Genetic determinants of ammonia-induced acute lung injury in mice.

In this study, a genetically diverse panel of 43 mouse strains was exposed to ammonia, and genome-wide association mapping was performed employing a single-nucleotide polymorphism (SNP) assembly. Transcriptomic analysis was used to help resolve the genetic determinants of ammonia-induced acute lung injury. The encoded proteins were prioritized based on molecular function, nonsynonymous SNP within a functional domain or SNP within the promoter region that altered expression. This integrative functional approach revealed 14 candidate genes that included Aatf, Avil, Cep162, Hrh4, Lama3, Plcb4, and Ube2cbp, which had significant SNP associations, and Aff1, Bcar3, Cntn4, Kcnq5, Prdm10, Ptcd3, and Snx19, which had suggestive SNP associations. Of these genes, Bcar3, Cep162, Hrh4, Kcnq5, and Lama3 are particularly noteworthy and had pathophysiological roles that could be associated with acute lung injury in several ways.

Enhanced therapeutic index of an antimicrobial peptide in mice by increasing safety and activity against multidrug-resistant bacteria.

The rising prevalence of antibiotic resistance underscores the urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs) are potentially effective therapeutics that disrupt bacterial membranes regardless of resistance to traditional antibiotics. We have developed engineered cationic AMPs (eCAPs) with broad activity against multidrug-resistant (MDR) bacteria, but stability remains an important concern. Therefore, we sought to enhance the clinical utility of eCAP WLBU2 in biological matrices relevant to respiratory infection. A designed substitution of d-Val for l-Val resulted in increased resistance to protease enzymatic degradation. We observed multiple gains of functions such as higher activity against bacteria in biofilm mode of growth, significantly lower toxicity to erythrocytes and white blood cells compared to WLBU2, with increased safety in mice. Direct airway delivery revealed a therapeutic index of >140 for the selected enantiomer compared to that of <35 for WLBU2. The data warrant clinical exploration by aerosolized delivery to mitigate MDR-related respiratory infection.

Enhanced efficacy of the engineered antimicrobial peptide WLBU2 via direct airway delivery in a murine model of Pseudomonas aeruginosa pneumonia.

OBJECTIVES: Pseudomonas aeruginosa is a common cause of pneumonia in patients with cystic fibrosis with the property to generate multidrug resistance against clinically used antibiotics. Antimicrobial peptides (AMPs) are a diverse group of effector molecules of the innate immune system that protect the host against pathogens. However, the lack of activity in common biological matrices has hampered efforts towards clinical development. In this study, we evaluated the therapeutic potential of the engineered AMP WLBU2 via direct airway delivery in a murine model of P. aeruginosa infection. METHODS: The human AMPs LL37 and WLBU2 were compared for (i) antibiofilm activity using P. aeruginosa on polarized human bronchial epithelial cells, and (ii) efficacy in P. aeruginosa pneumonia in mice using intratracheal instillation of bacteria and AMPs. RESULTS: WLBU2 (16 µM) prevents biofilm formation by up to 3-log compared with 1-log reduction by LL37. With a single dose of 1 µg (0.05 mg/kg) delivered intratracheally, the initial effect of LL37 was moderate and transitory, as bacterial load and inflammatory cytokines increased at 24 h with observed signs of disease such as lethargy and hypothermia, consistent with moribund state requiring euthanasia. In sharp contrast, WLBU2 reduced bacterial burden (by 2 logs) and bacteria-induced inflammation (leucocytic infiltrates, cytokine and chemokine gene expression) at 6 h and 24 h post-exposure, with no observed signs of disease or host toxicity. CONCLUSION: These promising results now establish a much lower minimum therapeutic dose of WLBU2 (a net gain of 80-fold) compared with the previously reported 4 mg/kg systemic minimum therapeutic dose, with significant implications for clinical development.

Functional genomic assessment of phosgene-induced acute lung injury in mice.

In this study, a genetically diverse panel of 43 mouse strains was exposed to phosgene and genome-wide association mapping performed using a high-density single nucleotide polymorphism (SNP) assembly. Transcriptomic analysis was also used to improve the genetic resolution in the identification of genetic determinants of phosgene-induced acute lung injury (ALI). We prioritized the identified genes based on whether the encoded protein was previously associated with lung injury or contained a nonsynonymous SNP within a functional domain. Candidates were selected that contained a promoter SNP that could alter a putative transcription factor binding site and had variable expression by transcriptomic analyses. The latter two criteria also required that ≥10% of mice carried the minor allele and that this allele could account for ≥10% of the phenotypic difference noted between the strains at the phenotypic extremes. This integrative, functional approach revealed 14 candidate genes that included Atp1a1, Alox5, Galnt11, Hrh1, Mbd4, Phactr2, Plxnd1, Ptprt, Reln, and Zfand4, which had significant SNP associations, and Itga9, Man1a2, Mapk14, and Vwf, which had suggestive SNP associations. Of the genes with significant SNP associations, Atp1a1, Alox5, Plxnd1, Ptprt, and Zfand4 could be associated with ALI in several ways. Using a competitive electrophoretic mobility shift analysis, Atp1a1 promoter (rs215053185) oligonucleotide containing the minor G allele formed a major distinct faster-migrating complex. In addition, a gene with a suggestive SNP association, Itga9, is linked to transforming growth factor ß1 signaling, which previously has been associated with the susceptibility to ALI in mice.

Integrative assessment of chlorine-induced acute lung injury in mice.

The genetic basis for the underlying individual susceptibility to chlorine-induced acute lung injury is unknown. To uncover the genetic basis and pathophysiological processes that could provide additional homeostatic capacities during lung injury, 40 inbred murine strains were exposed to chlorine, and haplotype association mapping was performed. The identified single-nucleotide polymorphism (SNP) associations were evaluated through transcriptomic and metabolomic profiling. Using ≥ 10% allelic frequency and ≥ 10% phenotype explained as threshold criteria, promoter SNPs that could eliminate putative transcriptional factor recognition sites in candidate genes were assessed by determining transcript levels through microarray and reverse real-time PCR during chlorine exposure. The mean survival time varied by approximately 5-fold among strains, and SNP associations were identified for 13 candidate genes on chromosomes 1, 4, 5, 9, and 15. Microarrays revealed several differentially enriched pathways, including protein transport (decreased more in the sensitive C57BLKS/J lung) and protein catabolic process (increased more in the resistant C57BL/10J lung). Lung metabolomic profiling revealed 95 of the 280 metabolites measured were altered by chlorine exposure, and included alanine, which decreased more in the C57BLKS/J than in the C57BL/10J strain, and glutamine, which increased more in the C57BL/10J than in the C57BLKS/J strain. Genetic associations from haplotype mapping were strengthened by an integrated assessment using transcriptomic and metabolomic profiling. The leading candidate genes associated with increased susceptibility to acute lung injury in mice included Klf4, Sema7a, Tns1, Aacs, and a gene that encodes an amino acid carrier, Slc38a4.

Haplotype association mapping of acute lung injury in mice implicates activin a receptor, type 1.

RATIONALE: Because acute lung injury is a sporadic disease produced by heterogeneous precipitating factors, previous genetic analyses are mainly limited to candidate gene case-control studies. OBJECTIVES: To develop a genome-wide strategy in which single nucleotide polymorphism associations are assessed for functional consequences to survival during acute lung injury in mice. METHODS: To identify genes associated with acute lung injury, 40 inbred strains were exposed to acrolein and haplotype association mapping, microarray, and DNA-protein binding were assessed. MEASUREMENTS AND MAIN RESULTS: The mean survival time varied among mouse strains with polar strains differing approximately 2.5-fold. Associations were identified on chromosomes 1, 2, 4, 11, and 12. Seven genes (Acvr1, Cacnb4, Ccdc148, Galnt13, Rfwd2, Rpap2, and Tgfbr3) had single nucleotide polymorphism (SNP) associations within the gene. Because SNP associations may encompass "blocks" of associated variants, functional assessment was performed in 91 genes within ± 1 Mbp of each SNP association. Using 10% or greater allelic frequency and 10% or greater phenotype explained as threshold criteria, 16 genes were assessed by microarray and reverse real-time polymerase chain reaction. Microarray revealed several enriched pathways including transforming growth factor-ß signaling. Transcripts for Acvr1, Arhgap15, Cacybp, Rfwd2, and Tgfbr3 differed between the strains with exposure and contained SNPs that could eliminate putative transcriptional factor recognition sites. Ccdc148, Fancl, and Tnn had sequence differences that could produce an amino acid substitution. Mycn and Mgat4a had a promoter SNP or 3'untranslated region SNPs, respectively. Several genes were related and encoded receptors (ACVR1, TGFBR3), transcription factors (MYCN, possibly CCDC148), and ubiquitin-proteasome (RFWD2, FANCL, CACYBP) proteins that can modulate cell signaling. An Acvr1 SNP eliminated a putative ELK1 binding site and diminished DNA-protein binding. CONCLUSIONS: Assessment of genetic associations can be strengthened using a genetic/genomic approach. This approach identified several candidate genes, including Acvr1, associated with increased susceptibility to acute lung injury in mice.

Endothelial dysfunction and claudin 5 regulation during acrolein-induced lung injury.

An integral membrane protein, Claudin 5 (CLDN5), is a critical component of endothelial tight junctions that control pericellular permeability. Breaching of endothelial barriers is a key event in the development of pulmonary edema during acute lung injury (ALI). A major irritant in smoke, acrolein can induce ALI possibly by altering CLDN5 expression. This study sought to determine the cell signaling mechanism controlling endothelial CLDN5 expression during ALI. To assess susceptibility, 12 mouse strains were exposed to acrolein (10 ppm, 24 h), and survival monitored. Histology, lavage protein, and CLDN5 transcripts were measured in the lung of the most sensitive and resistant strains. CLDN5 transcripts and phosphorylation status of forkhead box O1 (FOXO1) and catenin (cadherin-associated protein) beta 1 (CTNNB1) proteins were determined in control and acrolein-treated human endothelial cells. Mean survival time (MST) varied more than 2-fold among strains with the susceptible (BALB/cByJ) and resistant (129X1/SvJ) strains (MST, 17.3 ± 1.9 h vs. 41.4 ± 5.1 h, respectively). Histological analysis revealed earlier perivascular enlargement in the BALB/cByJ than in 129X1/SvJ mouse lung. Lung CLDN5 transcript and protein increased more in the resistant strain than in the susceptible strain. In human endothelial cells, 30 nM acrolein increased CLDN5 transcripts and increased p-FOXO1 protein levels. The phosphatidylinositol 3-kinase inhibitor LY294002 diminished the acrolein-induced increased CLDN5 transcript. Acrolein (300 nM) decreased CLDN5 transcripts, which were accompanied by increased FOXO1 and CTNNB1. The phosphorylation status of these transcription factors was consistent with the observed CLDN5 alteration. Preservation of endothelial CLDN5 may be a novel clinical approach for ALI therapy.

Epithelial reticulon 4B (Nogo-B) is an endogenous regulator of Th2-driven lung inflammation.

Nogo-B is a member of the reticulon family of proteins (RTN-4B) that is highly expressed in lung tissue; however, its function remains unknown. We show that mice with Th2-driven lung inflammation results in a loss of Nogo expression in airway epithelium and smooth muscle compared with nonallergic mice, a finding which is replicated in severe human asthma. Mice lacking Nogo-A/B (Nogo-KO) display an exaggerated asthma-like phenotype, and epithelial reconstitution of Nogo-B in transgenic mice blunts Th2-mediated lung inflammation. Microarray analysis of lungs from Nogo-KO mice reveals a marked reduction in palate lung and nasal clone (PLUNC) gene expression, and the levels of PLUNC are enhanced in epithelial Nogo-B transgenic mice. Finally, transgenic expression of PLUNC into Nogo-KO mice rescues the enhanced asthmatic-like responsiveness in these KO mice. These data identify Nogo-B as a novel protective gene expressed in lung epithelia, and its expression regulates the levels of the antibacterial antiinflammatory protein PLUNC.

Identification of multiple MAPK-mediated transcription factors regulated by tobacco smoke in airway epithelial cells.

Activation and regulation of transcription factors (TFs) are the major mechanisms regulating changes in gene expression upon environmental exposure. Tobacco smoke (TS) is a complex mixture of chemicals, each of which could act through different signal cascades, leading to the regulation of distinct TFs and alterations in subsequent gene expression. We proposed that TS exposure affects inflammatory gene expression at the transcriptional level by modulating the DNA binding activities of TFs. To investigate transcriptional regulation upon TS exposure, a protein/DNA array was applied to screen TFs that are affected by TS exposure. This array-based screening allowed us to simultaneously detect 244 different TFs. Our results indicated that multiple TFs were rapidly activated upon TS exposure. DNA-binding activity of differentially expressed TFs was confirmed by EMSA. Our results showed that at least 20 TFs displayed more than twofold expressional changes after smoke treatment. Ten smoke-induced TFs, including NF-kappaB, VDR, ISRE, and RSRFC4, were involved in MAPK signaling pathways. The NF-kappaB family, which is involved in inflammation-induced gene activation, was selected for further study to characterize TS exposure-induced transcriptional activation. Western blot analysis and immunofluorescence microscopy indicated that TS exposure induced phosphorylation of IkappaB and translocation of NF-kappaB p65/p50 heterodimers into the nucleus. This activity was abrogated by the MAPK inhibitors PD98059 and U0126. Our results confirmed that activation of MAPK signaling pathways by TS exposure increased transcriptional activity of NF-kappaB. These data provide a potential mechanism for TS-induced inflammatory gene expression.

Characterization of human mucin 5B gene expression in airway epithelium and the genomic clone of the amino-terminal and 5'-flanking region.

Human mucin (MUC) 5B gene expression in human airway epithelium was studied in both tissue sections and cultures of tracheobronchial epithelial (TBE) cells. In situ hybridization demonstrated that MUC5B message was expressed mainly in the mucous cells of submucosal glands of normal human airway tissues. Nevertheless, an elevated MUC5B message level could be seen in surface goblet cells from patients with airway diseases and inflammation. Regardless of the airway tissue sources, MUC5B message was regulated by all-trans-retinoic acid (RA) and culture conditions in both primary and passage-1 cultures of TBE cells. MUC5B message, to a lesser extent, was also found in the immortalized epithelial cell line HBE1, but not in BEAS-2B cells. To elucidate the molecular mechanism of MUC5B gene expression, a genomic clone was obtained and sequenced for the amino terminal and the 5'-flanking region of MUC5B gene. A luciferase reporter construct containing 4,169 base pairs of the 5'-flanking region of MUC5B gene demonstrated a cell type-specific basal promoter activity in transfection studies. Both RA and the air-liquid interface culture condition further enhanced this promoter activity. These results suggest that the 5'-flanking region of MUC5B gene contains cis-elements that are potentially involved in the regulation of MUC5B gene expression.

Characterization of a novel airway epithelial cell-specific short chain alcohol dehydrogenase/reductase gene whose expression is up-regulated by retinoids and is involved in the metabolism of retinol.

Multiple retinoic acid responsive cDNAs were isolated from a high density cDNA microarray membrane, which was developed from a cDNA library of human tracheobronchial epithelial cells. Five selected cDNA clones encoded the sequence of the same novel gene. The predicted open reading frame of the novel gene encoded a protein of 319 amino acids. The deduced amino acid sequence contains four motifs that are conserved in the short-chain alcohol dehydrogenase/reductase (SDR) family of proteins. The novel gene shows the greatest homology to a group of dehydrogenases that can oxidize retinol (retinol dehydrogenases). The mRNA of the novel gene was found in trachea, colon, tongue, and esophagus. In situ hybridization of airway tissue sections demonstrated epithelial cell-specific gene expression, especially in the ciliated cell type. Both all-trans-retinoic acid and 9-cis-retinoic acid were able to elevate the expression of the novel gene in primary human tracheobronchial epithelial cells in vitro. This elevation coincided with an enhanced retinol metabolism in these cultures. COS cells transfected with an expression construct of the novel gene were also elevated in the metabolism of retinol. The results suggested that the novel gene represents a new member of the SDR family that may play a critical role in retinol metabolism in airway epithelia as well as in other epithelia of colon, tongue, and esophagus.

Distribution of HSP70, protein kinase C, and spectrin is altered in lymphocytes during a fever-like hyperthermia exposure.

Many B and T lymphocytes display a significant heterogeneity with respect to the subcellular distribution of the cytoskeletal protein spectrin and protein kinase C (PKC), both of which often can be found in a large cytoplasmic aggregate in these cell types. In addition to spectrin and PKC, we recently have reported that HSP70 is also a component of this lymphocyte aggregate. Moreover, these three proteins can undergo dynamic and reversible changes in their localization causing "assembly" of the aggregate in response to various conditions associated with lymphocyte activation, indicating that this naturally occurring aggregate structure is sensitive to activation status. We show here that the same changes in HSP70/spectrin/PKC localization induced by PKC activation also can be caused, in vitro and in vivo, by a mild hyperthermia exposure, as occurs during a natural fever (39.5-40 degrees C, 2-12 hr). This mild heat exposure also triggers the activation of PKC, a major heat shock response, and lymphocyte proliferation. The increase in PKC activity, HSP70-spectrin-PKC aggregate formation, and heat shock protein expression resulting from exposure to fever-like hyperthermia are all inhibited by calphostin C, a specific inhibitor of PKC. These data demonstrate that changes observed during lymphocyte activation could be induced by a mild hyperthermia exposure occurring during a normal febrile episode.

Isolation and characterization of nucleolin gene as one of the vitamin A-responsive genes in airway epithelium by a palindromic primer-based mRNA differential display method.

A palindromic primer-based mRNA differential display method has been used to isolate various vitamin A-responsive genes from primary cultures of monkey tracheobronchial epithelial cells. This method, as compared with the original mRNA differential display (mDD) method described by Liang and Pardee, used only one arbitrarily designed primer instead of two in the polymerase chain reaction. The single-primer mDD method has several advantages over the two-primer mDD system, especially in the reamplification and the selection of 5'-end cDNA clone. To verify the usefulness of this approach, one of these differential display bands, M34, was initially chosen for further amplification and cloning. The clone derived from the M34 band has a DNA sequence with > 90% homology to the human nucleolin gene. Furthermore, DNA sequencing confirms that both 5' and 3' ends of the insert of M34 contain the invertly repetitive nucleotide sequence that was used to direct this cloning. Nucleolin is a multifunctional phosphoprotein that plays an important role in ribosome biogenesis and mRNA stability. Northern blot analysis demonstrated that in addition to the elevation by vitamin A, the level of nucleolin message is significantly higher in fetal than in adult tracheobronchial epithelial cultures. Furthermore, in situ hybridization demonstrated that the amount of nucleolin message is significantly higher in both basal and ciliated cell types than in mucous and intermediary cell types. These results support the feasibility that the single-primer mDD technique can be used to isolate vitamin A-responsive genes with a palindromic nature.

Hsp70 translocates into a cytoplasmic aggregate during lymphocyte activation.

The percentage of T and B lymphocytes expressing a distinct cytoplasmic aggregate enriched in spectrin, ankyrin, and in several other proteins including protein kinase C greatly increases following various activation protocols. Members of the 70 kDa family of heat shock proteins (hsp70) temporarily bind to and stabilize unfolded segments of other proteins, a function apparently required for proper protein folding and assembly. Considering the multiprotein and dynamic nature of the lymphocyte aggregate, the possibility that hsp70 also might be associated with components of this structure is considered here. Double immunofluorescence analysis indicates that hsp70 is a component of the lymphocyte aggregate and is coincident with spectrin in a subpopulation of freshly isolated, untreated lymphocytes from various murine tissues and in a T-lymphocyte hybridoma. When cell lysates of lymph node T cells are immunoprecipitated using an antibody against hsp70 or spectrin and then analyzed by Western blot utilizing the alternate antibody, it was found that hsp70 and spectrin coprecipitated with one another. Moreover, this coprecipitation could be abolished by addition of ATP. This latter observation was extended to lymphoid cells using a transient permeabilization procedure, and it was shown that addition of exogenous ATP results in the dissipation of the aggregate structure itself. Finally, conditions that result in T-cell activation and aggregate formation, i.e., treatment with the phorbol ester PMA or T-cell receptor cross-linking, also lead to the repositioning of hsp70 into the aggregate from a membrane/cytosolic locale in congruence with spectrin. These data suggest that hsp70 is an active component of the aggregate and that it may function in the interactions believed to occur in this unique activation-associated organelle.

The 170-kDa glucose-regulated stress protein is an endoplasmic reticulum protein that binds immunoglobulin.

Anoxia, glucose starvation, calcium ionophore A23187, EDTA, glucosamine, and several other conditions that adversely affect the function of the endoplasmic reticulum (ER) induce the synthesis of the glucose-regulated class of stress proteins (GRPs). The primary GRPs induced by these stresses migrate at 78 and 94 kDa (GRP78 and GRP94). In addition, another protein of approximately 150-170 kDa (GRP170) has been previously observed and is coordinately induced with GRP78 and GRP94. To characterize this novel stress protein, we have prepared an antisera against purified GRP170. Immunofluorescence, Endoglycosidase H sensitivity, and protease resistance of this protein in microsomes indicates that GRP170 is an ER lumenal glycoprotein retained in a pre-Golgi compartment. Immunoprecipitation of GRP170 with our antibody coprecipitates the GRP78 (also referred to as the B cell immunoglobulin-binding protein) and GRP94 members of this stress protein family in Chinese hamster ovary cells under stress conditions. ATP depletion, by immunoprecipitation in the presence of apyrase, does not affect the interaction between GRP78 and GRP170 but results in the coprecipitation of an unidentified 60-kDa protein. In addition, GRP170 is found to be coprecipitated with immunoglobulin (Ig) in four different B cell hybridomas expressing surface IgM, cytoplasmic Ig light chain only, cytoplasmic Ig heavy chain only, or an antigen specific secreted IgG. In addition, in IgM surface expressing WEHI-231 B cells, anti-IgM coprecipitates GRP78, GRP94, as well as GRP170; antibodies against GRP170 and GRP94 reciprocally coprecipitate GRP94/GRP170 as well as GRP78. Results suggest that this 170-kDa GRP is a retained ER lumenal glycoprotein that is constitutively present and that may play a role in immunoglobulin folding and assembly in conjunction or consecutively with GRP78 and GRP94.