Aglaia Ntokou

Intracellular microbes that deploy actin-based motility (ABM) as a virulence mechanism are phylogenetically diverse pathogens that cause a wide range of infections in animals and humans. These include bacteria that share pathogenic features such as the invasion of host cells, the rupture of intracellular compartments, and exploitation of host cytoskeletal proteins for cytosolic movement. The virulence factors that mediate ABM and the underlying molecular mechanisms differ substantially across genera and even within species when multiple proteins are encoded within a genome. Emerging evidence from these pathogens now reveals significant heterogeneity in bacterial actin-tail architecture, motility behaviors across distinct host-cell contexts, and susceptibility to immune restriction. This review provides an update on the recent advances in bacterial ABM, highlighting the emerging complexities of the interplay between host immunity and this important microbial virulence mechanism.

Interferon-gamma (IFN-γ) is a powerful transactivating signal eliciting hundreds of IFN-stimulated genes (ISGs) in humans to help combat infection. Most ISGs remain uncharacterized, and here we searched for actin-binding candidates that could potentially target intracellular pathogens to block their spread or promote immune cell migration into infected tissues. Dual RNA-Seq and in silico mining across 1,933 data sets discovered >225 actin-related genes; the most highly expressed was XIRP1 (xin actin binding repeat containing 1 protein), a new ISG with no reported immune function. We found XIRP1 induction required IFN-γ plus IL-1β or exposure to pathogenic Listeria, Shigella, or Salmonella in immune and non-immune cells. Within IFN-γ-activated human macrophages, the XIRP1 protein localized to actin-rich podosomes where it formed a dome-shaped cap facing the cytosol; genetic XIRP1 ablation led to significant actin loss from these structures. Within infected cells, XIRP1 was recruited onto cytosolic Listeria monocytogenes in an ActA-dependent manner. Live imaging found many listeriae were fully encapsulated by XIRP1 whereas incomplete XIRP1 coating allowed pathogen escape from the initial coat structure. Together, our results identify XIRP1 as a new podosome-associated ISG that targets cytosolic bacteria as part of the IFN-γ-induced defense program in humans.

BACKGROUND: TGF (transforming growth factor)-β pathway is central to blood-brain barrier development as it regulates cross talk between pericytes and endothelial cells. Murine embryos lacking TGFβ receptor Alk5 (activin receptor-like kinase 5) in brain pericytes (mutants) display endothelial cell hyperproliferation, abnormal vessel morphology, and gross germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH), leading to perinatal lethality. Mechanisms underlying how ALK5 signaling in pericytes noncell autonomously regulates endothelial cell behavior remain elusive. METHODS: Transcriptomic analysis of human brain pericytes with ALK5 silencing identified differential gene expression. Brain vascular cells isolated from mutant embryonic mice with GMH-IVH and preterm human IVH brain samples were utilized for target validation. Finally, pharmacological and genetic inhibition was used to study the therapeutic effects on GMH-IVH pathology. RESULTS: Herein, we establish that the TGFβ/ALK5 pathway robustly represses ANGPT2 (angiopoietin-2) in pericytes via epigenetic remodeling. TGFβ-driven SMAD (suppressor of mothers against decapentaplegic) 3/4 associates with TGIF1 (TGFβ-induced factor homeobox 1) and HDAC (histone deacetylase) 5 to form a corepressor complex at the Angpt2 promoter, resulting in promoter deacetylation and gene repression. Moreover, murine and human germinal matrix vessels display increased ANGPT2 expression during GMH-IVH. Isolation of vascular cells from murine germinal matrix identifies pericytes as a cellular source of excessive ANGPT2. In addition, mutant endothelial cells exhibit higher phosphorylated TIE2 (tyrosine protein kinase receptor). Pharmacological or genetic inhibition of ANGPT2 in mutants improves germinal matrix vessel morphology and attenuates GMH pathogenesis. Importantly, genetic ablation of Angpt2 in mutant pericytes prevents perinatal lethality, prolonging survival. CONCLUSIONS: This study demonstrates that TGFβ-mediated ANGPT2 repression in pericytes is critical for maintaining blood-brain barrier integrity and identifies pericyte-derived ANGPT2 as an important pathological target for GMH-IVH.

Aging is the predominant risk factor for atherosclerosis—the leading cause of death. Rare smooth muscle cell (SMC) progenitors clonally expand, giving rise to up to ~70% of atherosclerotic plaque cells; however, the effect of age on SMC clonality is not known. Our results indicate that aged bone marrow (BM)-derived cells noncell autonomously induce SMC polyclonality and worsen atherosclerosis. Indeed, in myeloid cells from aged mice and humans, TET2 levels are decreased, which epigenetically silences integrin β₃, resulting in increased tumor necrosis factor-α (TNFα) signaling. TNFα signals through TNF receptor 1 on SMCs to promote proliferation, and induces the recruitment and expansion of multiple SMC progenitors into the atherosclerotic plaque. Notably, integrin β₃ overexpression in aged BM preserves the dominance of the lineage of a single SMC progenitor and attenuates the plaque burden. Our results demonstrate a molecular mechanism of aged macrophage-induced SMC polyclonality and atherogenesis and suggest novel therapeutic strategies.

Obstructive arterial diseases, including supravalvular aortic stenosis (SVAS), atherosclerosis, and restenosis, share 2 important features: an abnormal or disrupted elastic lamellae structure and excessive smooth muscle cells (SMCs). However, the relationship between these pathological features is poorly delineated. SVAS is caused by heterozygous loss-of-function, hypomorphic, or deletion mutations in the elastin gene (ELN), and SVAS patients and elastin-mutant mice display increased arterial wall cellularity and luminal obstructions. Pharmacological treatments for SVAS are lacking, as the underlying pathobiology is inadequately defined. Herein, using human aortic vascular cells, mouse models, and aortic samples and SMCs derived from induced pluripotent stem cells of ELN-deficient patients, we demonstrated that elastin insufficiency induced epigenetic changes, upregulating the NOTCH pathway in SMCs. Specifically, reduced elastin increased levels of γ-secretase, activated NOTCH3 intracellular domain, and downstream genes. Notch3 deletion or pharmacological inhibition of γ-secretase attenuated aortic hypermuscularization and stenosis in Eln^–/– mutants. Eln^–/– mice expressed higher levels of NOTCH ligand JAGGED1 (JAG1) in aortic SMCs and endothelial cells (ECs). Finally, Jag1 deletion in SMCs, but not ECs, mitigated the hypermuscular and stenotic phenotype in the aorta of Eln^–/– mice. Our findings reveal that NOTCH3 pathway upregulation induced pathological aortic SMC accumulation during elastin insufficiency and provide potential therapeutic targets for SVAS.

Significance: Inward remodeling of arteries to reduce lumen diameter is a major factor in disease progression and morbidity in multiple vascular diseases, including hypertension and atherosclerosis. However, molecular mechanisms controlling inward arterial remodeling remain largely undefined. In this study, we identify endothelial MEKK3 as an unexpected regulator of inward remodeling via inhibition of TGFβ-Smad2/3 signaling. Genetic deletion of MEKK3 in adult endothelium results in induction of TGFβ-Smad2/3 signaling, endothelial-to-mesenchymal transition, and inward remodeling in both pulmonary and arterial circuits. The latter process results in pulmonary and systemic hypertension and accelerates atherosclerosis. These results provide a basis for understanding the inward artery remodeling that leads to reduced blood flow to affected tissues and exacerbates hypertension in vascular disease.

Excess macrophages and smooth muscle cells (SMCs) characterize many cardiovascular diseases, but crosstalk between these cell types is poorly defined. Pulmonary hypertension (PH) is a lethal disease in which lung arteriole SMCs proliferate and migrate, coating the normally unmuscularized distal arteriole. We hypothesized that increased macrophage platelet-derived growth factor–B (PDGF-B) induces pathological SMC burden in PH. Our results indicate that clodronate attenuates hypoxia-induced macrophage accumulation, distal muscularization, PH, and right ventricle hypertrophy (RVH). With hypoxia exposure, macrophage Pdgfb mRNA was upregulated in mice, and LysM‑Cre mice carrying floxed alleles for hypoxia-inducible factor 1a , hypoxia-inducible factor 2a , or Pdgfb had reduced macrophage Pdgfb and were protected against distal muscularization and PH. Conversely, LysM‑Cre von-Hippel Lindau ^fl/fl mice had increased macrophage Hifa and Pdgfb and developed distal muscularization, PH, and RVH in normoxia. Similarly, Pdgfb was upregulated in macrophages from human idiopathic or systemic sclerosis–induced pulmonary arterial hypertension patients, and macrophage-conditioned medium from these patients increased SMC proliferation and migration via PDGF-B. Finally, in mice, orotracheal administration of nanoparticles loaded with Pdgfb siRNA specifically reduced lung macrophage Pdgfb and prevented hypoxia-induced distal muscularization, PH, and RVH. Thus, macrophage-derived PDGF-B is critical for pathological SMC expansion in PH, and nanoparticle-mediated inhibition of lung macrophage PDGF-B has profound implications as an interventional strategy for PH.

Fibrosis is a macrophage-driven process of uncontrolled extracellular matrix accumulation. Neuronal guidance proteins such as netrin-1 promote inflammatory scarring. We found that macrophage-derived netrin-1 stimulates fibrosis through its neuronal guidance functions. In mice, fibrosis due to inhaled bleomycin engendered netrin-1–expressing macrophages and fibroblasts, remodeled adrenergic nerves, and augmented noradrenaline. Cell-specific knockout mice showed that collagen accumulation, fibrotic histology, and nerve-associated endpoints required netrin-1 of macrophage but not fibroblast origin. Adrenergic denervation; haploinsufficiency of netrin-1’s receptor, deleted in colorectal carcinoma; and therapeutic α₁ adrenoreceptor antagonism improved collagen content and histology. An idiopathic pulmonary fibrosis (IPF) lung microarray data set showed increased netrin-1 expression. IPF lung tissues were enriched for netrin-1⁺ macrophages and noradrenaline. A longitudinal IPF cohort showed improved survival in patients prescribed α₁ adrenoreceptor blockade. This work showed that macrophages stimulate lung fibrosis via netrin-1–driven adrenergic processes and introduced α₁ blockers as a potentially new fibrotic therapy.