Histone deacetylase 6 regulates endothelial MyD88-dependent canonical TLR signaling, lung inflammation, and alveolar remodeling in the developing lung

Abstract

Lung endothelial cell (EC) immune activation during bacterial sepsis contributes to acute lung injury and bronchopulmonary dysplasia in premature infants. The epigenetic regulators of sepsis-induced endothelial immune activation, lung inflammation, and alveolar remodeling remain unclear. Herein, we examined the role of the cytoplasmic histone deacetylase, HDAC6, in regulating EC Toll-like receptor 4 (TLR4) signaling and modulating sepsis-induced lung injury in a neonatal model of sterile sepsis. In human primary microvascular endothelial cells (HPMEC), lipopolysaccharide (LPS)-induced MAPK, IKK-β, and p65 phosphorylation as well as inflammatory cytokine expression were exaggerated with the HDAC6 inhibitor tubastatin A, and by dominant-negative HDAC6 with a mutated catalytic domain 2. Expression of HDAC6 wild-type protein suppressed LPS-induced myeloid differentiation primary response 88 (MyD88) acetylation, p65 (Lys310) acetylation, MyD88/TNF receptor-associated factor 6 (TRAF6) coimmunoprecipitation, and proinflammatory TLR4 signaling in HPMEC. In a neonatal mouse model of sepsis, the HDAC6 inhibitor tubastatin A amplified lung EC TLR4 signaling and vascular permeability. HDAC6 inhibition augmented LPS-induced MyD88 acetylation, MyD88/TRAF6 binding, p65 acetylation, canonical TLR4 signaling, and inflammation in the developing lung. Sepsis-induced decreases in the fibroblast growth factors FGF2 and FGF7 and increase in matrix metalloproteinase-9 were worsened with HDAC6 inhibition, while elastin expression was equally suppressed. Exaggerated sepsis-induced acute lung inflammation observed with HDAC6 inhibition worsened alveolar simplification evidenced by increases in mean linear intercepts and decreased radial alveolar counts. Our studies reveal that HDAC6 is a constitutive negative regulator of cytoplasmic TLR4 signaling in EC and the developing lung. The therapeutic efficacy of augmenting HDAC6 activity in neonatal sepsis to prevent lung injury needs to be evaluated.

FOSL1 is a novel mediator of endotoxin/lipopolysaccharide-induced pulmonary angiogenic signaling

“…In previous studies from our lab, a 25-fold lower dose of i.p. LPS, as in this study, did not result in significant lethality, but a more nuanced alteration of the angiogenic/vasculogenic phenotype with decreased lung EC population 51 . The dosing we used is more consistent with the clinical scenario in preterm infants, where most infants survive Gram-negative sepsis.…”

“…For all animal experiments, half the litter of pups were injected with LPS (2 mg/kg) and other half injected with sterile saline (controls) intra-peritoneally (i.p.) (Sigma, St Louis, MO) served as littermate controls, as previously performed 51 . We did not observe any mortality in our experiments.…”

“…Human FOSL1 cDNA was amplified and cloned into pIRES-EGFP-Puro (Addgene, Cambridge, MA) with 5′-ACTGCTAGCCACCATGACCTCAACCGGCCAGGATTCCA-3′ and 5′-TGAGAGCTCTTAGTGTGGGTGGGGCATATCCTCCCCAAA-3′ as performed previously 51 . HPMEC grown in 6-well tissue culture plates were transfected overnight with 2 µg of the indicated plasmids or empty plasmids (mock) with Lipofectamine 3,000 (Thermo-Fisher) as per the manufacturer’s protocol.…”

Toll-like receptor-mediated innate immunity against herpesviridae infection: a current perspective on viral infection signaling pathways

“…TLRs are mainly expressed on the membranes of immune cells including macrophages, dendritic cells, T cells, and B cells [17][18][19][20][21][22]. Moreover, TLRs are also found in non-immune cells, such as endothelial and epithelial cells, adipocytes, and cardiomyocytes [23][24][25][26][27]. TLRs predominantly occur on the cell surface, while TLRs 3, 7, 8, and 9 are expressed inside cells [3].…”

Recent advances in our understanding of the mechanisms of lung alveolarization and bronchopulmonary dysplasia

“…Additionally, the histone deacetylase 6 (Hdac6) inhibitor tubastatin A and the pan-histone deacetylase inhibitor trichostatin A worsened alveolar simplification in a neonatal sepsis (LPS-driven) model, a model in which alveolarization is also stunted (524), suggesting the utility of augmenting Hdac6 activity in the injured, developing lung (377). Along similar lines, intravitreal administration of bevacizumab, an irreversible VEGF inhibitor, worsened arrested alveologenesis in a hyperoxia-based BPD model in rats, highlighting the already established importance of VEGF in alveolarization (598).…”