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.

Impact of a Standardized, Pharmacist-Initiated "Test-Claim" Workflow for Anticipating Barriers to Accessing Discharge Antimicrobials.

Background: Inability to access and afford discharge oral antimicrobials may delay discharges or result in therapeutic failure. "Test-claims" have the potential to identify such barriers. Objective: This study evaluated discharge antimicrobial access and patient outcomes after implementation of a standardized, inpatient pharmacist-initiated antimicrobial discharge medication cost inquiry (aDMCI) process. Methods: This was an Institutional Review Board (IRB)-approved, pilot retrospective cohort study that included adults admitted for ≥72 hours from November 1, 2018, to February 28, 2019, and discharged on oral antimicrobials. Patients with a cost inquiry (aDMCI group) were compared with those without (standard-of-care, SOC, group). Primary endpoint was discharge delay. Secondary endpoints included percentage of patients discharged on suboptimal antimicrobials and medication errors from aDMCI. Results: 84 patients were included: 43 in SOC and 41 in aDMCI. Seventy-five antimicrobial cost inquiries were evaluated among 41 patients. There were no discharge delays or medication errors associated with the standardized "test-claim" (aDMCI) workflow. Patients in the SOC group had a greater Charlson Comorbidity Index (4 [2-6] vs 2 [1-4], P =0.004), were more likely to be immunosuppressed (24, 56% vs 12, 29%; P =0.014), and had longer hospitalization (8 [5-15] vs 6 [5-9] days, P =0.026). Primary access barriers were prior-authorization (8, 11%) and associated with linezolid and moxifloxacin cost inquiries. Most aDMCIs results were available in <24 hours (66, 88%). Conclusions: The aDMCI process is safe and offers an actionable transition of care tool that can identify barriers to accessing discharge medications while insulating patients from surprise out-of-pocket cost.

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.

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.

Congenital Deletion of Nedd4-2 in Lung Epithelial Cells Causes Progressive Alveolitis and Pulmonary Fibrosis in Neonatal Mice.

Recent studies found that expression of NEDD4-2 is reduced in lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and that the conditional deletion of Nedd4-2 in lung epithelial cells causes IPF-like disease in adult mice via multiple defects, including dysregulation of the epithelial Na+ channel (ENaC), TGFß signaling and the biosynthesis of surfactant protein-C proprotein (proSP-C). However, knowledge of the impact of congenital deletion of Nedd4-2 on the lung phenotype remains limited. In this study, we therefore determined the effects of congenital deletion of Nedd4-2 in the lung epithelial cells of neonatal doxycycline-induced triple transgenic Nedd4-2fl/fl/CCSP-rtTA2S-M2/LC1 mice, with a focus on clinical phenotype, survival, lung morphology, inflammation markers in BAL, mucin expression, ENaC function and proSP-C trafficking. We found that the congenital deletion of Nedd4-2 caused a rapidly progressive lung disease in neonatal mice that shares key features with interstitial lung diseases in children (chILD), including hypoxemia, growth failure, sterile pneumonitis, fibrotic lung remodeling and high mortality. The congenital deletion of Nedd4-2 in lung epithelial cells caused increased expression of Muc5b and mucus plugging of distal airways, increased ENaC activity and proSP-C mistrafficking. This model of congenital deletion of Nedd4-2 may support studies of the pathogenesis and preclinical development of therapies for chILD.

The biology of the ABCA3 lipid transporter in lung health and disease.

The lipid transporter, ATP-binding cassette class A3 (ABCA3), is a highly conserved multi-membrane-spanning protein that plays a critical role in the regulation of pulmonary surfactant homeostasis. Mutations in ABCA3 have been increasingly recognized as one of the causes of inherited pulmonary diseases. These monogenic disorders produce familial lung abnormalities with pathological presentations ranging from neonatal surfactant-deficiency-induced respiratory failure to childhood or adult diffuse parenchymal lung diseases for which specific treatment modalities remain limited. More than 200 ABCA3 mutations have been reported to date with approximately three quarters of patients presenting as compound heterozygotes. Recent advances in our understanding of the molecular basis underlying normal ABCA3 biosynthesis and processing and of the mechanisms of alveolar epithelial cell dysregulation caused by the expression of its mutant forms are beginning to emerge. These insights and the role of environmental factors and modifier genes are discussed in the context of the considerable variability in disease presentation observed in patients with identical ABCA3 gene mutations. Moreover, the opportunities afforded by an enhanced understanding of ABCA3 biology for targeted therapeutic strategies are addressed.

Lost after translation: insights from pulmonary surfactant for understanding the role of alveolar epithelial dysfunction and cellular quality control in fibrotic lung disease.

Dating back nearly 35 years ago to the Witschi hypothesis, epithelial cell dysfunction and abnormal wound healing have reemerged as central concepts in the pathophysiology of idiopathic pulmonary fibrosis (IPF) in adults and in interstitial lung disease in children. Alveolar type 2 (AT2) cells represent a metabolically active compartment in the distal air spaces responsible for pulmonary surfactant biosynthesis and function as a progenitor population required for maintenance of alveolar integrity. Rare mutations in surfactant system components have provided new clues to understanding broader questions regarding the role of AT2 cell dysfunction in the pathophysiology of fibrotic lung diseases. Drawing on data generated from a variety of model systems expressing disease-related surfactant component mutations [surfactant proteins A and C (SP-A and SP-C); the lipid transporter ABCA3], this review will examine the concept of epithelial dysfunction in fibrotic lung disease, provide an update on AT2 cell and surfactant biology, summarize cellular responses to mutant surfactant components [including endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and intrinsic apoptosis], and examine quality control pathways (unfolded protein response, the ubiquitin-proteasome system, macroautophagy) that can be utilized to restore AT2 homeostasis. This integrated response and its derangement will be placed in the context of cell stress and quality control signatures found in patients with familial or sporadic IPF as well as non-surfactant-related AT2 cell dysfunction syndromes associated with a fibrotic lung phenotype. Finally, the need for targeted therapeutic strategies for pulmonary fibrosis that address epithelial ER stress, its downstream signaling, and cell quality control are discussed.

A non-BRICHOS SFTPC mutant (SP-CI73T) linked to interstitial lung disease promotes a late block in macroautophagy disrupting cellular proteostasis and mitophagy.

Mutation of threonine for isoleucine at codon 73 (I73T) in the human surfactant protein C (hSP-C) gene (SFTPC) accounts for a significant portion of SFTPC mutations associated with interstitial lung disease (ILD). Cell lines stably expressing tagged primary translation product of SP-C isoforms were generated to test the hypothesis that deposition of hSP-C(I73T) within the endosomal system promotes disruption of a key cellular quality control pathway, macroautophagy. By fluorescence microscopy, wild-type hSP-C (hSP-C(WT)) colocalized with exogenously expressed human ATP binding cassette class A3 (hABCA3), an indicator of normal trafficking to lysosomal-related organelles. In contrast, hSP-C(I73T) was dissociated from hABCA3 but colocalized to the plasma membrane as well as the endosomal network. Cells expressing hSP-C(I73T) exhibited increases in size and number of cytosolic green fluorescent protein/microtubule-associated protein 1 light-chain 3 (LC3) vesicles, some of which colabeled with red fluorescent protein from the gene dsRed/hSP-C(I73T). By transmission electron microscopy, hSP-C(I73T) cells contained abnormally large autophagic vacuoles containing organellar and proteinaceous debris, which phenocopied ultrastructural changes in alveolar type 2 cells in a lung biopsy from a SFTPC I73T patient. Biochemically, hSP-C(I73T) cells exhibited increased expression of Atg8/LC3, SQSTM1/p62, and Rab7, consistent with a distal block in autophagic vacuole maturation, confirmed by flux studies using bafilomycin A1 and rapamycin. Functionally, hSP-C(I73T) cells showed an impaired degradative capacity for an aggregation-prone huntingtin-1 reporter substrate. The disruption of autophagy-dependent proteostasis was accompanied by increases in mitochondria biomass and parkin expression coupled with a decrease in mitochondrial membrane potential. We conclude that hSP-C(I73T) induces an acquired block in macroautophagy-dependent proteostasis and mitophagy, which could contribute to the increased vulnerability of the lung epithelia to second-hit injury as seen in ILD.

The cyclic peptide ecumicin targeting ClpC1 is active against Mycobacterium tuberculosis in vivo.

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development.

A nonaggregating surfactant protein C mutant is misdirected to early endosomes and disrupts phospholipid recycling.

Interstitial lung disease in both children and adults has been linked to mutations in the lung-specific surfactant protein C (SFTPC) gene. Among these, the missense mutation [isoleucine to threonine at codon 73 = human surfactant protein C (hSP-C(I73T) )] accounts for ∼30% of all described SFTPC mutations. We reported previously that unlike the BRICHOS misfolding SFTPC mutants, expression of hSP-C(I73T) induces lung remodeling and alveolar lipoproteinosis without a substantial Endoplasmic Reticulum (ER) stress response or ER-mediated intrinsic apoptosis. We show here that, in contrast to its wild-type counterpart that is directly routed to lysosomal-like organelles for processing, SP-C(I73T) is misdirected to the plasma membrane and subsequently internalized to the endocytic pathway via early endosomes, leading to the accumulation of abnormally processed proSP-C isoforms. Functionally, cells expressing hSP-C(I73T) demonstrated both impaired uptake and degradation of surfactant phospholipid, thus providing a molecular mechanism for the observed lipid accumulation in patients expressing hSP-C(I73T) through the disruption of normal phospholipid recycling. Our data provide evidence for a novel cellular mechanism for conformational protein-associated diseases and suggest a paradigm for mistargeted proteins involved in the disruption of the endosomal/lysosomal sorting machinery.

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.

Contamination level and location of recreational freshwater influence the ability to predict Escherichia coli concentration by qPCR targeting Bacteroides.

Fecal bacteria are common microbial contaminants in freshwater with the potential to cause human illness. Detection of these microbes have traditionally relied on microbial plating to enumerate colonies of fecal indicator bacteria (FIB) such as Escherichia coli (E. coli), which can take 24 h or longer to complete. Quantitative PCR (qPCR) is a rapid and sensitive method for detection of FIB in recreational water that could compliment or potentially substitute for microbial plating. In this study, we have isolated DNA from the beach water on the shoreline at three different locations of Lake Erie and subjected these samples to qPCR to examine the relative abundance of Bacteroides. These values were compared to colony forming units (CFU) of E. coli. The resultant linear regressions between these different measurements of microbe concentration were used to determine the efficacy of qPCR targeting Bacteroides at predicting E. coli concentrations that are relevant for decision making by recreational water managers. Our findings indicate that the ability of Bacteroides to serve as an early predictive tool for E. coli CFU concentration depends on sample location and level of bacterial contamination, but can be used in some cases to supplement recreational water quality measurement and consequential management.

Endoplasmic reticulum stress induced by surfactant protein C BRICHOS mutants promotes proinflammatory signaling by epithelial cells.

Chronic interstitial lung disease in both adults and children is associated with mutations of the surfactant protein C (SP-C) proprotein. Among these, mutations within the distal COOH propeptide, known as the BRICHOS domain, are associated with a severe disease phenotype. We showed that prolonged expression of the BRICHOS mutants, SP-C(Δexon4) and SP-C(L188Q), destabilizes endoplasmic reticulum (ER) quality-control mechanisms (the unfolded protein response, or UPR), resulting in the induction of ER stress signaling, an inhibition of the ubiquitin/proteasome system, and the activation of apoptotic pathways. Based on recent observations that the UPR and ER stress can be linked to the induction of proinflammatory signaling, we hypothesized that the epithelial cell dysfunction mediated by SP-C BRICHOS mutants would activate proinflammatory signaling pathways. In a test of this hypothesis, A549 and human embryonic kidney epithelial (HEK293) cells, transiently transfected with either SP-C(Δexon4) or SP-C(L188Q) mutants, each promoted the upregulation of multiple UPR response genes, including homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1) and GRP78. Commensurate with these results, increases in IL-8 secretion occurred and were accompanied by the activation of c-Jun N-terminal kinase (JNK)/activating protein-1 signaling. The stimulation of IL-8 cytokine release was completely attenuated by treatment with the JNK-specific inhibitor, SP600125. In addition, SP-C(Δexon4), but not SP-C(L188Q), activated NFκB. The treatment of SP-C(Δexon4) transfected cells with 4-phenylbutyric acid, a small molecule chaperone known to improve protein folding, blocked the activation of NFκB, but not the release of IL-8. Taken together, the results support the role of JNK signaling in mediating SP-C BRICHOS-induced cytokine release, and provide a link between SP-C BRICHOS mutants and proinflammatory cytokine signaling.

A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments.

The ATP binding cassette, class A (ABCA) proteins are homologous polytopic transmembrane transporters that function as lipid pumps at distinct subcellular sites in a variety of cells. Located within the N terminus of these transporters, there exists a highly conserved xLxxKN motif of unknown function. To define its role, human ABCA3 was employed as a primary model representing ABCA transporters, while mouse ABCA1 was utilized to support major findings. Transfection studies showed colocalization of both transporters with surfactant protein C (SP-C), a marker peptide for successful protein targeting to lysosomal-like organelles. In contrast, alanine mutation of xLxxKN resulted in endoplasmic reticulum retention. As proof of principle, swapping xLxxKN for the known lysosomal targeting motif of SP-C resulted in post-Golgi targeting of the SP-C chimera. However, these products failed to reach their terminal processing compartments, suggesting that the xLxxKN motif only serves as a Golgi exit signal. We propose a model whereby an N-terminal signal sequence, xLxxKN, directs ABCA transporters to a post-Golgi vesicular sorting station where additional signals may be required for selective delivery of individual transporters to final subcellular destinations.

Optimizing preoperative antibiotics in patients with ß-lactam allergies: A role for pharmacy.

PURPOSE: Patients with a reported ß-lactam allergy (BLA) are often given alternative perioperative antibiotic prophylaxis, increasing risk of surgical site infections (SSIs), acute kidney injury (AKI), and Clostridioides difficile infection (CDI). The purpose of this study was to implement and evaluate a pharmacist-led BLA clarification interview service in the preoperative setting. METHODS: A pharmacist performed BLA clarification telephone interviews before elective procedures from November 2018 to March 2019. On the basis of allergy history and a decision algorithm, first-line preoperative antibiotics, alternative antibiotics, or allergy testing referral was recommended. The pharmacist intervention (PI) group was compared to a standard of care (SOC) group who underwent surgery from November 2017 to March 2018. RESULTS: Eighty-seven patients were included, with 50 (57%) and 37 (43%) in the SOC and PI groups, respectively. The most common surgeries included orthopedic surgery in 41 patients (47%) and neurosurgery in 17 patients (20%). In the PI group, all BLA labels were updated after interview. Twenty-three patients were referred for allergy testing, 12 of the 23 (52%) completed BLA testing, and penicillin allergies were removed for 9 of the 12 patients. Overall, 28 of the 37 (76%) pharmacy antibiotic recommendations were accepted. Cefazolin use significantly increased from 28% to 65% after the intervention (P = 0.001). SSI occurred in 5 (10%) patients in the SOC group and no patients in the PI group (P = 0.051). All of these SSIs were associated with alternative antibiotics. Incidence of AKI and CDI was similar between the groups. No allergic reactions occurred in either group. CONCLUSION: Implementation of a pharmacy-driven BLA reconciliation significantly increased ß-lactam preoperative use without negative safety outcomes.

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.

Patient-specific iPSCs carrying an SFTPC mutation reveal the intrinsic alveolar epithelial dysfunction at the inception of interstitial lung disease.

Alveolar epithelial type 2 cell (AEC2) dysfunction is implicated in the pathogenesis of adult and pediatric interstitial lung disease (ILD), including idiopathic pulmonary fibrosis (IPF); however, identification of disease-initiating mechanisms has been impeded by inability to access primary AEC2s early on. Here, we present a human in vitro model permitting investigation of epithelial-intrinsic events culminating in AEC2 dysfunction, using patient-specific induced pluripotent stem cells (iPSCs) carrying an AEC2-exclusive disease-associated variant (SFTPCI73T). Comparing syngeneic mutant versus gene-corrected iPSCs after differentiation into AEC2s (iAEC2s), we find that mutant iAEC2s accumulate large amounts of misprocessed and mistrafficked pro-SFTPC protein, similar to in vivo changes, resulting in diminished AEC2 progenitor capacity, perturbed proteostasis, altered bioenergetic programs, time-dependent metabolic reprogramming, and nuclear factor κB (NF-κB) pathway activation. Treatment of SFTPCI73T-expressing iAEC2s with hydroxychloroquine, a medication used in pediatric ILD, aggravates the observed perturbations. Thus, iAEC2s provide a patient-specific preclinical platform for modeling the epithelial-intrinsic dysfunction at ILD inception.

Identification and absolute configuration of dihydroxy-arachidonic acids formed by oxygenation of 5S-HETE by native and aspirin-acetylated COX-2.

Biosynthesis of the prostaglandin endoperoxide by the cyclooxygenase (COX) enzymes is accompanied by formation of a small amount of 11R-hydroxyeicosatetraenoic acid (HETE), 15R-HETE, and 15S-HETE as by-products. Acetylation of COX-2 by aspirin abrogates prostaglandin synthesis and triggers formation of 15R-HETE as the sole product of oxygenation of arachidonic acid. Here, we investigated the formation of by-products of the transformation of 5S-HETE by native COX-2 and by aspirin-acetylated COX-2 using HPLC-ultraviolet, GC-MS, and LC-MS analysis. 5S,15S- dihydroxy (di)HETE, 5S,15R-diHETE, and 5S,11R-diHETE were identified as by-products of native COX-2, in addition to the previously described di-endoperoxide (5S,15S-dihydroxy-9S,11R,8S,12S-diperoxy-6E,13E-eicosadienoic acid) as the major oxygenation product. 5S,15R-diHETE was the only product formed by aspirin-acetylated COX-2. Both 5,15-diHETE and 5,11-diHETE were detected in CT26 mouse colon carcinoma cells as well as in lipopolysaccharide-activated RAW264.7 cells incubated with 5S-HETE, and their formation was attenuated in the presence of the COX-2 specific inhibitor, NS-398. Aspirin-treated CT26 cells gave 5,15-diHETE as the most prominent product formed from 5S-HETE. 5S,15S-diHETE has been described as a product of the cross-over of 5-lipoxygenase (5-LOX) and 15-LOX activities in elicited rat mononuclear cells and human leukocytes, and our studies implicate cross-over of the 5-LOX and COX-2 pathways as an additional biosynthetic route.

A non-BRICHOS surfactant protein c mutation disrupts epithelial cell function and intercellular signaling.

BACKGROUND: Heterozygous mutations of SFTPC, the gene encoding surfactant protein C (SP-C), cause sporadic and familial interstitial lung disease (ILD) in children and adults. The most frequent SFTPC mutation in ILD patients leads to a threonine for isoleucine substitution at position 73 (I73T) of the SP-C preprotein (proSP-C), however little is known about the cellular consequences of SP-CI73T expression. RESULTS: To address this, we stably expressed SP-CI73T in cultured MLE-12 alveolar epithelial cells. This resulted in increased intracellular accumulation of proSP-C processing intermediates, which matched proSP-C species recovered in bronchial lavage fluid from patients with this mutation. Exposure of SP-CI73T cells to drugs currently used empirically in ILD therapy, cyclophosphamide, azathioprine, hydroxychloroquine or methylprednisolone, enhanced expression of the chaperones HSP90, HSP70, calreticulin and calnexin. SP-CI73T mutants had decreased intracellular phosphatidylcholine level (PC) and increased lyso-PC level without appreciable changes of other phospholipids. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CI73T cells secreted into the medium soluble factors that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine influence of SP-CI73T on neighboring cells in the alveolar space. CONCLUSION: We show that I73T mutation leads to impaired processing of proSP-C in alveolar type II cells, alters their stress tolerance and surfactant lipid composition, and activates cells of the immune system. In addition, we show that some of the mentioned cellular aspects behind the disease can be modulated by application of pharmaceutical drugs commonly applied in the ILD therapy.