Epigenetics and Cyclooxygenase-2 Mediate Dysfunction in Alveolar Macrophages and Polymorphonuclear Neutrophils Post-bone Marrow Transplantation.

Infectious pulmonary complications limit the success of hematopoietic stem cell transplant (HSCT) therapy in both autologous and allogeneic patients. Susceptibility to pathogens, like Pseudomonas aeruginosa and Staphylococcus aureus, persists despite successful immune reconstitution. Despite high incidence of infectious pulmonary complications following HSCT, relatively little is known about the mechanisms promoting enhanced susceptibility. Alveolar macrophages (AMs) are the sentinel phagocytes in the lung and following infection polymorphonuclear neutrophils (PMNs) assist in bacterial clearance. Previous human studies implicate AM and PMN impairment post-HSCT. Using a murine syngeneic or allogeneic bone marrow transplant (BMT) model, our studies explore the mechanisms involved in promoting HSCT AM and PMN defects. We show that syngeneic BMT mice display increased susceptibility to both P. aeruginosa and S. aureus, which correlated with impaired AM function. Altered class A scavenger receptors impaired AM uptake of P. aeruginosa but not S. aureus, while defective bacterial killing conferred overall susceptibility to these pathogens. Syngeneic BMT AM susceptibility is promoted by upregulation of prostaglandin E2 (PGE2) and its rate-limiting enzyme, cyclooxygenase-2 (COX)-2. Studies exploring the etiology of enhanced COX-2 expression revealed a loss in DNA methylation of the COX-2 promoter mediated by transforming growth factor (TGF)-ß-induced miRNA-29b, resulting in elevated COX-2. Previous data show COX-2/PGE2 impaired PMN bacterial killing but had no effect on phagocytosis post-syngeneic BMT. Here we show that upregulation of COX-2/ PGE2 inhibits PMN extracellular trap (NET) formation post-syngeneic and allogeneic BMT, a novel finding that identifies PGE2 as a physiologically relevant inhibitor of NETosis. Together, these findings highlight the importance of epigenetic changes (DNA methylation and miRNA) in BMT AMs, as they directly and indirectly result in increased COX-2 expression and PGE2 production. Upregulation of this pathway establishes an immunosuppressive environment in the lung through the inhibition of AM and PMN functions. Moreover, these findings identify potential therapeutic avenues to further explore in HSCT patients.PHDImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/110388/1/rdomingo_1.pd

S100A8/A9 regulates CD11b expression and neutrophil recruitment during chronic tuberculosis

Neutrophil accumulation is associated with lung pathology during active tuberculosis (ATB). However, the molecular mechanism or mechanisms by which neutrophils accumulate in the lung and contribute to TB immunopathology are not fully delineated. Using the well-established mouse model of TB, our new data provide evidence that the alarmin S100A8/A9 mediates neutrophil accumulation during progression to chronic TB. Depletion of neutrophils or S100A8/A9 deficiency resulted in improved Mycobacterium tuberculosis (Mtb) control during chronic but not acute TB. Mechanistically, we demonstrate that, following Mtb infection, S100A8/A9 expression is required for upregulation of the integrin molecule CD11b specifically on neutrophils, mediating their accumulation during chronic TB disease. These findings are further substantiated by increased expression of S100A8 and S100A9 mRNA in whole blood in human TB progressors when compared with nonprogressors and rapidly decreased S100A8/A9 protein levels in the serum upon TB treatment. Furthermore, we demonstrate that S100A8/A9 serum levels along with chemokines are useful in distinguishing between ATB and asymptomatic Mtb-infected latent individuals. Thus, our results support targeting S100A8/A9 pathways as host-directed therapy for TB

Interleukin-17 limits hypoxia-inducible factor 1α and development of hypoxic granulomas during tuberculosis

Mycobacterium tuberculosis (Mtb) is a global health threat, compounded by the emergence of drug-resistant strains. A hallmark of pulmonary tuberculosis (TB) is the formation of hypoxic necrotic granulomas, which upon disintegration, release infectious Mtb. Furthermore, hypoxic necrotic granulomas are associated with increased disease severity and provide a niche for drug-resistant Mtb. However, the host immune responses that promote the development of hypoxic TB granulomas are not well described. Using a necrotic Mtb mouse model, we show that loss of Mtb virulence factors, such as phenolic glycolipids, decreases the production of the proinflammatory cytokine IL-17 (also referred to as IL-17A). IL-17 production negatively regulates the development of hypoxic TB granulomas by limiting the expression of the transcription factor hypoxia-inducible factor 1α (HIF1α). In human TB patients, HIF1α mRNA expression is increased. Through genotyping and association analyses in human samples, we identified a link between the single nucleotide polymorphism rs2275913 in the IL-17 promoter (-197G/G), which is associated with decreased IL-17 production upon stimulation with Mtb cell wall. Together, our data highlight a potentially novel role for IL-17 in limiting the development of hypoxic necrotic granulomas and reducing disease severity in TB

Hyperoxia prevents the dynamic neonatal increases in lung mesenchymal cell diversity

Abstract Rapid expansion of the pulmonary microvasculature through angiogenesis drives alveolarization, the final stage of lung development that occurs postnatally and dramatically increases lung gas-exchange surface area. Disruption of pulmonary angiogenesis induces long-term structural and physiologic lung abnormalities, including bronchopulmonary dysplasia, a disease characterized by compromised alveolarization. Although endothelial cells are primary determinants of pulmonary angiogenesis, mesenchymal cells (MC) play a critical and dual role in angiogenesis and alveolarization. Therefore, we performed single cell transcriptomics and in-situ imaging of the developing lung to profile mesenchymal cells during alveolarization and in the context of lung injury. Specific mesenchymal cell subtypes were present at birth with increasing diversity during alveolarization even while expressing a distinct transcriptomic profile from more mature correlates. Hyperoxia arrested the transcriptomic progression of the MC, revealed differential cell subtype vulnerability with pericytes and myofibroblasts most affected, altered cell to cell communication, and led to the emergence of Acta1 expressing cells. These insights hold the promise of targeted treatment for neonatal lung disease, which remains a major cause of infant morbidity and mortality across the world

Evidence for Phosphorylation-Dependent, Dynamic, Regulation of mGlu5 and Homer2 in Expression of Cocaine Aversion in Mice

Cocaine-induced changes in the expression of the glutamate-related scaffolding protein Homer2 influence this drug's psychostimulant and rewarding properties. In response to neuronal activity, Homer2 is phosphorylated on S117/S216 by calcium-calmodulin kinase IIα (CaMKIIα), which induces a rapid dissociation of mGlu5-Homer2 scaffolds. Herein, we examined the requirement for Homer2 phosphorylation in cocaine-induced changes in mGlu5-Homer2 coupling, to include behavioral sensitivity to cocaine. For this, mice with alanine point mutations at (S117/216)-Homer2 (Homer2AA/AA ) were generated, and we determined their affective, cognitive and sensorimotor phenotypes, as well as cocaine-induced changes in conditioned reward and motor hyperactivity. The Homer2AA/AA mutation prevented activity-dependent phosphorylation of S216 Homer2 in cortical neurons, but Homer2AA/AA mice did not differ from wild-type (WT) controls with respect to Morris maze performance, acoustic startle, spontaneous or cocaine-induced locomotion. Homer2AA/AA mice exhibited signs of hypoanxiety similar to the phenotype of transgenic mice with a deficit in signal-regulated mGluR5 phosphorylation (Grm5AA/AA ). However, opposite of Grm5AA/AA mice, Homer2AA/AA mice were less sensitive to the aversive properties of high-dose cocaine under both place-conditioning and taste-conditioning procedures. Acute injection with cocaine caused dissociation of mGluR5 and Homer2 in striatal lysates from WT, but not Homer2AA/AA mice, suggesting a molecular basis for the deficit in cocaine aversion. These findings indicate that CaMKIIα-dependent phosphorylation of Homer2 gates the negative motivational valence of high-dose cocaine via regulation of mGlu5 binding, furthering an important role for dynamic changes in mGlu5-Homer interactions in addiction vulnerability

Transforming Growth Factor-induced Protein Promotes NF-κB-mediated Angiogenesis during Postnatal Lung Development

Pulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that NF-κB promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NF-κB activation in the pulmonary vasculature are unknown. To identify mechanisms that activate proangiogenic NF-κB signaling in the developing pulmonary vasculature, proteomic analysis of the lung secretome was performed using two-dimensional difference gel electrophoresis. NF-κB activation and angiogenic function was assessed in primary pulmonary endothelial cells (PECs) and TGFBI (transforming growth factor-β-induced protein)-regulated genes identified using RNA sequencing. Alveolarization and pulmonary angiogenesis was assessed in wild-type and Tgfbi null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support. Secreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PECs. Proteomic analysis identified TGFBI as one protein highly expressed by the early alveolar lung that promoted PEC migration by activating NF-κB via αvβ3 integrins. RNA sequencing identified Csf3 as a TGFBI-regulated gene that enhances nitric oxide production in PECs. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization. Our studies identify TGFBI as a developmentally regulated protein that promotes NF-κB-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth