Aisha Nair

Essential Roles for Early Growth Response Transcription Factor Egr-1 in Tissue Fibrosis and Wound Healing

The early growth response gene (Egr-1) codes for a zinc finger transcription factor that has important roles in the regulation of cell growth, differentiation, and survival. Aberrant Egr-1 expression is implicated in carcinogenesis, inflammation, atherosclerosis, and ischemic injury. We reported previously that normal fibroblasts stimulated by transforming growth factor-ss showed rapid and transient induction of Egr-1. Moreover, we observed that tissue expression of Egr-1 was elevated in patients with scleroderma, which suggests that Egr-1 may be involved in tissue repair and fibrosis. Here, we investigated matrix remodeling and wound healing in mice harboring gain of function or loss of function mutations of Egr-1. Using the model of bleomycin-induced scleroderma, we found that the early influx of inflammatory cells into the skin and lungs, and the subsequent development of fibrosis in these organs, were markedly attenuated in Egr-1 null mice. Furthermore, full-thickness incisional skin wound healing was impaired, and skin fibroblasts lacking Egr-1 showed reduced migration and myofibroblast transdifferentiation in vitro. In contrast, transgenic mice with fibroblast-specific Egr-1 overexpression showed exuberant tissue repair, with enhanced collagen accumulation and increased tensile strength of incisional wounds. Together, these results point to the fundamental role that Egr-1 plays in the regulation of transforming growth factor-ss-dependent physiological and pathological matrix remodeling.

Hypercapnia impairs lung neutrophil function and increases mortality in murine Pseudomonas pneumonia

Hypercapnia, an elevation of the level of carbon dioxide (CO2) in blood and tissues, is a marker of poor prognosis in chronic obstructive pulmonary disease and other pulmonary disorders. We previously reported that hypercapnia inhibits the expression of TNF and IL-6 and phagocytosis in macrophages in vitro. In the present study, we determined the effects of normoxic hypercapnia (10% CO2, 21% O2, and 69% N2) on outcomes of Pseudomonas aeruginosa pneumonia in BALB/c mice and on pulmonary neutrophil function. We found that the mortality of P. aeruginosa pneumonia was increased in 10% CO2-exposed compared with air-exposed mice. Hypercapnia increased pneumonia mortality similarly in mice with acute and chronic respiratory acidosis, indicating an effect unrelated to the degree of acidosis. Exposure to 10% CO2 increased the burden of P. aeruginosa in the lungs, spleen, and liver, but did not alter lung injury attributable to pneumonia. Hypercapnia did not reduce pulmonary neutrophil recruitment during infection, but alveolar neutrophils from 10% CO2-exposed mice phagocytosed fewer bacteria and produced less H2O2 than neutrophils from air-exposed mice. Secretion of IL-6 and TNF in the lungs of 10% CO2-exposed mice was decreased 7 hours, but not 15 hours, after the onset of pneumonia, indicating that hypercapnia inhibited the early cytokine response to infection. The increase in pneumonia mortality caused by elevated CO2 was reversible when hypercapnic mice were returned to breathing air before or immediately after infection. These results suggest that hypercapnia may increase the susceptibility to and/or worsen the outcome of lung infections in patients with severe lung disease.

A mechanism of benefit of soy genistein in asthma: inhibition of eosinophil p38‐dependent leukotriene synthesis

Background Dietary intake of the soy isoflavone genistein is associated with reduced severity of asthma, but the mechanisms responsible for this effect are unknown.

Hypercapnia Inhibits Autophagy and Bacterial Killing in Human Macrophages by Increasing Expression of Bcl-2 and Bcl-xL

Hypercapnia, the elevation of CO2 in blood and tissue, commonly develops in patients with advanced lung disease and severe pulmonary infections, and it is associated with high mortality. We previously reported that hypercapnia alters expression of host defense genes, inhibits phagocytosis, and increases the mortality of Pseudomonas pneumonia in mice. However, the effect of hypercapnia on autophagy, a conserved process by which cells sequester and degrade proteins and damaged organelles that also plays a key role in antimicrobial host defense and pathogen clearance, has not previously been examined. In the present study we show that hypercapnia inhibits autophagy induced by starvation, rapamycin, LPS, heat-killed bacteria, and live bacteria in the human macrophage. Inhibition of autophagy by elevated CO2 was not attributable to acidosis. Hypercapnia also reduced macrophage killing of Pseudomonas aeruginosa. Moreover, elevated CO2 induced the expression of Bcl-2 and Bcl-xL, antiapoptotic factors that negatively regulate autophagy by blocking Beclin 1, an essential component of the autophagy initiation complex. Furthermore, small interfering RNA targeting Bcl-2 and Bcl-xL and the small molecule Z36, which blocks Bcl-2 and Bcl-xL binding to Beclin 1, prevented hypercapnic inhibition of autophagy and bacterial killing. These results suggest that targeting the Bcl-2/Bcl-xL–Beclin 1 interaction may hold promise for ameliorating hypercapnia-induced immunosuppression and improving resistance to infection in patients with advanced lung disease and hypercapnia.

Focused Screening Identifies Evoxine as a Small Molecule That Counteracts CO2-Induced Immune Suppression

Patients with severe lung disease may develop hypercapnia, elevation of the levels of CO2 in the lungs and blood, which is associated with increased risk of death, often from infection. To identify compounds that ameliorate the adverse effects of hypercapnia, we performed a focused screen of 8832 compounds using a CO2-responsive luciferase reporter in Drosophila S2* cells. We found that evoxine, a plant alkaloid, counteracts the CO2-induced transcriptional suppression of antimicrobial peptides in S2* cells. Strikingly, evoxine also inhibits hypercapnic suppression of interleukin-6 and the chemokine CCL2 expression in human THP-1 macrophages. Evoxine’s effects are selective, since it does not prevent hypercapnic inhibition of phagocytosis by THP-1 cells or CO2-induced activation of AMPK in rat ATII pulmonary epithelial cells. The results suggest that hypercapnia suppresses innate immune gene expression by definable pathways that are evolutionarily conserved and demonstrate for the first time that specific CO2 effects can be targeted pharmacologically.

A role for heat shock factor 1 in hypercapnia-induced inhibition of inflammatory cytokine expression

Hypercapnia, elevated levels of CO2 in the blood, is a known marker for poor clinical prognosis and is associated with increased mortality in patients hospitalized with both bacterial and viral pneumonias. Although studies have established a connection between elevated CO2 levels and poor pneumonia outcomes, a mechanistic basis of this association has not yet been established. We previously reported that hypercapnia inhibits expression of key NF‐κB‐regulated, innate immune cytokines, TNF‐α, and IL‐6, in LPS‐stimulated macrophages in vitro and in mice during Pseudomonas pneumonia. The transcription factor heat shock factor 1 (HSF1) is important in maintaining proteostasis during stress and has been shown to negatively regulate NF‐κB activity. In this study, we tested the hypothesis that HSF1 activation in response to hypercapnia results in attenuated NF‐κB‐regulated gene expression. We found that hypercapnia induced the protein expression and nuclear accumulation of HSF1 in primary murine alveolar macrophages and in an alveolar macrophage cell line (MH‐S). In MH‐S cells treated with short interfering RNA targeting Hsf1, LPS‐induced IL‐6 and TNF‐α release were elevated during exposure to hypercapnia. Pseudomonas‐infected Hsf1+/+ (wild‐type) mice, maintained in a hypercapnic environment, showed lower levels of IL‐6 and TNF‐α in bronchoalveolar lavage fluid and IL‐1β in lung tissue than did infected mice maintained in room air. In contrast, infected Hsf1+/• mice exposed to either hypercapnia or room air had similarly elevated levels of those cytokines. These results suggest that hypercapnia‐mediated inhibition of NF‐κB cytokine production is dependent on HSF1 expression and/or activation.—Lu, Z., Casalino‐Matsuda, S. M., Nair, A., Buchbinder, A., Budinger, G. R. S., Sporn, P. H. S., Gates, K. L. A role for heat shock factor 1 in hypercapnia‐induced inhibition of inflammatory cytokine expression. FASEB J. 32, 3614–3622 (2018). www.fasebj.org

Hypercapnia Alters Expression of Immune Response, Nucleosome Assembly and Lipid Metabolism Genes in Differentiated Human Bronchial Epithelial Cells

Hypercapnia, the elevation of CO2 in blood and tissues, commonly occurs in severe acute and chronic respiratory diseases, and is associated with increased risk of mortality. Recent studies have shown that hypercapnia adversely affects innate immunity, host defense, lung edema clearance and cell proliferation. Airway epithelial dysfunction is a feature of advanced lung disease, but the effect of hypercapnia on airway epithelium is unknown. Thus, in the current study we examined the effect of normoxic hypercapnia (20% CO2 for 24 h) vs normocapnia (5% CO2), on global gene expression in differentiated normal human airway epithelial cells. Gene expression was assessed on Affymetrix microarrays, and subjected to gene ontology analysis for biological process and cluster-network representation. We found that hypercapnia downregulated the expression of 183 genes and upregulated 126. Among these, major gene clusters linked to immune responses and nucleosome assembly were largely downregulated, while lipid metabolism genes were largely upregulated. The overwhelming majority of these genes were not previously known to be regulated by CO2. These changes in gene expression indicate the potential for hypercapnia to impact bronchial epithelial cell function in ways that may contribute to poor clinical outcomes in patients with severe acute or advanced chronic lung diseases.

72 MAST CELLS AND EOSINOPHILS STIMULATE MYOFIBROBLAST DIFFERENTIATION BY SIGNALING THROUGH THE TRANSFORMING GROWTH FACTOR b TYPE I (ALK5) RECEPTOR.

Rationale Mast cells and eosinophils synthesize various mediators, including transforming growth factor b (TGF-b) and cysteinyl leukotrienes (cysLTs), that may promote subepithelial fibrosis in asthma. We sought to determine the roles of TGF-b and cysLTs in myofibroblast differentiation stimulated by coculture of fibroblasts with mast cells or eosinophils. Methods IMR-90 human lung fibroblasts were serum starved and cocultured for 48 hours with either LAD-2 human mast cells or freshly isolated human blood eosinophils in the absence or presence of SB431542 (10 M) to block signaling through the TGF-b type I (ALK5) receptor; MK886 (1 M) to block leukotriene synthesis; MK571 (100 nM) to block the cysLT1 receptor; or Bay u9773 (3 M) to block the cysLT1 and cysLT2 receptors. a-Smooth muscle actin (a-SMA) expression was assessed as an index of myofibroblast differentiation. Results Mast cells and eosinophils stimulated 2.50.2 (n = 5, p < .005)- and 1.80.2 (n = 5, p < .0001)-fold increases, respectively, in fibroblast a-SMA expression, determined by immunoblot analysis. In comparison, TGF-b (2.5 ng/mL) increased a-SMA expression by 5.50.5-fold (n = 6, p < .0001). SB431542 inhibited the increase in a-SMA expression stimulated by mast cells, eosinophils and TGF-b by 6,019%, 8,221% and 9,210%, respectively (all p < .0001). By contrast, none of the leukotriene pathway inhibitors significantly affected a-SMA expression. Immunofluorescence microscopy for a-SMA confirmed the immunoblot results. Conclusion: Our data indicate that mast cells and eosinophils stimulate differentiation of myofibroblasts by TGF-b signaling through the ALK5 receptor and that cysLTs are less important in this process. Inhibition of ALK5 receptor signaling may be a useful strategy to interrupt airway remodeling in asthma. Funded by NIH R01HL072891, Crane Asthma Center of Northwestern University.

44 AMBIENT PARTICULATE MATTER INDUCES MYOFIBROBLAST DIFFERENTIATION VIA MACROPHAGE-DEPENDENT TRANSFORMING GROWTH FACTOR b TYPE I (ALK5) SIGNALING.

Rationale Increased levels of ambient particulate matter have been associated with increased pulmonary morbidity and mortality. To investigate if particulate matter induces airway remodeling, we studied the effects of particulate matter (< 10 μm in diameter) collected from Dusseldorf, Germany (DPM), on fibroblast to myofibroblast differentiation. Methods Human fetal lung fibroblasts (IMR-90) were grown to subconfluence, serum-starved for 48 hours, and exposed to either TGF-b1 (2 ng/mL), DPM, or conditioned medium from human monocytic (THP-1) cells exposed to DPM. The role of TGF-b1 signaling was assessed by the addition of SB431542 (10 μM), a TGF-b type I (ALK5) receptor inhibitor. After 48 hours, cells were lysed and analyzed by immunoblot for a-smooth muscle actin (aSMA), a marker of myofibroblast differentiation. Results Direct exposure of IMR-90 cells to DPM did not increase aSMA expression. However, conditioned medium of THP-1 cells exposed to DPM induced increased aSMA expression in fibroblasts. This increase was blocked by the ALK5 inhibitor SB431542. Conclusions Ambient particulate matter triggers macrophage-dependent induction of myofibroblast differentiation via ALK5 receptor signaling. We speculate that particulate matter induces airway remodeling.

65 THE SOY ISOFLAVONE GENISTEIN BLOCKS TRANSFORMING GROWTH FACTOR b1-STIMULATED LUNG FIBROBLAST TO MYOFIBROBLAST TRANSFORMATION.

Rationale Genistein is a dietary isoflavone and a broad-spectrum tyrosine kinase inhibitor contained in soy products. An epidemiologic study of asthma revealed that subjects with high consumption of dietary soy isoflavones had better lung function than those with lower intake. In a guinea pig model of allergic asthma, genistein reduced methacholine-induced bronchoconstriction. To explore the mechanisms underlying these observations, we tested genisteins ability to block lung myofibroblast differentiation, a key phenotypic change in asthmatic airway remodeling. Methods Human fetal lung fibroblasts (IMR-90) were grown to subconfluence in DMEM containing 10% FBS, serum-deprived for 24 hours, and treated with genistein (10 μM) for an additional 24 hurs. Cells were then stimulated with TGF-b1 (2 ng/mL) for 24 hours, and a-smooth muscle actin (aSMA) expression, a marker of the myofibroblast phenotype, was assessed by both immunoblot and immunofluorescence microscopy. To determine the intracellular mechanism of inhibitory actions by genistein, we also assessed phosphorylation of Smad2 by immunoblot in the presence and absence of genistein after 30-minute stimulation with TGF-b1. Results Treatment with genistein resulted in 54.9 ± 17.1% reduction in TGF-b1 induced expression of aSMA. Immunofluorescence microscopy revealed a decrease in aSMA staining intensity and stress fiber formation in genistein-treated cells. In addition, Smad2 phosphorylation was inhibited by 71.1 ± 16.7% in genistein-treated cells. Conclusions These results demonstrate that genistein interferes with TGF-b1-stimulated myofibroblast differentiation and suggest that tyrosine kinase inhibition may have a role in modulation of asthmatic airway remodeling. Funded by NIH RO1HL072891, T32HL076139, The CHEST Foundation-GSK Clinical Research Trainee Award.