Abstract
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 and 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 were still able to exit quiescence in response to other stress stimuli, such as LPS. We found that DKO HSCs and progenitors had impaired sensing of inflammatory signals ex vivo, and that levels of IL-1β and MIG were higher in the bone marrow (BM) after LPS than after 5-FU administration. Furthermore, exogenous in vivo administration of IL-1β could induce cell cycle entry of DKO HSCs. Our findings have clinical implications for the use of PI3K inhibitors in combination with chemotherapy.
Authors
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
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
Abstract
Parkinson’s disease (PD) is primarily a nonfamilial, age-related disorder caused by α-synuclein accumulation and the progressive loss of dopamine neurons in the substantia nigra pars compacta (SNc). GPCR-cAMP signaling has been linked to a reduction in human PD 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 potentially 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 PD, 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 PD 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 PD. Therefore, RGS6 is a key regulator of D2R-Gi/o signaling in SNc dopamine neurons, protecting against PD 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
Abstract
The ang1-tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (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 midgestation lethality due to heart defects associated with a hyperplastic but simplified trabecular meshwork (fewer but thicker trabeculae). Reduced proliferation and production of endocardial cells 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 cardiomyocytes, which is associated with upregulation of bone morphogenetic protein 10, increased retinoic acid (RA) signaling, and extracellular signal-regulated protein kinases 1 and 2 hyperphosphorylation in the myocardium. Intriguingly, myocardial phenotypes in conditional knockout hearts could be partially rescued by inhibiting in utero RA signaling with pan-RA receptor antagonist BMS493. These findings reveal 2 complementary functions of endocardial Tie2 during ventricular chamber formation: ensuring normal trabeculation by supporting endocardial cell proliferation and sprouting and preventing hypertrabeculation via suppression of RA signaling in trabecular cardiomyocytes.
Authors
Xianghu Qu, Cristina Harmelink, H. Scott Baldwin
Abstract
Recombinant adeno-associated virus–mediated (rAAV-mediated) gene delivery can efficiently target muscle tissues to serve as “biofactories” for secreted proteins in prophylactic and therapeutic scenarios. Nevertheless, efficient rAAV-mediated gene delivery is often limited by host immune responses against the transgene product. The development of strategies to prevent antitransgene immunity is therefore crucial. The use of endogenous microRNA-mediated (miRNA-mediated) regulation to detarget transgene expression from antigen-presenting cells (APCs) has shown promise for reducing immunogenicity. However, the mechanisms underlying miRNA-mediated modulation of antitransgene immunity by APC detargeting are not fully understood. Using the highly immunogenic ovalbumin (OVA) protein as a proxy for foreign antigens, we show that rAAV vectors containing miR142-binding sites efficiently repress costimulatory signals in DCs, significantly blunt the cytotoxic T cell response, allow for sustained transgene expression in skeletal myoblasts, and attenuate clearance of transduced muscle cells in mice. Furthermore, the blunting of humoral immunity against circulating OVA correlates with detargeting of OVA expression from APCs. This demonstrates that incorporating APC-specific miRNA-binding sites into rAAV vectors provides an effective strategy for reducing transgene-specific immune response. This approach holds promise for clinical applications where the safe and efficient delivery of a prophylactic or therapeutic protein is desired.
Authors
Yuanyuan Xiao, Manish Muhuri, Shaoyong Li, Wanru Qin, Guangchao Xu, Li Luo, Jia Li, Alexander J. Letizia, Sean K. Wang, Ying Kai Chan, Chunmei Wang, Sebastian P. Fuchs, Dan Wang, Qin Su, M. Abu Nahid, George M. Church, Michael Farzan, Li Yang, Yuquan Wei, Ronald C. Desrosiers, Christian Mueller, Phillip W.L. Tai, Guangping Gao
Abstract
Excess dietary salt contributes to inflammation and hypertension via poorly understood mechanisms. Antigen-presenting cells including 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 subpressor 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 diet–fed mice to germ-free mice predisposed them to increased inflammation and hypertension. These findings provide potentially novel insights 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. Simons, 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
Abstract
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 affect 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 both factors in a potentially novel model (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 insertion-deletion/substitution ratio and number 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 KrasG12D p53–/– sarcomas did not harbor recurrent oncogenic mutations; rather, they exhibited amplifications of specific oncogenes: Kras and Myc in KrasG12D p53–/– sarcomas and Met and Yap1 for radiation-induced sarcomas. These results reveal that different initiating events drive oncogenesis through distinct mechanisms.
Authors
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
Abstract
Alteration of innate immune cells in the lungs can promote loss of peripheral tolerance that leads to autoimmune responses in cigarette smokers. Development of autoimmunity in smokers with emphysema is also strongly linked to the expansion of autoreactive T helper (Th) cells expressing interferon γ (Th1), and interleukin 17A (Th17). However, the mechanisms responsible for enhanced self-recognition and reduced immune tolerance in smokers with emphysema remain less clear. Here we show that C1q, a component of the complement protein 1 complex (C1), is downregulated in lung CD1a+ antigen-presenting cells (APCs) isolated from emphysematous human and mouse lung APCs after chronic cigarette smoke exposure. C1q potentiated the function of APCs to differentiate CD4+ T cells to regulatory T cells (Tregs), while it inhibited Th17 cell induction and proliferation. Mice deficient in C1q that were exposed to chronic smoke exhibited exaggerated lung inflammation marked by increased Th17 cells, whereas reconstitution of C1q in the lungs enhanced Treg abundance, dampened smoke-induced lung inflammation, and prevented the development of emphysema. Our findings demonstrate that cigarette smoke–mediated loss of C1q could play a key role in reduced peripheral tolerance, which could be explored to treat emphysema.
Authors
Xiaoyi Yuan, Cheng-Yen Chang, Ran You, Ming Shan, Bon Hee Gu, Matthew C. Madison, Gretchen Diehl, Sarah Perusich, Li-Zhen Song, Lorraine Cornwell, Roger D. Rossen, Rick Wetsel, Rajapakshe Kimal, Cristian Coarfa, Holger K. Eltzschig, David B. Corry, Farrah Kheradmand
Abstract
The mechanisms regulating translation and splicing are not well understood. We provide insight into a new regulator of translation, 2-oxoglutarate and iron dependent oxygenase domain–containing protein 1 (OGFOD1), which is a prolyl-hydroxylase that catalyzes the posttranslational hydroxylation of Pro62 in the small ribosomal protein S23. We show that deletion of OGFOD1 in an in vitro model of human cardiomyocytes decreases translation of specific proteins (e.g., RNA-binding proteins) and alters splicing. RNA-Seq showed poor correlation between changes in mRNA and protein synthesis, suggesting that posttranscriptional regulation was the primary cause for the observed differences. We found that loss of OGFOD1 and the resultant alterations in protein translation modulated the cardiac proteome, shifting it toward higher protein amounts of sarcomeric proteins, such as cardiac troponins, titin, and cardiac myosin-binding protein C. Furthermore, we found a decrease of OGFOD1 during cardiomyocyte differentiation. These results suggest that loss of OGFOD1 modulates protein translation and splicing, thereby leading to alterations in the cardiac proteome, and highlight the role of altered translation and splicing in regulating the proteome.
Authors
Andrea Stoehr, Leslie Kennedy, Yanqin Yang, Sajni Patel, Yongshun Lin, Kaari L. Linask, Maria Fergusson, Jun Zhu, Marjan Gucek, Jizhong Zou, Elizabeth Murphy
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. Although 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 clamp studies. 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, Dieter Egli, Mitchell A. Lazar, Domenico Accili
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