RIPK1 kinase-dependent inflammation and cell death contribute to the pathogenesis of COPD.

BACKGROUND: Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD. METHODS: We assessed RIPK1 expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patients via immunohistochemistry. Next, we assessed the consequences of genetic and pharmacological inhibition of RIPK1 kinase activity in experimental COPD, using Ripk1 S25D/S25D kinase-deficient mice and the RIPK1 kinase inhibitor GSK'547. RESULTS: RIPK1 expression increased in alveolar type 1 (AT1), AT2, ciliated and neuroendocrine cells in human COPD. RIPK1 protein levels were significantly increased in airway epithelium of COPD patients compared with never-smokers and smokers without airflow limitation. In mice, exposure to cigarette smoke (CS) increased Ripk1 expression similarly in AT2 cells, and further in alveolar macrophages and T-cells. Genetic and/or pharmacological inhibition of RIPK1 kinase activity significantly attenuated airway inflammation upon acute and subacute CS exposure, as well as airway remodelling, emphysema, and apoptotic and necroptotic cell death upon chronic CS exposure. Similarly, pharmacological RIPK1 kinase inhibition significantly attenuated elastase-induced emphysema and lung function decline. Finally, RNA-seq on lung tissue of CS-exposed mice revealed downregulation of cell death and inflammatory pathways upon pharmacological RIPK1 kinase inhibition. CONCLUSIONS: RIPK1 kinase inhibition is protective in experimental models of COPD and may represent a novel promising therapeutic approach.

An intestinal organoid-based platform that recreates susceptibility to T-cell-mediated tissue injury.

A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T-cell-mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that cocultured allogeneic T cells kill Atg16L1-mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T-cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.

The 24-hour respiratory quotient predicts energy intake and changes in body mass.

To define the relationship between the respiratory quotient (RQ) and energy intake (EI) and to determine the impact of spontaneous locomotor activity (LMA) in the development of diet-induced obesity (DIO), we fed C57BL/6 mice a high-fat diet (HFD) for either 4 days or 17 wk and analyzed them using indirect calorimetry. Importantly, changes in body mass during calorimetry (DeltaM(b)) significantly covaried with RQ and EI; adjusting the data for DeltaM(b) permitted an analysis of the energy-balanced state. The 24-h RQ strongly predicted 24-h EI, and the slope of this relationship was diet dependent (HFD or chow) but independent of the HFD feeding period. Early-stage DIO was characterized by dark-period hyperphagia and fat storage, offset by greater light-period lipid oxidation; later stage DIO mice had a milder hyperphagia and lower substrate flexibility. Consequently, whereas 24-h RQ equaled the food quotient of the HFD in both early- and late-stage DIO, the range of RQ values was negatively correlated with, and mostly explained by, 24-h EI only in late-stage DIO. Lean and early-stage DIO mice had similar LMA values that were reduced in late-stage DIO. However, LMA significantly explained variance in total energy expenditure (EE) in only early-stage DIO mice. This indicated that the link between LMA and EE was a transient adaptive response to early DIO, whereas the later loss of LMA did not explain body weight gain in C57BL/6 DIO mice.

Enhanced gastrointestinal motility with orally active ghrelin receptor agonists.

The orexigenic peptide ghrelin has been shown to have prokinetic activity in the gastrointestinal (GI) system of several species, including humans. In this series of experiments, we have evaluated the prokinetic activity of novel, small-molecule ghrelin receptor (GhrR) agonists after parenteral and peroral dosing in mice and rats. Gastric emptying, small intestinal transport, and fecal output were determined after intraperitoneal and intracerebroventricular dosing of GhrR agonists, using ghrelin as a positive control. These same parameters were evaluated after oral gavage dosing of the synthetic agonists. Regardless of dose route, GhrR agonist treatment increased gastric emptying, small intestinal transit, and fecal output. However, fecal output was only increased by GhrR agonist treatment if mice were able to feed during the stimulatory period. Thus, GhrR agonists can stimulate upper GI motility, and the orexigenic action of the compounds can indirectly contribute to prokinetic activity along the entire GI tract. The orexigenic and prokinetic effects of either ghrelin or small-molecule GhrR agonists were selective for the GhrR because they were absent when evaluated in GhrR knockout mice. We next evaluated the efficacy of the synthetic GhrR agonists dosed in a model of opiate-induced bowel dysfunction induced by a single injection of morphine. Oral dosing of a GhrR agonist normalized GI motility in opiate-induced dysmotility. These data demonstrate the potential utility of GhrR agonists for treating gastrointestinal hypomotility disorders.

Discovery and characterization of aminopiperidinecoumarin melanin concentrating hormone receptor 1 antagonists.

4-(1-Benzo[1,3]dioxol-5-ylmethylpiperidine-4-ylmethyl)-6-chlorochromen-2-one (7) is a potent, orally bioavailable melanin concentrating hormone receptor 1 (MCHr1) antagonist that causes dose-dependent weight loss in diet-induced obese mice. Further evaluation of 7 in an anesthetized dog model of cardiovascular safety revealed adverse hemodynamic effects at a plasma concentration comparable to the minimally effective therapeutic concentration. These results highlight the need for scrutiny of the cardiovascular safety profile of MCHr1 antagonists.

Central effects of long-term relaxin expression in the rat.

A recombinant adenovirus containing the human H2 preprorelaxin (hH2) cDNA and a reporter gene was coinjected with a transactivator virus (Ad-tTA) into the lateral cerebral ventricles of female rats. Cardiovascular effects were measured over a 21-day period. Circulating vasopressin in the periphery was significantly greater (P < .0001) in the relaxin-treated group throughout the experimental period, compared with controls. There was a significant decrease in plasma osmolality (P < .05) by approximately 10 mmol/L in the treated group by day 14. Immunofluorescence for hH2 present in cryosections showed rAd transduction and hH2 expression from ependymal cells of the ventricular system. Adenovirus-mediated delivery of hH2 to the brain is capable of producing bioactive relaxin that affects cardiovascular parameters.

The prolactin-releasing peptide receptor (GPR10) regulates body weight homeostasis in mice.

To identify new drug targets for the treatment of obesity, we employed a degenerate reverse transcriptasepolymerase chain reaction technique to isolate novel members of the G-protein coupled receptor superfamily from mouse hypothalamus. One of our clones was found to encode a protein with 90% amino acid identity to human GPR10, which was previously identified as the receptor for prolactin-releasing peptide (PrRP) and has been implicated in lactation, the regulation of food intake and other physiological functions. To investigate the role of GPR10 in food intake and body weight homeostasis, we generated mice carrying a targeted deletion of the GPR10 gene. First, using these knockout animals, we confirmed that GPR10 is the principle receptor for PrRP in the mouse hypothalamus because deletion of GPR10 completely abolished PrRP binding to isolated hypothalamic cell membranes. Second, we investigated the effect of normal and high-fat diets on energy intake, body weight, and glucose homeostasis in wild-type and GPR10 knockout mice. After fasting and refeeding, food intake in knockout animals was unchanged relative to control littermates. However, beginning at 16 wk of age on a normal diet, knockout mice became hyperphagic, obese, and showed significant increases in body fat and the levels of leptin and insulin, as well as decreased glucose tolerance. This metabolic profile was similar to the effect of a high-fat diet on wild-type animals. Our findings provide direct evidence that GPR10 is the receptor for PrRP and that it is involved in the regulation of energy balance in mice. GPR10 knockout mice will also prove useful for investigating other proposed activities for PrRP.

Adenovirus-mediated expression of human prorelaxin promotes the invasive potential of canine mammary cancer cells.

This study reports the characterization of a recombinant adenoviral vector containing a tetracycline-regulatable promoter, driving the bicistronic expression of the human H2 preprorelaxin (hH2) cDNA and enhanced green fluorescent protein, via an internal ribosomal entry site. An hH2 ELISA was used to measure the secreted levels of recombinant hH2 in transfected canine (CF33.Mt) and human (MDA-MB-435) mammary cancer cell lines over a 6-d period; secreted peptide peaked on d 2 and 4 for the canine and human cell types, respectively. An unprocessed hH2 immunoreactive form of approximately 18 kDa was identified by Western blotting analysis and confirmed by mass spectrometry, suggesting that prorelaxin remains unprocessed in these cell types. The biological activity of the adenovirally expressed human prorelaxin was measured in the established human monocytic cell line THP-1 cAMP ELISA and in an in vitro Transwell cell migration system. Exogenous recombinant hH2 and adenovirally-mediated delivery of prorelaxin to CF33.Mt cells conferred a significant migratory action in the cells, compared with controls. Cell proliferation assays were performed to discount the possibility that the effect of relaxin was mitogenic. Thus, we have demonstrated that prorelaxin has the ability to facilitate cell migration processes exclusive of its ability to stimulate cell proliferation. In validating this adenovirus-based system, we have created a potential tool for further exploration of the physiology of relaxin in mammalian systems.

CARD-8 protein, a new CARD family member that regulates caspase-1 activation and apoptosis.

Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis, NFkappaB activation, and cytokine regulation. In this study we identified a novel human protein, CARD-8, which contains a C-terminal CARD domain with high similarity to the CARD domain of caspase-1/ICE. We demonstrate that CARD-8 interacts physically with caspase-1 and negatively regulates caspase-1-dependent IL-1beta generation in the THP-1 monocytic cell line. CARD-8 binds also to ICEBERG and pseudo-ICE, two other recently identified proteins, which bind to the CARD domain of caspase-1 and negatively regulate its activity. Reverse transcriptase-PCR analysis revealed that CARD-8 is expressed mainly in monocytes, placenta, lymph nodes, and spleen. This pattern of expression is consistent with caspase-1 expression in the same cells and tissues. CARD-8 was also found to negatively regulate NF-kappaB activation by TNF-alpha stimulation and by ectopically expressed RICK, suggesting that this protein may control cell survival. Consistent with these results, stable expression of CARD-8 in U937 or THP-1 cells sensitizes the cells to differentiation-induced apoptosis. Overexpression of CARD-8 can also induce apoptosis in transfected cells. The results suggest that CARD-8 represents a new signaling molecule involved in the regulation of caspase-1 and NF-kappaB activation.