Tuberculosis patients and mice infected with live Mycobacterium tuberculosis (Mtb) accumulate high numbers of myeloid-derived suppressor cells (MDSCs). Here, we hypothesized that also dead Mtb vaccines may induce MDSCs that could impair the efficacy of vaccination. We found that repeated injections of Mtb vaccines (heat-killed Mtb in Incomplete Freund's Adjuvant, like Montanide) but not single or control vaccines without Mtb strongly expanded CD11b+ myeloid cells in the spleen, that suppressed T cell proliferation and killing ex vivo. Dead Mtb vaccination induced the generation of CD11b+ Ly-6Chigh CD115+ iNOS/Nos2+ monocytic MDSCs (M-MDSCs) upon application of inflammatory or microbial activation signals. In vivo these M-MDSCs positioned strategically in the spleen by infiltrating the splenic bridging channels and white pulp areas. Notably, within 6 to 24 hours in a Nos2-dependent fashion they produced NO to rapidly kill conventional and plasmacytoid dendritic cells (cDCs, pDCs) while, surprisingly, sparing T cells in vivo. Thus, we demonstrate that Mtb vaccine induced M-MDSCs to not directly suppress T cell in vivo but, instead, M-MDSCs directly target DC subpopulations thereby indirectly suppressing effector T cell responses. Collectively, we demonstrate that Mtb booster vaccines induce M-MDSCs in the spleen that can be activated to kill DCs cautioning to thoroughly investigate MDSC formation in individuals after Mtb vaccination in clinical trials.
Eliana Ribechini, Ina Eckert, Andreas Beilhack, Nelita Du Plessis, Gerhard Walzl, Ulrike Schleicher, Uwe Ritter, Manfred B. Lutz
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.
Cibele Rocha-Resende, Carla Weinheimer, Geetika Bajpai, Luigi Adamo, Scot J. Matkovich, Joel Schilling, Philip M. Barger, Kory J. Lavine, Douglas L. Mann
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.
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
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.
Anna Zychlinsky Scharff, Matthieu Rousseau, Livia Lacerda Mariano, Tracy Canton, Camila Rosat Consiglio, Matthew L. Albert, Magnus Fontes, Darragh Duffy, Molly A. Ingersoll
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.
Lalita Oonthonpan, Adam J. Rauckhorst, Lawrence R. Gray, Audrey C. Boutron, Eric B. Taylor
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.
Zili Luo, Katelin E. Ahlers-Dannen, Mackenzie M. Spicer, Jianqi Yang, Stephanie Alberico, Hanna E. Stevens, Nandakumar S. Narayanan, Rory A. Fisher
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.
Taiyi Kuo, Manashree Damle, Bryan J. González, Dietrich Egli, Mitchell A. Lazar, Domenico Accili
Many cytokines and chemokines that are important for hematopoiesis activate the PI3K signaling pathway. Because this pathway is frequently mutated and activated in cancer, PI3K inhibitors have been developed for the treatment of several malignancies, and are now being tested in the clinic in combination with chemotherapy. However, the role of PI3K in adult hematopoietic stem cells (HSCs), particularly during hematopoietic stress, is still unclear. We previously showed that the individual PI3K catalytic isoforms P110α or P110β have dispensable roles in HSC function, suggesting redundancy between PI3K isoforms in HSCs. We now demonstrate that simultaneous deletion of P110α and P110δ in double knockout (DKO) HSCs uncovers their redundant requirement in HSC cycling after 5-fluorouracil (5-FU) chemotherapy administration. In contrast, DKO HSCs are still able to exit quiescence in response to other stress stimuli, such as LPS. We found that DKO HSCs and progenitors have impaired sensing of inflammatory signals ex vivo, and that levels of IL1-β and MIG are higher in the bone marrow after LPS than after 5-FU administration. Furthermore, exogenous in vivo administration of IL1-β can induce cell cycle entry of DKO HSCs. Our findings have important clinical implications for the use of PI3K inhibitors in combination with chemotherapy.
Shayda Hemmati, Taneisha Sinclair, Meng Tong, Boris Bartholdy, Rachel O. Okabe, Kristina Ames, Leanne Ostrodka, Tamanna Haque, Imit Kaur, Taylor S. Mills, Anupriya Agarwal, Eric M. Pietras, Jean J. Zhao, Thomas M. Roberts, Kira Gritsman
Cancer development is influenced by hereditary mutations, somatic mutations due to random errors in DNA replication, or external factors. It remains unclear how distinct cell-intrinsic and -extrinsic factors impact oncogenesis within the same tissue type. We investigated murine soft tissue sarcomas generated by oncogenic alterations (KrasG12D activation and p53 deletion), carcinogens (3-methylcholanthrene [MCA] or ionizing radiation), and in a novel model combining both factors (MCA plus p53 deletion). Whole-exome sequencing demonstrated distinct mutational signatures in individual sarcoma cohorts. MCA-induced sarcomas exhibited high mutational burden and predominantly G-to-T transversions, while radiation-induced sarcomas exhibited low mutational burden and a distinct genetic signature characterized by C-to-T transitions. The indel to substitution ratio and amount of gene copy number variations were high for radiation-induced sarcomas. MCA-induced tumors generated on a p53-deficient background showed the highest genomic instability. MCA-induced sarcomas harbored mutations in putative cancer-driver genes that regulate MAPK signaling (Kras and Nf1) and the Hippo pathway (Fat1 and Fat4). In contrast, radiation-induced sarcomas and KrasG12Dp53–/– sarcomas did not harbor recurrent oncogenic mutations, rather they exhibited amplifications of specific oncogenes: Kras and Myc in KrasG12Dp53–/– sarcomas, and Met and Yap1 for radiation-induced sarcomas. These results reveal that different initiating events drive oncogenesis through distinct mechanisms.
Chang-Lung Lee, Yvonne M. Mowery, Andrea R. Daniel, Dadong Zhang, Alexander B. Sibley, Joe R. Delaney, Amy J. Wisdom, Xiaodi Qin, Xi Wang, Isibel Caraballo, Jeremy Gresham, Lixia Luo, David Van Mater, Kouros Owzar, David G. Kirsch
The ang1-Tie2 pathway is required for normal vascular development, but its molecular effectors are not well-defined during cardiac ontogeny. Here we show that endocardial specific attenuation of Tie2 results in mid-gestation lethality due to heart defects associated with a hyperplastic but simplified trabecular meshwork (fewer but thicker trabeculae). Reduced proliferation and production of endocardial cells (ECs) following endocardial loss of Tie2 results in decreased endocardial sprouting required for trabecular assembly and extension. The hyperplastic trabeculae result from enhanced proliferation of trabecular cardiomyocyte (CMs), which is associated with upregulation of Bmp10, increased retinoic acid (RA) signaling, and Erk1/2 hyperphosphorylation in the myocardium. Intriguingly, myocardial phenotypes in Tie2-cko hearts could be partially rescued by inhibiting in utero RA signaling with pan-retinoic acid receptor antagonist BMS493. These findings reveal two complimentary functions of endocardial Tie2 during ventricular chamber formation: ensuring normal trabeculation by supporting EC proliferation and sprouting, and preventing hypertrabeculation via suppression of RA signaling in trabecular CMs.
Xianghu Qu, Cristina Harmelink, H. Scott Baldwin
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