Abstract
Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.
Authors
Jessica B. Sarthi, Annie M. Trumbull, Shayda M. Abazari, Vincent van Unen, Joshua E. Chan, Yanfen Jiang, Jesse Gammons, Marc O. Anderson, Onur Cil, Calvin J. Kuo, Zachary M. Sellers
Abstract
Loss of ferroptosis contributes to the development of human cancer, and restoration of ferroptosis has been demonstrated as a potential therapeutic strategy in cancer treatment. However, the mechanisms of how ferroptosis escape contributes to ovarian cancer (OV) development are not well elucidated. Here, we show that ferroptosis negative regulation signatures correlated with the tumorigenesis of OV and were associated with poor prognosis, suggesting that restoration of ferroptosis represents a potential therapeutic strategy in OV. High-throughput drug screening with a kinase inhibitor library identified MEK inhibitors as ferroptosis inducers in OV cells. We further demonstrated that MEK inhibitor–resistant OV cells were less vulnerable to trametinib-induced ferroptosis. Mechanistically, mTOR/eIF4E binding protein 1 (4EBP1) signaling promoted solute carrier family 7 member 11 (SLC7A11) protein synthesis, leading to ferroptosis inhibition in MEK inhibitor–resistant cells. Dual inhibition of MEK and mTOR/4EBP1 signaling restrained the protein synthesis of SLC7A11 via suppression of the mTOR/4EBP1 axis to reactivate ferroptosis in resistant cells. Together, these findings provide a promising therapeutic option for OV treatment through ferroptosis restoration by the combined inhibition of MEK and mTOR/4EBP1 pathways.
Authors
Jiaxin Yin, Jianfeng Chen, Jing Han Hong, Yulin Huang, Rong Xiao, Shini Liu, Peng Deng, Yichen Sun, Kelila Xin Ye Chai, Xian Zeng, Jason Yongsheng Chan, Peiyong Guan, Yali Wang, Peili Wang, Chongjie Tong, Qiang Yu, Xiaojun Xia, Choon Kiat Ong, Bin Tean Teh, Ying Xiong, Jing Tan
Abstract
Sleep disturbance usually accompanies anxiety disorders and exacerbates their incidence rates. The precise circuit mechanisms remain poorly understood. Here, we found that glutamatergic neurons in the posteroventral medial amygdala (MePVGlu neurons) are involved in arousal and anxiety-like behaviors. Excitation of MePVGlu neurons not only promoted wakefulness but also increased anxiety-like behaviors. Different projections of MePVGlu neurons played various roles in regulating anxiety-like behaviors and sleep-wakefulness. MePVGlu neurons promoted wakefulness through the MePVGlu/posteromedial cortical amygdaloid area (PMCo) pathway and the MePVGlu/bed nucleus of the stria terminals (BNST) pathway. In contrast, MePVGlu neurons increased anxiety-like behaviors through the MePVGlu/ventromedial hypothalamus (VMH) pathway. Chronic sleep disturbance increased anxiety levels and reduced reparative sleep, accompanied by the enhanced excitability of MePVGlu/PMCo and MePVGlu/VMH circuits but suppressed responses of glutamatergic neurons in the BNST. Inhibition of the MePVGlu neurons could rescue chronic sleep deprivation–induced phenotypes. Our findings provide important circuit mechanisms for chronic sleep disturbance–induced hyperarousal response and obsessive anxiety-like behavior and are expected to provide a promising strategy for treating sleep-related psychiatric disorders and insomnia.
Authors
Ying Li, Yuchen Deng, Yifei Zhang, Dan Xu, Xuefen Zhang, Yue Li, Yidan Li, Ming Chen, Yuxin Wang, Jiyan Zhang, Like Wang, Yufeng Cang, Peng Cao, Linlin Bi, Haibo Xu
Abstract
Glucocorticoid synthesis by adrenal glands (AGs) is regulated by the hypothalamic-pituitary-adrenal axis to facilitate stress responses when the host is exposed to stimuli. Recent studies implicate macrophages as potential steroidogenic regulators, but the molecular mechanisms by which AG macrophages exert such influence remain unclear. In this study, we investigated the role of AG macrophages in response to cold challenge or atherosclerotic inflammation as physiologic models of acute or chronic stress. Using single-cell RNA sequencing, we observed dynamic AG macrophage polarization toward classical activation and lipid-associated phenotypes following acute or chronic stimulation. Among transcriptional alterations induced in macrophages, triggering receptor expressed on myeloid cells 2 (Trem2) was highlighted because of its upregulation following stress. Conditional deletion of macrophage Trem2 revealed a protective role in stress responses. Mechanistically, Trem2 deletion led to increased AG macrophage death, abolished the TGF-β–producing capacity of AG macrophages, and resulted in enhanced glucocorticoid production. In addition, enhanced glucocorticoid production was replicated by blockade of TGF-β signaling. Together, these observations suggest that AG macrophages restrict steroidogenesis through Trem2 and TGF-β, which opens potential avenues for immunotherapeutic interventions to resolve stress-related disorders.
Authors
Yingzheng Xu, Michael T. Patterson, Bastien Dolfi, Alisha Zhu, Adeline Bertola, Patricia R. Schrank, Alexandre Gallerand, Ainsley E. Kennedy, Hannah Hillman, Lynn Dinh, Sia Shekhar, Samuel Tollison, Tyler D. Bold, Stoyan Ivanov, Jesse W. Williams
Abstract
Osteoporotic fractures are a major complication of long-term glucocorticoid therapy. Glucocorticoids transiently increase bone resorption, but they predominantly inhibit bone formation and induce osteocyte apoptosis, leading to bone loss. Current treatments of glucocorticoid-induced osteoporosis aim mainly at reducing bone resorption and are, therefore, inadequate. We previously showed that signaling via the NO/cGMP/protein kinase G pathway plays a key role in skeletal homeostasis. Here, we show that pharmacological PKG activation with the guanylyl cyclase-1 activator cinaciguat or expression of a constitutively active, mutant PKG2R242Q restored proliferation, differentiation, and survival of primary mouse osteoblasts exposed to dexamethasone. Cinaciguat treatment of WT mice or osteoblast-specific expression of PKG2R242Q in transgenic mice prevented dexamethasone-induced loss of cortical bone mass and strength. These effects of cinaciguat and PKG2R242Q expression were due to preserved bone formation parameters and osteocyte survival. The basis for PKG2’s effects appeared to be through recovery of Wnt/β-catenin signaling, which was suppressed by glucocorticoids but critical for proliferation, differentiation, and survival of osteoblast-lineage cells. Cinaciguat reduced dexamethasone activation of osteoclasts, but this did not occur in the PKG2R242Q transgenic mice, suggesting a minor role in osteoprotection. We propose that existing PKG-targeting drugs could represent a novel therapeutic approach to prevent glucocorticoid-induced osteoporosis.
Authors
Shyamsundar Pal China, Hema Kalyanaraman, Shunhui Zhuang, Justin A. Cabriales, Robert L. Sah, Renate B. Pilz
Abstract
A defining feature of systemic lupus erythematosus (SLE) is loss of tolerance to self-DNA, and deficiency of DNASE1L3, the main enzyme responsible for chromatin degradation in blood, is also associated with SLE. This association can be found in an ultrarare population of pediatric patients with DNASE1L3 deficiency who develop SLE, adult patients with loss-of-function variants of DNASE1L3 who are at a higher risk for SLE, and patients with sporadic SLE who have neutralizing autoantibodies against DNASE1L3. To mitigate the pathogenic effects of inherited and acquired DNASE1L3 deficiencies, we engineered a long-acting enzyme biologic with dual DNASE1/DNASE1L3 activity that is resistant to DNASE1 and DNASE1L3 inhibitors. Notably, we found that the biologic prevented the development of lupus in Dnase1–/–Dnase1L3–/– double-knockout mice and rescued animals from death in pristane-induced lupus. Finally, we confirmed that the human isoform of the enzyme biologic was not recognized by autoantibodies in SLE and efficiently degraded genomic and mitochondrial cell–free DNA, as well as microparticle DNA, in SLE plasma. Our findings suggest that autoimmune diseases characterized by aberrant DNA accumulation, such as SLE, can be effectively treated with a replacement DNASE tailored to bypass pathogenic mechanisms, both genetic and acquired, that restrict DNASE1L3 activity.
Authors
Paul R. Stabach, Dominique Sims, Eduardo Gomez-Bañuelos, Sandra Zehentmeier, Kris Dammen-Brower, Andrew Bernhisel, Sophia Kujawski, Sam G. Lopez, Michelle Petri, Daniel W. Goldman, Ethan R. Lester, Quan Le, Tayyaba Ishaq, Hana Kim, Shivani Srivastava, Deepika Kumar, Joao P. Pereira, Kevin J. Yarema, Fotios Koumpouras, Felipe Andrade, Demetrios T. Braddock
Abstract
Cystic fibrosis (CF) is a genetic disorder that disrupts CF transmembrane conductance regulator (CFTR) anion channels and impairs airway host defenses. Airway inflammation is ubiquitous in CF, and suppressing it has generally been considered to improve outcomes. However, the role of inflammation in people taking CFTR modulators, small-molecule drugs that restore CFTR function, is not well understood. We previously showed that inflammation enhances the efficacy of CFTR modulators. To further elucidate this relationship, we treated human ΔF508-CF epithelia with TNF-α and IL-17, two inflammatory cytokines that are elevated in CF airways. TNF-α+IL-17 enhanced CFTR modulator–evoked anion secretion through mechanisms that raise intracellular Cl– (Na+/K+/2Cl– cotransport) and HCO3– (carbonic anhydrases and Na+/HCO3– cotransport). This enhancement required p38 MAPK signaling. Importantly, CFTR modulators did not affect CF airway surface liquid viscosity under control conditions but prevented the rise in viscosity in epithelia treated with TNF-α+IL-17. Finally, antiinflammatory drugs limited CFTR modulator responses in TNF-α+IL-17–treated epithelia. These results provide critical insights into mechanisms by which inflammation increases responses to CFTR modulators. They also suggest an equipoise between potential benefits and limitations of suppressing inflammation in people taking modulators, call into question current treatment approaches, and highlight a need for additional studies.
Authors
Tayyab Rehman, Alejandro A. Pezzulo, Andrew L. Thurman, Rachel L. Zemans, Michael J. Welsh
Abstract
The excessive formation and release of neutrophil extracellular traps (NETs) in sepsis may represent a substantial mechanism contributing to multiorgan damage, which is associated with a poor prognosis. However, the precise role of NETs in mediating the transition from innate immunity to adaptive immunity during the progression of inflammation and sepsis remains incompletely elucidated. In this study, we provide evidence that, despite a reduction in the number of CD4+ T cells in the late stage of sepsis, there is a notable upregulation in the proportion of Tregs. Mechanistically, we have identified that NETs can induce metabolic reprogramming of naive CD4+ T cells through the Akt/mTOR/SREBP2 pathway, resulting in enhanced cholesterol metabolism, thereby promoting their conversion into Tregs and augmenting their functional capacity. Collectively, our findings highlight the potential therapeutic strategy of targeting intracellular cholesterol normalization for the management of immunosuppressed patients with sepsis.
Authors
Yuxin Shi, Dan Wu, Yanghanzhao Wang, Yuwen Shao, Fu Zeng, Di Zhou, Hao Zhang, Changhong Miao
Abstract
Parasympathetic dysfunction after chronic myocardial infarction (MI) is known to predispose ventricular tachyarrhythmias (ventricular tachycardia/ventricular fibrillation [VT/VF]). VT/VF after MI is more common in males than females. The mechanisms underlying the decreased vagal tone and the associated sex difference in the occurrence of VT/VF after MI remain elusive. In this study, using optogenetic approaches, we found that responses of glutamatergic vagal afferent neurons were impaired following chronic MI in male mice, leading to reduced reflex efferent parasympathetic function. Molecular analyses of vagal ganglia demonstrated reduced glutamate levels, accompanied by decreased mitochondrial function and impaired redox status in infarcted males versus sham animals. Interestingly, infarcted females demonstrated reduced vagal sensory impairment, associated with greater vagal ganglia glutamate levels and decreased vagal mitochondrial dysfunction and oxidative stress compared with infarcted males. Treatment with 17β-estradiol mitigated this pathological remodeling and improved vagal neurotransmission in infarcted male mice. These data suggest that a decrease in efferent vagal tone following MI results from reduced glutamatergic afferent vagal signaling that may be due to impaired redox homeostasis in the vagal ganglia, which subsequently leads to pathological remodeling in a sex-dependent manner. Importantly, estrogen prevents pathological remodeling and improves parasympathetic function following MI.
Authors
Asokan Devarajan, Kerry Wang, Zulfiqar A. Lokhandwala, Maryam Emamimeybodi, Kassandra Shannon, John D. Tompkins, Andrea L. Hevener, Aldons J. Lusis, E. Dale Abel, Marmar Vaseghi
Abstract
Antibody-mediated depletion studies have demonstrated that CD8+ T cells are required for effective immune control of SIV. However, this approach is potentially confounded by several factors, including reactive CD4+ T cell proliferation, and provides no information on epitope specificity, a likely determinant of CD8+ T cell efficacy. We circumvented these limitations by selectively depleting CD8+ T cells specific for the Gag epitope CTPYDINQM (CM9) via the administration of immunotoxin-conjugated tetrameric complexes of CM9/Mamu-A*01. Immunotoxin administration effectively depleted circulating but not tissue-localized CM9-specific CD8+ T cells, akin to the bulk depletion pattern observed with antibodies directed against CD8. However, we found no evidence to indicate that circulating CM9-specific CD8+ T cells suppressed viral replication in Mamu-A*01+ rhesus macaques during acute or chronic progressive infection with a pathogenic strain of SIV. This observation extended to macaques with established infection during and after continuous antiretroviral therapy. In contrast, natural controller macaques experienced dramatic increases in plasma viremia after immunotoxin administration, highlighting the importance of CD8+ T cell–mediated immunity against CM9. Collectively, these data showed that CM9-specific CD8+ T cells were necessary but not sufficient for robust immune control of SIV in a nonhuman primate model and, more generally, validated an approach that could inform the design of next-generation vaccines against HIV-1.
Authors
Jennifer Simpson, Carly E. Starke, Alexandra M. Ortiz, Amy Ransier, Sam Darko, Sian Llewellyn-Lacey, Christine M. Fennessey, Brandon F. Keele, Daniel C. Douek, David A. Price, Jason M. Brenchley
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