Christoph Reinhardt

BACKGROUND: Endothelial dysfunction is an early event in atherosclerosis development and is centrally linked with insufficient endothelial NO production. However, chronically increased NO levels, including NO from other cellular sources, may induce endothelial dysfunction. Here, we studied how chronically elevated NO production from erythrocytes, achieved by genetic deletion of ARG1 (arginase-1), impacts smooth muscle cell (SMC) lipid accumulation and atherosclerosis progression. METHODS: Primary aortic SMCs from mice lacking ARG1 in red blood cell (RBC.ARG1-knockout [KO]) were subjected to RNA-sequencing, lipidomic, metabolic, and molecular analyses; atherosclerosis burden was quantified en face and at the aortic root. RESULTS: Increased lipid droplet formation in SMCs from RBC.ARG1-KO mice was observed using brightfield and electron microscopy and confirmed by Oil Red O and boron-dipyrromethene lipid dye staining. RNA sequencing revealed the simultaneous overexpression of genes regulating lipid uptake ( Cd36 ), catabolism ( Cpt1a ), and de novo lipogenesis ( Acaca , Fasn [ Fatty acid synthase ]) in RBC.ARG1-KO SMCs, and inhibiting fatty acid translocase (CD36), ACC (acetyl-CoA [coenzyme A] carboxylase), or fatty acid synthase prevented the lipid accumulation in RBC.ARG1-KO SMCs. Increased expression of CD36 downstream of NO and overactivated sGC (soluble guanylyl cyclase)-cyclic guanosine monophosphate signaling was identified as a mediator of increased lipid uptake in RBC.ARG1-KO SMCs. Loss of PDE (phosphodiesterase) 2A, coupling cyclic guanosine monophosphate with cyclic adenosine monophosphate and PKA (protein kinase A) activation, was also observed, resulting in AMPK (5’ AMP-activated protein kinase) inhibition, thus unlocking acetyl-CoA carboxylase, catalyzing the rate-limiting step in fatty acid synthesis. Inhibiting PDE2A recapitulated the RBC.ARG1-KO SMC phenotype, while inhibiting PKA or ATP generation from cyclic adenosine monophosphate abrogated the lipid droplet accumulation in RBC.ARG1-KO SMCs. Increased Oil Red O-positive aortic atherosclerosis burden in hypercholesterolemic apolipoprotein E–deficient RBC.ARG1-KO mice was confirmed by histology and elevated levels of polyunsaturated long-chain cholesterol esters in aortic atheroma by mass spectrometry lipidomics. CONCLUSIONS: Our findings show the importance of erythrocyte-derived NO for metabolically reprogramming SMCs toward increased fatty acid uptake and lipogenesis, and identify PDE2A as a molecular switch linking chronically activated NO signaling with lipid accumulation and atheroma progression.

Polyphosphates are evolutionarily conserved linear chains of phosphate residues present in all living cells. Bacteria accumulate polyphosphates under stress and starvation for energy and phosphate storage, protein folding, and stress adaptation. During infection, bacteria release polyphosphates that may impair host responses, although the exact mechanisms remain elusive. In this study, polyphosphates were found to be elevated in bronchoalveolar lavage fluids from patients with Legionnaires' disease, in Legionella pneumophila cultured alone, or during infection of bone marrow‐derived macrophages (BMDMs) from C57BL/6J mice. We performed RNA‐sequencing of infected BMDMs co‐incubated with or without long‐chain, bacterial‐type polyphosphates. Among nearly 500 differentially expressed genes, Il12b (p40) showed the strongest suppression among highly expressed genes. IL‐12p40 protein release was dose‐dependently reduced by long‐chain polyphosphates (~P _i 700), but not by short‐chain, mammalian‐type polyphosphates (~P _i 70), indicating a specific bacterial mechanism targeting innate immune signaling. In contrast, IL‐18, processed by inflammasome activation and synergizing functionally with IL‐12, was not consistently suppressed. Polyphosphates predominantly inhibited LPS/TLR4 signaling, with Legionella ‐induced IL‐12 relying on the MyD88 pathway, but not TRIF. RAGE and P2Y1, previously implicated in polyphosphate biology, were not required for IL‐12 suppression. However, PI3K/AKT signaling appeared to mediate polyphosphate effects, which were reversed by the PI3K inhibitors, LY294002, copanlisib, and eganelisib. Finally, long‐chain polyphosphates suppressed IL‐12 release also from human monocyte‐derived macrophages exposed to L. pneumophila or LPS. In summary, our findings identify a selective inhibition of IL‐12 by long‐chain bacterial polyphosphates, suggesting that these molecules act as bacterial effectors capable of suppressing protective innate immune responses.

Bacterial sepsis remains a devastating clinical problem. Here, we describe a protective role for the recently discovered acid-sensitive, proton-activated chloride channel, PACC1 (PAC/ASOR/TMEM206), during sepsis. Initially, we found PACC1 was enriched in healthy human and mouse mononuclear phagocytes, particularly macrophages, and differentially regulated by inflammatory stimuli, suggesting PACC1 involvement in innate immunity. To further investigate, we generated de novo Pacc1 knockout ( ^−/− ) mice, which presented without major immunologic abnormalities at baseline. Compared to wild-type (WT), Pacc1 ^−/− myeloid cells showed normal phagocytic uptake of acid-insensitive Escherichia coli BioParticles , but impaired development of the acidifying phagolysosome using acid-sensitive E. coli BioParticles. Transcriptomic profiling of Pacc1 ^−/− macrophages revealed dysregulated phagolysosomal and cytokine networks (e.g., interferons). Because phagolysosomal bacterial clearance is essential to resolve infection, we challenged Pacc1 ^−/− mice with intraperitoneal gram-negative E. coli sepsis. Pacc1 ^−/− mice displayed increased bacterial burden, immune cell infiltration, inflammation, and lethality. In contrast, phagocytosis-independent E. coli lipopolysaccharide (LPS)-induced endotoxemia yielded comparable WT and Pacc1 ^−/− survival, as well as similar inflammatory responses. Finally, we engineered Pacc1 -floxed ( ^fl/fl ) mice crossed with a myeloid lineage Cre-deleter strain to interrogate myeloid cell–intrinsic PACC1 in vivo. Consistent with a predominate role for PACC1 during phagocytosis and bacterial clearance in these cells, LysM-Cre/Pacc1 ^fl/fl mice exhibited impaired E. coli sepsis survival but indifferent endotoxemia phenotypes. In conclusion, PACC1 links sterilizing phagolysosomal activity with immune networks in sepsis pathobiology.

Glucagon-like peptide-1 (GLP-1) and glucose-induced insulinotropic polypeptide (GIP) receptor agonists have revolutionized obesity therapy, but causes of obesity-associated dysregulation of endogenous incretin production remain incompletely understood. Here we show that intestinal transmembrane serine protease 2 (TMPRSS2) plays a pivotal role in deregulating anti-diabetic GLP-1 production in obesity. TMPRSS2 is widely coexpressed in intestinal epithelial cells along with its signaling target protease-activated receptor 2 (PAR2). In addition to its role in regulating coagulation protease–mediated adipose tissue inflammation, PAR2 signaling in the gut controls postprandial GIP secretion. TMPRSS2, but not the epithelial cell–expressed proteases FXa or matriptase, activates PAR2 and thereby promotes postprandial GIP release. Accordingly, a PAR2-mutant mouse resistant to TMPRSS2 cleavage is protected from GIP upregulation and diet-induced obesity. In the context of obesity, TMPRSS2 also attenuates bioavailability of the ghrelin pathway and thereby suppresses GLP-1–mediated control of glucose homeostasis. Pharmacological inhibition or genetic deletion of TMPRSS2 restores ghrelin signaling–dependent GLP-1 secretion and GLP-1’s anti-diabetic effects on nutritional glucose homeostasis. Thus, epithelial cell–expressed TMPRSS2, which critically contributes to the lung pathology in SARS-CoV-2 infection, emerges as an intestinal incretin regulator and a potential link between infection and chronic cardiometabolic diseases. GLP-1 based therapeutics revolutionized anti-diabetic and anti-obesity treatments. The current study identifies epithelial TMPRSS2 as a critical regulator of GLP-1 in obesity induced diabetes.

Bacteroides thetaiotaomicron ( B. theta ) dominates the gut microbiome of most mammals. This strictly anaerobic gut symbiont colonizes the mucus layer of host intestinal epithelial cells in both healthy and diseased conditions. Reduced neuronal and vagal afferent innervation observed in germ-free mice was found to be normalized by colonization with B. theta . In addition to deficits in gut innervation, germ-free mice have been reported to have reduced neuronal number and neurotransmitter levels in the brain. Here, we investigated the hallmarks of Alzheimer’s disease (AD) in the brain of germ-free mice compared to mice mono-colonized with B. theta . We analyzed the number of mature neurons, neurotransmitter transporters, amyloid precursor protein processing, and inflammatory status in three brain regions: the hippocampus, prefrontal cortex (PFC), and cerebellum. The hippocampus and the PFC are regions thought to be highly susceptible to pathogenesis, whereas the cerebellum is thought to be only mildly affected. Interestingly, secretion of neuroprotective sAPPα decreased in hippocampus and remained unchanged in PFC, while levels were increased in the cerebellum in response to bacterial colonization. In addition, the number of presynaptic boutons increased in the hippocampus but remained unaffected in the cerebellum. IMPORTANCE: The gut microbiome has been reported to not only contribute to diseases of the gastrointestinal tract but also to interfere with and potentially even initiate diseases of other organ systems, such as the brain. Interference with the gut microbiome has been shown to elicit cognitive changes, for example, in rodent models of AD. Colonization with the common gut microbe B. theta not only affected the brain per se in our study but also showed specific brain region-dependent effects related to AD. This implies that evaluating the impact the microbiome might have on brain disorders needs a much more detailed investigation in the future with spatial and also potentially time resolution.

Bacterial sepsis remains a devastating clinical problem. Here, we describe a protective role for the recently discovered acid-sensitive, proton-activated chloride channel, PACC1 (PAC/ASOR/TMEM206), during sepsis. Initially, we found PACC1 was enriched in healthy human and mouse mononuclear phagocytes, particularly macrophages, and differentially regulated by inflammatory stimuli, suggesting PACC1 involvement in innate immunity. To further investigate, we generated de novo Pacc1 knockout ( ^-/- ) mice, which presented without major immunologic abnormalities at baseline. Compared to wildtype (WT), Pacc1 ^-/- myeloid cells showed normal phagocytic uptake of acid-insensitive Escherichia coli BioParticles , but impaired development of the acidifying phagolysosome using acid-sensitive E. coli BioParticles. Transcriptomic profiling of Pacc1 ^-/- macrophages revealed dysregulated phagolysosomal and cytokine networks (e.g., interferons). Because phagolysosomal bacterial clearance is essential to resolve infection, we challenged Pacc1 ^-/- mice with intraperitoneal gram-negative E. coli sepsis. Pacc1 ^-/- mice displayed increased bacterial burden, immune cell infiltration, inflammation, and lethality. In contrast, phagocytosis-independent E. coli lipopolysaccharide (LPS)-induced endotoxemia yielded comparable WT and Pacc1 ^-/- survival, as well as similar inflammatory responses. Finally, we engineered Pacc1 -floxed ( ^fl/fl ) mice crossed with a myeloid lineage Cre-deleter strain to interrogate myeloid cell-intrinsic PACC1 in vivo . Consistent with a predominate role for PACC1 during phagocytosis and bacterial clearance in these cells, LysM-Cre/Pacc1 ^fl/fl mice exhibited impaired E. coli sepsis survival but indifferent endotoxemia phenotypes. In conclusion, PACC1 links sterilizing phagolysosomal activity with immune networks in sepsis pathobiology. Significance Statement: Bacterial sepsis remains a major global health burden. Here, we report an essential role for the recently discovered acid-sensitive chloride channel, PACC1 (PAC/ASOR/TMEM206), in protective host defense during bacterial infection and sepsis. PACC1 is highly expressed in human and mouse phagocytic myeloid cells, particularly macrophages, where it regulates phagocytic bacterial clearance and inflammatory responses. Using de novo generated mice, we show that global or myeloid cell-targeted deletion of PACC1 impairs development of phagolysosomal acidification, confers susceptibility to bacterial infection and excessive inflammation, and undermines host defense. These findings warrant further investigation of PACC1 in sepsis pathobiology.