Michael F. Beers

Endoplasmic reticulum stress enhances fibrotic remodeling in the lungs

Evidence of endoplasmic reticulum (ER) stress has been found in lungs of patients with familial and sporadic idiopathic pulmonary fibrosis. We tested whether ER stress causes or exacerbates lung fibrosis by (i) conditional expression of a mutant form of surfactant protein C (L188Q SFTPC) found in familial interstitial pneumonia and (ii) intratracheal treatment with the protein misfolding agent tunicamycin. We developed transgenic mice expressing L188Q SFTPC exclusively in type II alveolar epithelium by using the Tet-On system. Expression of L188Q SFTPC induced ER stress, as determined by increased expression of heavy-chain Ig binding protein (BiP) and splicing of X-box binding protein 1 (XBP1) mRNA, but no lung fibrosis was identified in the absence of a second profibrotic stimulus. After intratracheal bleomycin, L188Q SFTPC-expressing mice developed exaggerated lung fibrosis and reduced static lung compliance compared with controls. Bleomycin-treated L188Q SFTPC mice also demonstrated increased apoptosis of alveolar epithelial cells and greater numbers of fibroblasts in the lungs. With a complementary model, intratracheal tunicamycin treatment failed to induce lung remodeling yet resulted in augmentation of bleomycin-induced fibrosis. These data support the concept that ER stress produces a dysfunctional epithelial cell phenotype that facilitates fibrotic remodeling. ER stress pathways may serve as important therapeutic targets in idiopathic pulmonary fibrosis.

The three R’s of lung health and disease: repair, remodeling, and regeneration

All tissues and organs can be classified according to their ability to repair and regenerate during adult homeostasis and after injury. Some exhibit a high rate of constant cell turnover, while others, such as the lung, exhibit only low-level cell regeneration during normal adult homeostasis but have the ability to rapidly regenerate new cells after injury. Lung regeneration likely involves both activation of progenitor cells as well as cell replacement through proliferation of remaining undamaged cells. The pathways and factors that control this process and its role in disease are only now being explored. In this Review, we will discuss the connection between pathways required for lung development and how the lung responds to injury and disease, with a particular emphasis on recent studies describing the role for the epithelium in repair and regeneration.

Interstitial lung disease in a baby with a de novo mutation in the SFTPC gene

Mutations in the surfactant protein C gene (SFTPC) were recently reported in patients with interstitial lung disease. In a 13‐month-old infant with severe respiratory insufficiency, a lung biopsy elicited combined histological patterns of nonspecific interstitial pneumonia and pulmonary alveolar proteinosis. Immunohistochemical and biochemical analyses showed an intra-alveolar accumulation of surfactant protein (SP)‐A, precursors of SP‐B, mature SP‐B, aberrantly processed proSP‐C, as well as mono- and dimeric SP‐C. Sequencing of genomic DNA detected a de novo heterozygous missense mutation of the SFTPC gene (g.1286T>C) resulting in a substitution of threonine for isoleucine (I73T) in the C‐terminal propeptide. At the ultrastructural level, abnormal transport vesicles were detected in type‐II pneumocytes. Fusion proteins, consisting of enhanced green fluorescent protein and wild-type or mutant proSP‐C, were used to evaluate protein trafficking in vitro. In contrast to wild-type proSP‐C, mutant proSP‐C was routed to early endosomes when transfected into A549 epithelial cells. In contrast to previously reported mutations, the I73T represents a new class of surfactant protein C gene mutations, which is marked by a distinct trafficking, processing, palmitoylation, and secretion of the mutant and wild-type surfactant protein C. This report heralds the emerging diversity of phenotypes associated with the expression of mutant surfactant C proteins.

S-nitrosylation of surfactant protein-D controls inflammatory function.

The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D) have been implicated in the regulation of the innate immune system within the lung. In particular, SP-D appears to have both pro- and anti-inflammatory signaling functions. At present, the molecular mechanisms involved in switching between these functions remain unclear. SP-D differs in its quaternary structure from SP-A and the other members of the collectin family, such as C1q, in that it forms large multimers held together by the N-terminal domain, rather than aligning the triple helix domains in the traditional “bunch of flowers” arrangement. There are two cysteine residues within the hydrophobic N terminus of SP-D that are critical for multimer assembly and have been proposed to be involved in stabilizing disulfide bonds. Here we show that these cysteines exist within the reduced state in dodecameric SP-D and form a specific target for S-nitrosylation both in vitro and by endogenous, pulmonary derived nitric oxide (NO) within a rodent acute lung injury model. S-nitrosylation is becoming increasingly recognized as an important post-translational modification with signaling consequences. The formation of S-nitrosothiol (SNO)-SP-D both in vivo and in vitro results in a disruption of SP-D multimers such that trimers become evident. SNO-SP-D but not SP-D, either dodecameric or trimeric, is chemoattractive for macrophages and induces p38 MAPK phosphorylation. The signaling capacity of SNO-SP-D appears to be mediated by binding to calreticulin/CD91. We propose that NO controls the dichotomous nature of this pulmonary collectin and that posttranslational modification by S-nitrosylation causes quaternary structural alterations in SP-D, causing it to switch its inflammatory signaling role. This represents new insight into both the regulation of protein function by S-nitrosylation and NOs role in innate immunity.

Deletion of exon 4 from human surfactant protein C results in aggresome formation and generation of a dominant negative.

Human surfactant protein C (hSP-C) is synthesized by the alveolar type 2 cell as a 197 amino acid integral membrane proprotein and proteolytically processed to a secreted 3.7 kDa mature form. Although the SP-C null mouse possesses a non-lethal phenotype, a heterozygous substitution of A for G in the first base of intron 4 of the human SP-C gene (c.460+1A>G) has been reported in association with familial interstitial lung disease and absence of mature protein. This mutation produces a splice deletion of exon 4 (ΔExon4) resulting in removal of a positionally conserved cysteine in the C-terminal flanking propeptide. Based on a prior study showing that an identical deletion in the rat isoform diverted mutant protein to stable aggregates, we hypothesized that expression of the ΔExon4 mutation would result in disruption of intracellular trafficking of both mutant and wild-type proSP-C. We tested this in vitro using fusion proteins of EGFP conjugated either to wild-type SP-C (EGFP/hSP-C1-197) or to SP-C deleted of Exon4 (EGFP/hSP-CΔExon4). Fluorescence microscopy showed that EGFP/hSP-C1-197 transfected into A549 cells was expressed in a punctuate pattern in CD63 (+) cytoplasmic vesicles, whereas EGFP/hSP-CΔExon4 accumulated in ubiquitinated perinuclear inclusions linked to the microtubule organizing center. A similar juxtanuclear pattern was observed following transfection of SP-C cDNA lacking only cysteine residues in the C-terminal propeptide encoded by Exon 4 (EGFP/hSP-CC120/121G). To evaluate whether mutant proSP-C could function as a dominant negative, EGFP/hSP-CΔExon4 was cotransfected with HA-tagged hSP-C1-197 and resulted in the restriction of both forms to perinuclear compartments. Addition of Na+ 4-phenylbutyrate, a facilitator of trafficking of other misfolded proteins, attenuated the aggregation of EGFP/hSP-CΔExon4. We conclude that c.460+1A>G mutation of human SP-C results in disruption of disulfide-mediated folding encoded by Exon 4 leading to diversion of unprocessed proSP-C to aggresomes. The heterotypic oligomerization of hSP-C1-197 and hSP-CΔExon4 provides a molecular mechanism for the dominant-negative effect observed in vivo.

Role of Endoplasmic Reticulum Stress in Epithelial–Mesenchymal Transition of Alveolar Epithelial Cells: Effects of Misfolded Surfactant Protein

Endoplasmic reticulum (ER) stress has been implicated in alveolar epithelial type II (AT2) cell apoptosis in idiopathic pulmonary fibrosis. We hypothesized that ER stress (either chemically induced or due to accumulation of misfolded proteins) is also associated with epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). ER stress inducers, thapsigargin (TG) or tunicamycin (TN), increased expression of ER chaperone, Grp78, and spliced X-box binding protein 1, decreased epithelial markers, E-cadherin and zonula occludens-1 (ZO-1), increased the myofibroblast marker, α-smooth muscle actin (α-SMA), and induced fibroblast-like morphology in both primary AECs and the AT2 cell line, RLE-6TN, consistent with EMT. Overexpression of the surfactant protein (SP)-C BRICHOS mutant SP-C(ΔExon4) in A549 cells increased Grp78 and α-SMA and disrupted ZO-1 distribution, and, in primary AECs, SP-C(ΔExon4) induced fibroblastic-like morphology, decreased ZO-1 and E-cadherin and increased α-SMA, mechanistically linking ER stress associated with mutant SP to fibrosis through EMT. Whereas EMT was evident at lower concentrations of TG or TN, higher concentrations caused apoptosis. The Src inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4]pyramidine) (PP2), abrogated EMT associated with TN or TG in primary AECs, whereas overexpression of SP-C(ΔExon4) increased Src phosphorylation, suggesting a common mechanism. Furthermore, increased Grp78 immunoreactivity was observed in AT2 cells of mice after bleomycin injury, supporting a role for ER stress in epithelial abnormalities in fibrosis in vivo. These results demonstrate that ER stress induces EMT in AECs, at least in part through Src-dependent pathways, suggesting a novel role for ER stress in fibroblast accumulation in pulmonary fibrosis.

ANGPT2 Genetic Variant Is Associated with Trauma-associated Acute Lung Injury and Altered Plasma Angiopoietin-2 Isoform Ratio

RATIONALE Acute lung injury (ALI) acts as a complex genetic trait, yet its genetic risk factors remain incompletely understood. Large-scale genotyping has not previously been reported for ALI. OBJECTIVES To identify ALI risk variants after major trauma using a large-scale candidate gene approach. METHODS We performed a two-stage genetic association study. We derived findings in an African American cohort (n = 222) using a cardiopulmonary disease-centric 50K single nucleotide polymorphism (SNP) array. Genotype and haplotype distributions were compared between subjects with ALI and without ALI, with adjustment for clinical factors. Top performing SNPs (P < 10(-4)) were tested in a multicenter European American trauma-associated ALI case-control population (n = 600 ALI; n = 2,266 population-based control subjects) for replication. The ALI-associated genomic region was sequenced, analyzed for in silico prediction of function, and plasma was assayed by ELISA and immunoblot. MEASUREMENTS AND MAIN RESULTS Five SNPs demonstrated a significant association with ALI after adjustment for covariates in Stage I. Two SNPs in ANGPT2 (rs1868554 and rs2442598) replicated their significant association with ALI in Stage II. rs1868554 was robust to multiple comparison correction: odds ratio 1.22 (1.06-1.40), P = 0.0047. Resequencing identified predicted novel splice sites in linkage disequilibrium with rs1868554, and immunoblots showed higher proportion of variant angiopoietin-2 (ANG2) isoform associated with rs1868554T (0.81 vs. 0.48; P = 0.038). CONCLUSIONS An ANGPT2 region is associated with both ALI and variation in plasma angiopoietin-2 isoforms. Characterization of the variant isoform and its genetic regulation may yield important insights about ALI pathogenesis and susceptibility.

Nonspecific Interstitial Pneumonia, Alveolar Proteinosis, and Abnormal Proprotein Trafficking Resulting from a Spontaneous Mutation in the Surfactant Protein C Gene

Human surfactant protein C (hSP-C1–197) is synthesized as a 197 amino acid proprotein and cleaved to a mature 3.7 kD form. Although interstitial lung disease in patients with mutations of the hSP-C gene is becoming increasingly recognized, the mechanisms linking molecular events with clinical pathogenesis are not fully defined. We describe a full-term infant with respiratory insufficiency associated with a spontaneous heterozygous mutation resulting in a substitution of lysine for glutamic acid at position 66 (= E66K) of the proximal hSP-C COOH flanking propeptide. Lung histology and biochemical studies of the index patient (hSP-CE66K) revealed nonspecific interstitial pneumonia, increased alveolar total phospholipid lacking phosphatidylglycerol, and increased surfactant protein A. Localization of proSP-C from lung sections prepared from this patient using immunofluorescence and immunogold electron microscopy revealed abnormal proSP-C staining in endosomal-like vesicles of type II cells distinct from SP-B. To evaluate the effect of the E66K substitution on intracellular trafficking of proSP-C, fusion proteins consisting of enhanced green fluorescent protein (EGFP) and hSP-C1–197 (wild type) or mutant hSP-CE66K were generated and transfected into A549 cells. EGFP/hSP-C1–197 was expressed within CD-63-positive, EEA-1-negative vesicles, whereas EGFP/hSP-CE66K localized to EEA-1 positive vesicles. The E66K substitution is representative of a new class of SP-C mutation associated with interstitial lung disease that is diverted from the normal biosynthetic pathway. We propose that, similar to other storage disorders, lung injury results from induction of a toxic gain of function induced by the mutant product that is subject to genetic modifiers and environmental influences.

Comprehensive characterisation of pulmonary and serum surfactant protein D in COPD

BackgroundPulmonary surfactant protein D (SP-D) is considered as a candidate biomarker for the functional integrity of the lung and for disease progression, which can be detected in serum. The origin of SP-D in serum and how serum concentrations are related to pulmonary concentrations under inflammatory conditions is still unclear.MethodsIn a cross-sectional study comprising non-smokers (n = 10), young - (n = 10), elderly smokers (n = 20), and smokers with COPD (n = 20) we simultaneously analysed pulmonary and serum SP-D levels with regard to pulmonary function, exercise, repeatability and its quaternary structure by native gel electrophoresis. Statistical comparisons were conducted by ANOVA and post-hoc testing for multiple comparisons; repeatability was assessed by Bland-Altman analysis.ResultsIn COPD, median (IQR) pulmonary SP-D levels were lower (129(68) ng/ml) compared to smokers (young: 299(190), elderly: 296(158) ng/ml; p < 0.01) and non-smokers (967(708) ng/ml; p < 0.001). The opposite was observed in serum, with higher concentrations in COPD (140(89) ng/ml) as compared to non-smokers (76(47) ng/ml; p < 0.01). SP-D levels were reproducible and correlated with the degree of airway obstruction in all smokers. In addition, smoking lead to disruption of the quaternary structure.ConclusionsPulmonary and serum SP-D levels are stable markers influenced by smoking and related to airflow obstruction and disease state. Smaller subunits of pulmonary SP-D and the rapid increase of serum SP-D levels in COPD due to exercise support the translocation hypothesis and its use as a COPD biomarker.Trial registrationno interventional trial

IL-4 and IL-13 Form a Negative Feedback Circuit with Surfactant Protein-D in the Allergic Airway Response

The innate immune molecule surfactant protein-D (SP-D) plays an important regulatory role in the allergic airway response. In this study, we demonstrate that mice sensitized and challenged with either Aspergillus fumigatus (Af) or OVA have increased SP-D levels in their lung. SP-D mRNA and protein levels in the lung also increased in response to either rIL-4 or rIL-13 treatment. Type II alveolar epithelial cell expression of IL-4Rs in mice sensitized and challenged with Af, and in vitro induction of SP-D mRNA and protein by IL-4 and IL-13, but not IFN-γ, suggested a direct role of IL-4R-mediated events. The regulatory function of IL-4 and IL-13 was further supported in STAT-6-deficient mice as well as in IL-4/IL-13 double knockout mice that failed to increase SP-D production upon allergen challenge. Interestingly, addition of rSP-D significantly inhibited Af-driven Th2 cell activation in vitro whereas mice lacking SP-D had increased numbers of CD4+ cells with elevated IL-13 and thymus- and activation-regulated chemokine levels in the lung and showed exaggerated production of IgE and IgG1 following allergic sensitization. We propose that allergen exposure induces elevation in SP-D protein levels in an IL-4/IL-13-dependent manner, which in turn, prevents further activation of sensitized T cells. This negative feedback regulatory circuit could be essential in protecting the airways from inflammatory damage after allergen inhalation.