Immunomodulatory role of non-neuronal cholinergic signaling in myocardial injury

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Abstract

Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the non-neuronal cholinergic system is not well understood. To address the immunomodulatory role of the non-neuronal cholinergic system in the heart we used a previously validated diphtheria toxin (DT)-induced cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or Pyridostigmine Bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIlowCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared to diluent treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with ACh, nicotine and muscarine. Viewed together, these findings reveal a previously unappreciated immunomodulatory role for the non-neuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.

Authors

Cibele Rocha-Resende, Carla Weinheimer, Geetika Bajpai, Luigi Adamo, Scot J. Matkovich, Joel Schilling, Philip M. Barger, Kory J. Lavine, Douglas L. Mann

Large-scale lipidomics identifies associations between plasma sphingolipids and T2DM incidence

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Abstract

BACKGROUND. Sphingolipids (SPs) are ubiquitous, structurally diverse molecules that include ceramides, sphingomyelins, and sphingosines. They are involved in various pathologies including obesity and type 2 diabetes mellitus (T2DM). Therefore, it is likely that perturbations in plasma concentrations of SPs are associated with disease. Identifying these associations may reveal useful biomarkers or provide insight into disease processes. METHODS. We performed a lipidomics evaluation of molecularly-distinct SPs in the plasma of 2,302 ethnically-Chinese Singaporeans using electrospray ionization mass spectrometry coupled with liquid chromatography. SP profiles were compared to clinical and biochemical characteristics, and subjects were evaluated by follow-up visits for 11 years. RESULTS. We found that ceramides correlate positively but hexosylceramides correlate negatively with body mass index (BMI) and homeostatic model assessment of insulin resistance (HOMA-IR). Furthermore, SPs with a d16:1 sphingoid backbone correlate more positively with BMI and HOMA-IR, while d18:2 SPs correlate less positively, relative to canonical d18:1 SPs. We also found that higher concentrations of two distinct sphingomyelins were associated with a higher risk of T2DM (HR 1.45, 95% CI 1.18–1.78 for SM d16:1/C18:0; and HR 1.40, 95% CI 1.17–1.68 for SM d18:1/C18:0). CONCLUSION. We identified significant associations between SPs and obesity/T2DM characteristics, specifically, that of hexosylceramides, d16:1 SPs, and d18:2 SPs. This suggests that the balance of SP metabolism, rather than ceramide accumulation, is associated with the pathology of obesity. We further identified two specific SPs that may represent prognostic biomarkers for T2DM. FUNDING. Funding sources are listed in the Acknowledgements section

Authors

Wee Siong Chew, Federico Torta, Shanshan Ji, Hyungwon Choi, Husna Begum, Xueling Sim, Chin Meng Khoo, Eric Yin Hao Khoo, Wei-Yi Ong, Rob M. Van Dam, Markus R. Wenk, E. Shyong Tai, Deron R. Herr

High dietary salt-induced dendritic cell activation underlies microbial dysbiosis-associated hypertension

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Abstract

Excess dietary salt contributes to inflammation and hypertension via poorly understood mechanisms. Antigen presenting cells including dendritic cells (DCs) play a key role in regulating intestinal immune homeostasis in part by surveying the gut epithelial surface for pathogens. Previously, we found that highly reactive γ-ketoaldehydes or isolevuglandins (IsoLGs) accumulate in DCs and act as neoantigens, promoting an autoimmune-like state and hypertension. We hypothesized that excess dietary salt alters the gut microbiome leading to hypertension and this is associated with increased immunogenic IsoLG-adduct formation in myeloid antigen presenting cells. To test this hypothesis, we performed fecal microbiome analysis and measured blood pressure of healthy human volunteers with salt intake above or below the American Heart Association recommendations. We also performed 16S rRNA analysis on cecal samples of mice fed normal or high salt diets. In humans and mice, high salt intake was associated with changes in the gut microbiome reflecting an increase in Firmicutes, Proteobacteria and genus Prevotella bacteria. These alterations were associated with higher blood pressure in humans and predisposed mice to vascular inflammation and hypertension in response to a sub-pressor dose of angiotensin II. Mice fed a high salt diet exhibited increased intestinal inflammation including the mesenteric arterial arcade and aorta, with a marked increase in the B7 ligand CD86 and formation of IsoLG-protein adducts in CD11c+ myeloid cells. Adoptive transfer of fecal material from conventionally housed high salt-fed mice to germ-free mice predisposed them to increased intestinal inflammation and hypertension. These findings provide novel insight into the mechanisms underlying inflammation and hypertension associated with excess dietary salt and may lead to interventions targeting the microbiome to prevent and treat this important disease.

Authors

Jane F. Ferguson, Luul A. Aden, Natalia R. Barbaro, Justin P. Van Beusecum, Liang Xiao, Alan J. Simmons, Cassandra Warden, Lejla Pasic, Lauren E. Himmel, Mary K. Washington, Frank L. Revetta, Shilin Zhao, Shivani Kumaresan, Matthew B. Scholz, Zhengzheng Tang, Guanhua Chen, Muredach P. Reilly, Annet Kirabo

Genomic distinctions between metastatic lower and upper tract urothelial carcinoma revealed through rapid autopsy

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Abstract

Background: Little is known about the genomic differences between metastatic urothelial carcinoma (LTUC) and upper tract urothelial carcinoma (UTUC). We compare genomic features of primary and metastatic UTUC and LTUC tumors in a cohort of patients with end stage disease. Methods: We performed whole exome sequencing on matched primary and metastatic tumor samples (N=37) from 7 patients with metastatic UC collected via rapid autopsy. Inter- and intra-patient mutational burden, mutational signatures, predicted deleterious mutations, and somatic copy alterations (sCNV) were analyzed. Results: We investigated 3 patients with UTUC (3 primary samples, 13 metastases) and 4 patients with LTUC (4 primary samples, 17 metastases). We found that sSNV burden was higher in metastatic LTUC compared to UTUC. Moreover, the APOBEC mutational signature was pervasive in metastatic LTUC and less so in UTUC. Despite a lower overall sSNV burden, UTUC displayed greater inter- and intra-individual genomic distances at the copy number level between primary and metastatic tumors than LTUC. Our data also indicate that metastatic UTUC lesions can arise from small clonal populations present in the primary cancer. Importantly, putative druggable mutations were found across patients with the majority shared across all metastases within a patient. Conclusions: Metastatic UTUC demonstrated a lower overall mutational burden but greater structural variability compared to LTUC. Our findings suggest that metastatic UTUC displays a greater spectrum of copy number divergence from LTUC. Importantly, we identified druggable lesions shared across metastatic samples, which demonstrate a level of targetable homogeneity within individual patients.

Authors

Brian R. Winters, Navonil De Sarkar, Sonali Arora, Hamid Bolouri, Sujata Jana, Funda Vakar-Lopez, Heather H. Cheng, Michael T. Schweizer, Evan Y. Yu, Petros Grivas, John K. Lee, Lori Kollath, Sarah K. Holt, Lisa McFerrin, Gavin Ha, Peter S. Nelson, Robert B. Montgomery, Jonathan L. Wright, Hung-Ming Lam, Andrew C. Hsieh

Sex differences in IL-17 contribute to chronicity in male versus female urinary tract infection

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Abstract

Sex-based differences influence incidence and outcome of infectious disease. Women have a significantly greater incidence of urinary tract infection (UTI) than men, yet, conversely, male UTI is more persistent with greater associated morbidity. Mechanisms underlying these sex-based differences are unknown, in part due to a lack of experimental models. We optimized a model to transurethrally infect male mice and directly compared UTI in both sexes. Although both sexes were initially equally colonized by uropathogenic E. coli, only male and testosterone-treated female mice remained chronically infected for up to 4 weeks. Female mice had more robust innate responses, including higher IL-17 expression, and increased γδ T cells and group 3 innate lymphoid cells in the bladder following infection. Accordingly, neutralizing IL-17 abolished resolution in female mice, identifying a cytokine pathway necessary for bacterial clearance. Our findings support the concept that sex-based responses to UTI contribute to impaired innate immunity in males and provide a rationale for non-antibiotic-based immune targeting to improve the response to UTI.

Authors

Anna Zychlinsky Scharff, Matthieu Rousseau, Livia Lacerda Mariano, Tracy Canton, Camila Rosat Consiglio, Matthew L. Albert, Magnus Fontes, Darragh Duffy, Molly A. Ingersoll

High protein feeding induces de novo lipogenesis in healthy humans: a randomised 3-way crossover study

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Abstract

BACKGROUND. Dietary changes have led to a growing prevalence of Type 2 diabetes and non-alcoholic fatty liver disease. A hallmark of both disorders is hepatic lipid accumulation, derived in part from increased de novo lipogenesis. Despite high protein diets being popular for weight loss to tackle these metabolic disorders, the effect of dietary protein on de novo lipogenesis is poorly studied. We aimed to characterise the effect of dietary protein on de novo lipid synthesis. METHODS. Herein, we use a 3-way crossover interventional study in healthy males to determine the effect of high protein feeding on de novo lipogenesis as well as in vitro models to determine the effects of specific amino acids on fatty acid synthesis. The primary outcome was change in de novo lipogenesis-associated triglycerides in response to protein feeding. RESULTS. We demonstrate that high protein feeding, rich in glutamate, increases de novo lipogenesis-associated triglycerides in plasma (2-fold compared to Control; p < 0.0001) and liver-derived very low-density lipoprotein particles (1.8 fold; p < 0.0001) in samples from human subjects (n = 9 per group). In hepatocytes, we show that glutamate derived carbon is incorporated into palmitate and subsequently into triglycerides. In addition, supplementation with glutamate, glutamine and leucine, but not lysine increases synthesised triglyceride content in cells and decreases glucose uptake. Glutamate, glutamine and leucine increase activation of protein kinase B, suggesting that these amino acids induce de novo lipogenesis via the insulin signalling cascade. CONCLUSION. These findings provide mechanistic insight into how select amino acids may induce de novo lipogenesis and insulin resistance, suggesting that high protein feeding to tackle diabetes and obesity requires greater consideration.

Authors

Evelina Charidemou, Tom Ashmore, Xuefei Li, Ben D. McNally, James A. West, Sonia Liggi, Matthew Harvey, Elise Orford, Julian L. Griffin

Two human patient mitochondrial pyruvate carrier mutations reveal distinct molecular mechanisms of dysfunction

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Abstract

The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix and linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient C289T and T236A MPC1 alleles disrupt MPC function. MPC1 C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and a full length point mutant (R97W). MPC1 T236A encodes a full length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from two human patient MPC1 mutations. They also illustrate an efficient gene pass-through system for mechanistically investigating human inborn errors in pyruvate metabolism.

Authors

Lalita Oonthonpan, Adam J. Rauckhorst, Lawrence R. Gray, Audrey C. Boutron, Eric B. Taylor

Platelet bioenergetics correlate with muscle energetics and are altered in older adults

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Abstract

BACKGROUND. Physical function decreases with age, and though bioenergetic alterations contribute to this decline, the mechanisms by which mitochondrial function changes with age remains unclear. This is partially because human mitochondrial studies require highly invasive procedures, such as muscle biopsies, to obtain live tissue with functional mitochondria. However, recent studies demonstrate that circulating blood cells are potentially informative in identifying systemic bioenergetic changes. Here, we hypothesize that human platelet bioenergetics reflect bioenergetics measured in muscle biopsies. METHODS & RESULTS. We demonstrate that maximal and ATP-linked respiratory rate measured in isolated platelets from older adults (86–93 years) correlates significantly with maximal respiration (r = 0.595; P = 0.003) measured by muscle biopsy respirometry and maximal ATP production (r = 0.643; P = 0.004) measured by 31P-MRS respectively, in the same individuals. Comparison of platelet bioenergetics in this aged cohort to platelets from younger adults (18–35 years) shows aged adults demonstrate lower basal and ATP-linked respiration. Platelets from older adults also show enhanced proton leak, which is likely due to increased protein levels of uncoupling protein 2, and correlates with increased gate speed in this cohort (r = 0.58; P = 0.0019). While no significant difference in glycolysis was observed in older adults compared to younger adults, platelet glycolytic rate correlated with fatigability (r = 0.44; P = 0.016). CONCLUSIONS. These data advance the mechanistic understanding of age-related changes in mitochondrial function. Further, they suggest that measuring platelet bioenergetics provides a potential supplement or surrogate for muscle biopsy measurement and may be a valuable tool to study mitochondrial involvement in age-related decline of physical function.

Authors

Andrea C. Braganza, Catherine G. Corey, Adam J. Santanasto, Giovanna Distefano, Paul M. Coen, Nancy W. Glynn, Seyed-Mehdi Nouraie, Bret H. Goodpaster, Anne B. Newman, Sruti Shiva

Age-dependent nigral dopaminergic neurodegeneration and α-synuclein accumulation in RGS6-deficient mice

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Abstract

Parkinson’s is primarily a non-familial, age-related disorder caused by α-synuclein accumulation and the progressive loss of dopamine neurons in the substantia nigra pars compacta (SNc). G protein-coupled receptor (GPCR)-cAMP signaling has been linked to a reduction in human Parkinson’s incidence and α-synuclein expression. Neuronal cAMP levels are controlled by GPCRs coupled to Gs or Gi/o, which increase or decrease cAMP, respectively. Regulator of G protein signaling 6 (RGS6) powerfully inhibits Gi/o signaling. Therefore, we hypothesized that RGS6 suppresses D2 autoreceptor- Gi/o signaling in SNc dopamine neurons promoting neuronal survival and reducing α-synuclein expression. Here we provide novel evidence that RGS6 critically suppresses late-age-onset SNc dopamine neuron loss and α-synuclein accumulation. RGS6 is restrictively expressed in human SNc dopamine neurons and, despite their loss in Parkinson’s, all surviving neurons express RGS6. RGS6-/- mice exhibit hyperactive D2 autoreceptors with reduced cAMP signaling in SNc dopamine neurons. Importantly, RGS6-/- mice recapitulate key sporadic Parkinson’s hallmarks, including: SNc dopamine neuron loss, reduced nigrostriatal dopamine, motor deficits, and α-synuclein accumulation. To our knowledge, Rgs6 is the only gene whose loss phenocopies these features of human Parkinson’s. Therefore, RGS6 is a key regulator of D2R-Gi/o signaling in SNc dopamine neurons, protecting against Parkinson’s neurodegeneration and α-synuclein accumulation.

Authors

Zili Luo, Katelin E. Ahlers-Dannen, Mackenzie M. Spicer, Jianqi Yang, Stephanie Alberico, Hanna E. Stevens, Nandakumar S. Narayanan, Rory A. Fisher

Induction of α cell-restricted Gc in dedifferentiating β cells contributes to stress-induced β-cell dysfunction

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Abstract

Diabetic β cell failure is associated with β cell dedifferentiation. To identify effector genes of dedifferentiation, we integrated analyses of histone methylation as a surrogate of gene activation status and RNA expression in β cells sorted from mice with multiparity-induced diabetes. Interestingly, only a narrow subset of genes demonstrated concordant changes to histone methylation and RNA levels in dedifferentiating β cells. Notable among them was the α cell signature gene Gc, encoding a vitamin D-binding protein. While diabetes was associated with Gc induction, Gc-deficient islets did not induce β cell dedifferentiation markers and maintained normal ex vivo insulin secretion in the face of metabolic challenge. Moreover, Gc-deficient mice exhibited a more robust insulin secretory response than normal controls during hyperglycemic clamps. The data are consistent with a functional role of Gc activation in β cell dysfunction, and indicate that multiparity-induced diabetes is associated with altered β cell fate.

Authors

Taiyi Kuo, Manashree Damle, Bryan J. González, Dietrich Egli, Mitchell A. Lazar, Domenico Accili