and tissue. Dexamethasone treatment (1 mg/kg daily, i.p.) was utilized to investigate corticosteroid efficacy in highly eosinophilic exacerbations induced by mutant SP-CI73T. Dexamethasone successfully reduced total and eosinophil (CD11b+SigF+CD11c-) counts at 14 days and was linked to reduced evidence of structural damage and perivascular infiltrate. Together, these results illustrate the deleterious role of eosinophils in inflammatory events preceding lung fibrosis and demonstrate the efficacy of corticosteroid treatment in highly eosinophilic exacerbations induced by mutant SP-CI73T.

The common ABCA3E292V variant disrupts AT2 cell quality control and increases susceptibility to lung injury and aberrant remodeling.

ATP-binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in pulmonary surfactant function. The substitution of valine for glutamic acid at codon 292 (E292V) produces a hypomorphic variant that accounts for a significant portion of ABCA3 mutations associated with lung disorders spanning from neonatal respiratory distress syndrome and childhood interstitial lung disease to diffuse parenchymal lung disease (DPLD) in adults including pulmonary fibrosis. The mechanisms by which this and similar ABCA3 mutations disrupt alveolar type 2 (AT2) cell homeostasis and cause DPLD are largely unclear. The present study, informed by a patient homozygous for the E292V variant, used an in vitro and a preclinical murine model to evaluate the mechanisms by which E292V expression promotes aberrant lung injury and parenchymal remodeling. Cell lines stably expressing enhanced green fluorescent protein (EGFP)-tagged ABCA3 isoforms show a functional deficiency of the ABCA3E292V variant as a lipid transporter. AT2 cells isolated from mice constitutively homozygous for ABCA3E292V demonstrate the presence of small electron-dense lamellar bodies, time-dependent alterations in macroautophagy, and induction of apoptosis. These changes in AT2 cell homeostasis are accompanied by a spontaneous lung phenotype consisting of both age-dependent inflammation and fibrillary collagen deposition in alveolar septa. Older ABCA3E292V mice exhibit increased vulnerability to exogenous lung injury by bleomycin. Collectively, these findings support the hypothesis that the ABCA3E292V variant is a susceptibility factor for lung injury through effects on surfactant deficiency and impaired AT2 cell autophagy.

Role of CCR2+ Myeloid Cells in Inflammation Responses Driven by Expression of a Surfactant Protein-C Mutant in the Alveolar Epithelium.

Acute inflammatory exacerbations (AIE) represent precipitous deteriorations of a number of chronic lung conditions, including pulmonary fibrosis (PF), chronic obstructive pulmonary disease and asthma. AIEs are marked by diffuse and persistent polycellular alveolitis that profoundly accelerate lung function decline and mortality. In particular, excess monocyte mobilization during AIE and their persistence in the lung have been linked to poor disease outcome. The etiology of AIEs remains quite uncertain, but environmental exposure and genetic predisposition/mutations have been identified as two contributing factors. Guided by clinical evidence, we have developed a mutant model of pulmonary fibrosis leveraging the PF-linked missense isoleucine to threonine substitution at position 73 [I73T] in the alveolar type-2 cell-restricted Surfactant Protein-C [SP-C] gene [SFTPC]. With this toolbox at hand, the present work investigates the role of peripheral monocytes during the initiation and progression of AIE-PF. Genetic ablation of CCR2+ monocytes (SP-CI73TCCR2KO) resulted in improved lung histology, mouse survival, and reduced inflammation compared to SP-CI73TCCR2WT cohorts. FACS analysis of CD11b+CD64-Ly6Chi monocytes isolated 3 d and 14 d after SP-CI73T induced injury reveals dynamic transcriptional changes associated with "Innate Immunity' and 'Extracellular Matrix Organization' signaling. While immunohistochemical and in situ hybridization analysis revealed comparable levels of tgfb1 mRNA expression localized primarily in parenchymal cells found nearby foci of injury we found reduced effector cell activation (C1q, iNOS, Arg1) in SP-CI73TCCR2KO lungs as well as partial colocalization of tgfb1 mRNA expression in Arg1+ cells. These results provide a detailed picture of the role of resident macrophages and recruited monocytes in the context of AIE-PF driven by alveolar epithelial dysfunction.

In utero gene editing for monogenic lung disease.

Monogenic lung diseases that are caused by mutations in surfactant genes of the pulmonary epithelium are marked by perinatal lethal respiratory failure or chronic diffuse parenchymal lung disease with few therapeutic options. Using a CRISPR fluorescent reporter system, we demonstrate that precisely timed in utero intra-amniotic delivery of CRISPR-Cas9 gene editing reagents during fetal development results in targeted and specific gene editing in fetal lungs. Pulmonary epithelial cells are predominantly targeted in this approach, with alveolar type 1, alveolar type 2, and airway secretory cells exhibiting high and persistent gene editing. We then used this in utero technique to evaluate a therapeutic approach to reduce the severity of the lethal interstitial lung disease observed in a mouse model of the human SFTPCI73T mutation. Embryonic expression of SftpcI73T alleles is characterized by severe diffuse parenchymal lung damage and rapid demise of mutant mice at birth. After in utero CRISPR-Cas9-mediated inactivation of the mutant SftpcI73T gene, fetuses and postnatal mice showed improved lung morphology and increased survival. These proof-of-concept studies demonstrate that in utero gene editing is a promising approach for treatment and rescue of monogenic lung diseases that are lethal at birth.

Epithelial Expression of an Interstitial Lung Disease-Associated Mutation in Surfactant Protein-C Modulates Recruitment and Activation of Key Myeloid Cell Populations in Mice.

Patients with idiopathic pulmonary fibrosis (IPF) often experience precipitous deteriorations, termed "acute exacerbations" (AE), marked by diffuse alveolitis and altered gas exchange, resulting in a significant loss of lung function or mortality. The missense isoleucine to threonine substitution at position 73 (I73T) in the alveolar type 2 cell-restricted surfactant protein-C (SP-C) gene (SFTPC) has been linked to clinical IPF. To better understand the sequence of events that impact AE-IPF, we leveraged a murine model of inducible SP-CI73T (SP-CI73T/I73TFlp+/- ) expression. Following administration of tamoxifen to 8-12-wk-old mice, an upregulation of SftpcI73T initiated a diffuse lung injury marked by increases in bronchoalveolar lavage fluid (BALF) protein and histochemical evidence of CD45+ and CD11b+ cell infiltrates. Flow cytometry of collagenase-digested lung cells revealed a transient, early reduction in SiglecFhiCD11blowCD64hiCD11chi macrophages, countered by the sequential accumulation of SiglecFloCD11b+CD64-CD11c-CCR2+Ly6C+ immature macrophages (3 d), Ly6G+ neutrophils (7 d), and SiglecFhiCD11bhiCD11clo eosinophils (2 wk). By mRNA analysis, BALF cells demonstrated a time-dependent phenotypic shift from a proinflammatory (3 d) to an anti-inflammatory/profibrotic activation state, along with serial elaboration of monocyte and eosinophil recruitment factors. The i.v. administration of clodronate effectively reduced total BALF cell numbers, CCR2+ immature macrophages, and eosinophil influx while improving survival. In contrast, resident macrophage depletion from the intratracheal delivery of clodronate liposomes enhanced SftpcI73T -induced mortality. These results using SftpcI73T mice provide a detailed ontogeny for AE-IPF driven by alveolar epithelial dysfunction that induces a polycellular inflammation initiated by the early influx of proinflammatory CCR2+Ly6Chi immature macrophages.

An SFTPC BRICHOS mutant links epithelial ER stress and spontaneous lung fibrosis.

Alveolar type 2 (AT2) cell endoplasmic reticulum (ER) stress is a prominent feature in adult and pediatric interstitial lung disease (ILD and ChILD), but in vivo models linking AT2 cell ER stress to ILD have been elusive. Based on a clinical ChILD case, we identified a critical cysteine residue in the surfactant protein C gene (SFTPC) BRICHOS domain whose mutation induced ER stress in vitro. To model this in vivo, we generated a knockin mouse model expressing a cysteine-to-glycine substitution at codon 121 (C121G) in the Sftpc gene. SftpcC121G expression during fetal development resulted in a toxic gain-of-function causing fatal postnatal respiratory failure from disrupted lung morphogenesis. Induced SftpcC121G expression in adult mice resulted in an ER-retained pro-protein causing AT2 cell ER stress. SftpcC121G AT2 cells were a source of cytokines expressed in concert with development of polycellular alveolitis. These cytokines were subsequently found in a high-dimensional proteomic screen of bronchoalveolar lavage fluid from ChILD patients with the same class of SFTPC mutations. Following alveolitis resolution, SftpcC121G mice developed spontaneous pulmonary fibrosis and restrictive lung impairment. This model provides proof of concept linking AT2 cell ER stress to fibrotic lung disease coupled with translationally relevant biomarkers.

Expression of mutant Sftpc in murine alveolar epithelia drives spontaneous lung fibrosis.

Epithelial cell dysfunction is postulated as an important component in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mutations in the surfactant protein C (SP-C) gene (SFTPC), an alveolar type II (AT2) cell-restricted protein, have been found in sporadic and familial IPF. To causally link these events, we developed a knockin mouse model capable of regulated expression of an IPF-associated isoleucine-to-threonine substitution at codon 73 (I73T) in Sftpc (SP-CI73T). Tamoxifen-treated SP-CI73T cohorts developed rapid increases in SftpcI73T mRNA and misprocessed proSP-CI73T protein accompanied by increased early mortality (days 7-14). This acute phase was marked by diffuse parenchymal lung injury, tissue infiltration by monocytes, polycellular alveolitis, and elevations in bronchoalveolar lavage and AT2 mRNA content of select inflammatory cytokines. Resolution of alveolitis (2-4 weeks), commensurate with a rise in TGF-ß1, was followed by aberrant remodeling marked by collagen deposition, AT2 cell hyperplasia, α-smooth muscle actin-positive (α-SMA-positive) cells, and restrictive lung physiology. The translational relevance of the model was supported by detection of multiple IPF biomarkers previously reported in human cohorts. These data provide proof of principle that mutant SP-C expression in vivo causes spontaneous lung fibrosis, strengthening the role of AT2 cell dysfunction as a key upstream driver of IPF pathogenesis.

Aberrant lung remodeling in a mouse model of surfactant dysregulation induced by modulation of the Abca3 gene.

The lipid transporter, ATP binding cassette class A3 (ABCA3), plays a critical role in the biogenesis of alveolar type 2 (AT2) cell lamellar bodies (LBs). A relatively large number of mutations in the ABCA3 gene have been identified in association with diffuse parenchymal lung disease (DPLD), the most common of which is a missense mutation (valine substitution for lysine at residue 292 (ABCA3E292V)) that leads to functional impairment of the transporter in vitro. The consequences of ABCA3E292V gene expression in vivo are unknown. To address this question, we developed mouse models expressing ABCA3E292V knocked-in to the endogenous mouse locus. The parental (F1) mouse line (mAbca3E292V) that retained an intronic pgk-Neo selection cassette (inserted in reverse orientation) (mAbca3E292V-rNeo) demonstrated an allele dependent extracellular surfactant phospholipid (PL) deficiency. We hypothesize that this PL deficiency leads to aberrant parenchymal remodeling contributing to the pathophysiology of the DPLD phenotype. Compared to wild type littermates, baseline studies of mice homozygous for the pgk-Neo insert (mAbca3E292V-rNeo+/+) revealed nearly 50% reduction in bronchoalveolar lavage (BAL) PL content that was accompanied by quantitative reduction in AT2 LB size with a compensatory increase in LB number. The phenotypic alteration in surfactant lipid homeostasis resulted in an early macrophage predominant alveolitis which peaked at 8 weeks of age. This was followed by age-dependent development of histological DPLD characterized initially by peribronchial inflammatory cell infiltration and culminating in both an emphysema-like phenotype (which included stereologically quantifiable reductions in both alveolar septal surface area and volume of septal wall tissue) plus foci of trichrome-positive collagen deposition together with substantial proliferation of hyperplastic AT2 cells. In addition to spontaneous lung remodeling, mABCA3E292V-rNeo mice were rendered more vulnerable to exogenous injury. Three weeks following intratracheal bleomycin challenge, mAbca3-rNeo mice demonstrated allele-dependent susceptibility to bleomycin including enhanced weight loss, augmented airspace destruction, and increased fibrosis. Removal of the rNeo cassette from mAbca3 alleles resulted in restoration of BAL PL content to wild-type levels and an absence of changes in lung histology up to 32 weeks of age. These results support the importance of surfactant PL homeostasis as a susceptibility factor for both intrinsic and exogenously induced lung injury/remodeling.

Disruption of N-linked glycosylation promotes proteasomal degradation of the human ATP-binding cassette transporter ABCA3.

The lipid transport protein, ABCA3, expressed in alveolar type 2 (AT2) cells, is critical for surfactant homeostasis. The first luminal loop of ABCA3 contains three putative N-linked glycosylation sites at residues 53, 124, and 140. A common cotranslational modification, N-linked glycosylation, is critical for the proper expression of glycoproteins by enhancing folding, trafficking, and stability through augmentation of the endoplasmic reticulum (ER) folding cycle. To understand its role in ABCA3 biosynthesis, we utilized EGFP-tagged fusion constructs with either wild-type or mutant ABCA3 cDNAs that contained glutamine for asparagine substitutions at the putative glycosylation motifs. In A549 cells, inhibition of glycosylation by tunicamycin increased the electrophoretic mobility (Mr) and reduced the expression level of wild-type ABCA3 in a dose-dependent manner. Fluorescence imaging of transiently transfected A549 or primary human AT2 cells showed that although single motif mutants exhibited a vesicular distribution pattern similar to wild-type ABCA3, mutation of N124 and N140 residues resulted in a shift toward an ER-predominant distribution. By immunoblotting, the N53 mutation exhibited no effect on either the Mr or ABCA3 expression level. In contrast, substitutions at N124 or N140, as well a N124/N140 double mutation, resulted in increased electrophoretic mobility indicative of a glycosylation deficiency accompanied by reduced overall expression levels. Diminished steady-state levels of glycan-deficient ABCA3 isoforms were rescued by treatment with the proteasome inhibitor MG132. These results suggest that cotranslational N-linked glycosylation at N124 and N140 is critical for ABCA3 stability, and its disruption results in protein destabilization and proteasomal degradation.

Immune reconstitution during Pneumocystis lung infection: disruption of surfactant component expression and function by S-nitrosylation.

Pneumocystis pneumonia (PCP), the most common opportunistic pulmonary infection associated with HIV infection, is marked by impaired gas exchange and significant hypoxemia. Immune reconstitution disease (IRD) represents a syndrome of paradoxical respiratory failure in patients with active or recently treated PCP subjected to immune reconstitution. To model IRD, C57BL/6 mice were selectively depleted of CD4(+) T cells using mAb GK1.5. Following inoculation with Pneumocystis murina cysts, infection was allowed to progress for 2 wk, GK1.5 was withdrawn, and mice were followed for another 2 or 4 wk. Flow cytometry of spleen cells demonstrated recovery of CD4(+) cells to >65% of nondepleted controls. Lung tissue and bronchoalveolar lavage fluid harvested from IRD mice were analyzed in tandem with samples from CD4-depleted mice that manifested progressive PCP for 6 wks. Despite significantly decreased pathogen burdens, IRD mice had persistent parenchymal lung inflammation, increased bronchoalveolar lavage fluid cellularity, markedly impaired surfactant biophysical function, and decreased amounts of surfactant phospholipid and surfactant protein (SP)-B. Paradoxically, IRD mice also had substantial increases in the lung collectin SP-D, including significant amounts of an S-nitrosylated form. By native PAGE, formation of S-nitrosylated SP-D in vivo resulted in disruption of SP-D multimers. Bronchoalveolar lavage fluid from IRD mice selectively enhanced macrophage chemotaxis in vitro, an effect that was blocked by ascorbate treatment. We conclude that while PCP impairs pulmonary function and produces abnormalities in surfactant components and biophysics, these responses are exacerbated by IRD. This worsening of pulmonary inflammation, in response to persistent Pneumocystis Ags, is mediated by recruitment of effector cells modulated by S-nitrosylated SP-D.

Surfactant protein D protects against acute hyperoxic lung injury.

RATIONALE: Surfactant protein D (SP-D) is a member of the collectin family of soluble, innate, host defense molecules with demonstrated immunomodulatory properties in vitro. Constitutive absence of SP-D in mice is associated with lung inflammation, alteration in surfactant lipid homeostasis, and increased oxidative-nitrative stress. OBJECTIVES: To test the hypothesis that SP-D would protect against acute lung injury from hyperoxia in vivo. METHODS: Transgenic mice overexpressing rat SP-D constitutively (SP-D OE) or conditionally via regulation with doxycycline (SP-D Dox-on) were subjected to continuous hyperoxic challenge for up to 14 days. MEASUREMENTS AND MAIN RESULTS: Compared with littermate control mice (wild-type [WT]), SP-D OE mice exposed to 80% O(2) demonstrated substantially increased survival accompanied by significant reductions in wet to dry lung ratios and bronchoalveolar lavage (BAL) protein. Although SP-D OE and WT mice exhibited a twofold increase in total BAL cells and neutrophilia in response to hyperoxia, the SP-D OE group had lower levels of BAL proinflammatory cytokines and chemokines, including IL-6, tumor necrosis factor-alpha, and monocyte chemotactic protein-1; increased mRNA levels of the transcription factor NF-E2 related factor-2 (NRF-2) and phase 2 antioxidants hemoxygenase-1 (HO-1), glutathione peroxidase-2 (GPx-2) and NAD(P)H quinone oxidoreductase-1 (Nqo-1); and decreases in lung tissue thiobarbituric acid-reactive substances. As proof of principle, the protective role of SP-D on hyperoxic injury was confirmed as SP-D Dox-on mice exposed to 85% O(2) demonstrated increased mortality upon withdrawal of doxycycline. CONCLUSIONS: Local expression of SP-D protects against hyperoxic lung injury through modulation of proinflammatory cytokines and antioxidant enzymatic scavenger systems.

Selective inhibition of inducible NO synthase activity in vivo reverses inflammatory abnormalities in surfactant protein D-deficient mice.

Surfactant protein D (SP-D)-deficient (SP-D-/-) mice exhibit early development of emphysema. Previously we have shown that SP-D deficiency results in increased production and activity of inducible NO synthase (iNOS). In this study, we examined whether treatment with the iNOS inhibitor 1400W could inhibit the inflammatory phenotype. Mice were treated with 1400W systemically for 7 wk from 3 wk of age. Treatment reduced total lung NO synthase activity to 14.7+/-6.1% of saline-treated 10-wk-old SP-D-/- littermates. Long-term administration of 1400W reduced lung inflammation and cellular infiltration; and significantly attenuated the increased levels of matrix metalloproteinases 2 and 9, chemokines (KC, TARC), and cytokines (IFN-gamma) seen in bronchoalveolar lavage (BAL) of SP-D-/- mice. Abrogation of these levels was associated with decreasing BAL chemotactic activity for RAW cells. Two weeks of treatment with 1400W reduced total lung NO synthase (NOS) activity to 12.7+/-6.3% of saline-treated SP-D-/- mice. Short-term iNOS inhibition resulted in attenuation of pulmonary inflammation within SP-D-/- mice as shown by decreases in total BAL cell count (63+/-6% of SP-D-/- control), macrophage size (>25 microm) within the BAL (62+/-10% of SP-D-/- control), and a percentage of BAL macrophages producing oxidants (76+/-9% of SP-D-/- control). These studies showed that s.c. delivery of 1400W can be achieved in vivo and can attenuate the inflammatory processes within SP-D deficiency. Our results represent the first report linking defects in the innate immune system in the lung with alterations in NO homeostasis.

SP-D-deficient mice are resistant to hyperoxia.

Surfactant protein D (SP-D), a member of the collectin superfamily, modulates pulmonary inflammatory responses and innate immunity. Disruption of the SP-D gene in mice induces peribronchiolar inflammation, accumulation of large, foamy macrophages, increased bronchoalveolar lavage (BAL) phospholipid, and pulmonary emphysema. We hypothesized that absence of SP-D aggravates hyperoxia-induced injury. To test this, SP-D-deficient (SP-D-/-) and wild-type (SP-D+/+) mice were exposed to 80% or 21% oxygen. Paradoxically, during 14 days of hyperoxia, SP-D-/- mice had 100% survival vs. 30% in SP-D+/+. The survival advantage in SP-D-/- mice was accompanied by lower histopathological injury scores at days 5 and 14, although total BAL cells (8.2 +/- 1.4 x 10(5) in SP-D-/- vs. 4.04 +/- 0.25 x 10(5) in SP-D+/+ mice) and neutrophils (1.2 +/- 0.4 x 10(5) vs. 0.03 +/- 0.02 x 10(5) in SP-D-/- and SP-D+/+, respectively) were increased. In addition, BAL protein and lung-to-body weight ratios were similarly elevated in both groups after 3, 5, and 14 days of continuous exposure. Biochemically, in contrast to SP-D+/+, SP-D-/- mice had higher levels of surfactant phospholipid and SP-B at baseline and 5 days after hyperoxia accompanied by a preservation of surfactant biophysical activity. From a multiplex assay of nine cytokines, we found elevated levels of IL-13 in BAL fluid of normoxic SP-D-/- mice compared with SP-D+/+. We conclude that the resistance of SP-D-deficient mice to hyperoxia reflects homeostatic changes in the SP-D-/- phenotype involving both phospholipid and SP-B-mediated induced resistance of surfactant to inactivation as well as changes in the immunomodulatory BAL cytokine profile.

Surfactant protein-A inhibits Aspergillus fumigatus-induced allergic T-cell responses.

BACKGROUND: The pulmonary surfactant protein (SP)-A has potent immunomodulatory activities but its role and regulation during allergic airway inflammation is unknown. METHODS: We studied changes in SP-A expression in the bronchoalveolar lavage (BAL) using a murine model of single Aspergillus fumigatus (Af) challenge of sensitized animals. RESULTS: SP-A protein levels in the BAL fluid showed a rapid, transient decline that reached the lowest values (25% of controls) 12 h after intranasal Af provocation of sensitized mice. Decrease of SP-A was associated with influx of inflammatory cells and increase of IL-4 and IL-5 mRNA and protein levels. Since levels of SP-A showed a significant negative correlation with these BAL cytokines (but not with IFN-gamma), we hypothesized that SP-A exerts an inhibitory effect on Th2-type immune responses. To study this hypothesis, we used an in vitro Af-rechallenge model. Af-induced lymphocyte proliferation of cells isolated from sensitized mice was inhibited in a dose-dependent manner by addition of purified human SP-A (0.1-10 microg/ml). Flow cytometric studies on Af-stimulated lymphocytes indicated that the numbers of CD4+ (but not CD8+) T cells were significantly increased in the parental population and decreased in the third and fourth generation in the presence of SP-A. Further, addition of SP-A to the tissue culture inhibited Af-induced IL-4 and IL-5 production suggesting that SP-A directly suppressed allergen-stimulated CD4+ T cell function. CONCLUSION: We speculate that a transient lack of this lung collectin following allergen exposure of the airways may significantly contribute to the development of a T-cell dependent allergic immune response.