T cells play an important role in acute kidney injury (AKI). Metabolic programming of T cells regulates their function, is a rapidly emerging field, and is unknown in AKI. We induced ischemic AKI in C57B6 mice and collected kidneys and spleens at multiple time points. T cells were isolated and analyzed by an immune-metabolic assay. Unbiased machine learning analyses identified a distinct T cell subset with reduced VDAC1 and mTOR expression in post-AKI kidneys. Ischemic kidneys showed higher expression of trimethylation of histone H3 lysine 27 (H3K27Me3) and glutaminase. Splenic T cells from post-AKI mice had higher expression of GLUT1, hexokinase II, and CPT1a. Human nonischemic and ischemic kidney tissue displayed similar findings to mouse kidneys. Given a convergent role for glutamine in T cell metabolic pathways and the availability of a relatively safe glutamine antagonist JHU083, effects on AKI were evaluated. JHU083 attenuated renal injury and reduced T cell activation and proliferation in ischemic and nephrotoxic AKI, whereas T cell-deficient mice were not protected by glutamine blockade. In vitro hypoxia demonstrated upregulation of glycolysis-related enzymes. T cells undergo metabolic reprogramming during AKI, and reconstitution of metabolism by targeting T cell glutamine pathway could be a promising novel therapeutic approach.
Kyungho Lee, Elizabeth A. Thompson, Sepideh Gharaie, Chirag H. Patel, Johanna T. Kurzhagen, Phillip M. Pierorazio, Lois J. Arend, Ajit G. Thomas, Sanjeev Noel, Barbara S. Slusher, Hamid Rabb
cyclic GMP-AMP synthase (cGAS) is a DNA sensor and responsible for inducing an anti-tumor immune response. Recent studies reveal cGAS is frequently inhibited in cancer, and therapeutic targets to promote anti-tumor cGAS function remain elusive. SRC is a proto-oncogene tyrosine kinase and is expressed at elevated levels in numerous cancers. Here, we demonstrate that SRC expression in primary and metastatic bladder cancer negatively correlates with innate immune gene expression and immune cell infiltration. We determine that SRC restricts cGAS signaling in human cell lines through SRC small molecule inhibitors, depletion, and overexpression. cGAS and SRC interact in cells and in vitro, while SRC directly inhibits cGAS enzymatic activity and DNA binding in a kinase-dependent manner. SRC phosphorylates cGAS, and inhibition of cGAS Y248 phosphorylation partially reduces SRC inhibition. Collectively, our study demonstrates that cGAS anti-tumor signaling is hindered by the proto-oncogene SRC and describes how cancer-associated proteins can regulate the innate immune system.
William Dunker, Shivam A. Zaver, Jose Mario Bello Pineda, Cameron J. Howard, Robert K. Bradley, Joshua J. Woodward
Fasting is associated with increased susceptibility to hypoglycemia in people with type 1 diabetes, thereby making it a significant health risk. To date, the relationship between fasting and insulin-induced hypoglycemia has not been well characterized, so our objective was to determine whether insulin-independent factors, such as counterregulatory hormone responses, are adversely impacted by fasting in healthy control subjects. Counterregulatory responses to insulin-induced hypoglycemia were measured in twelve healthy people during two metabolic studies. During one study, subjects ate breakfast and lunch, after which they underwent a two-hour bout of insulin-induced hypoglycemia (FED). During the other study, subjects remained fasted prior to hypoglycemia (FAST). As expected, hepatic glycogen concentrations were lower in FAST, and associated with diminished peak glucagon levels and reduced endogenous glucose production (EGP) during hypoglycemia. Accompanying lower EGP in FAST was a reduction in peripheral glucose utilization, and a resultant reduction in the amount of exogenous glucose required to maintain glycemia. These data suggest that whereas a fasting-induced lowering of glucose utilization could potentially delay the onset of insulin-induced hypoglycemia, subsequent reductions in glucagon levels and EGP are likely to encumber recovery from it. As a result of this diminished metabolic flexibility in response to fasting, susceptibility to hypoglycemia could be enhanced in patients with type 1 diabetes under similar conditions.
Shana O. Warner, Yufei Dai, Nicole Sheanon, Michael V. Yao, Rebecca L. Cason, Shahriar Arbabi, Shailendra B. Patel, Diana M. Lindquist, Jason J. Winnick
Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS gene encoding sacsin, a huge protein highly expressed in cerebellar Purkinje cells (PCs). ARSACS patients, as well as mouse models, display early degeneration of PCs, but the underlying mechanisms remain unexplored, with no available treatments. In this work, we demonstrated aberrant calcium (Ca2+) homeostasis and its impact on PC degeneration in ARSACS. Mechanistically, we found pathological elevation in Ca2+-evoked responses in Sacs-/- PCs, as the result of defective mitochondria and ER trafficking to distal dendrites and strong downregulation of key Ca2+ buffer-proteins. Alteration of cytoskeletal linkers, that we identified as specific sacsin interactors, likely account for faulty organellar trafficking in Sacs-/- cerebellum. Based on this pathogenetic cascade, we treated Sacs-/- mice with Ceftriaxone, a repurposed drug which exerts neuroprotection by limiting neuronal glutamatergic stimulation, and thus Ca2+ fluxes into PCs. Ceftriaxone treatment significantly improved motor performances of Sacs-/- mice, at both pre- and post-symptomatic stages. We correlated this effect to restored Ca2+ homeostasis, which arrests PC degeneration and attenuates secondary neuroinflammation. These findings disclose new key steps in ARSACS pathogenesis and support further optimization of Ceftriaxone in pre-clinical and clinical settings for the treatment of ARSACS patients.
Andrea Del Bondio, Fabiana Longo, Daniele De Ritis, Erica Spirito, Paola Podini, Bernard Brais, Angela Bachi, Angelo Quattrini, Francesca Maltecca
BACKGROUND. Current studies suggest mitochondrial dysfunction is a major contributor to impaired physical performance and exercise intolerance in chronic kidney disease (CKD). We conducted a clinical trial of coenzyme Q10 (CoQ10) and nicotinamide riboside (NR) to determine their impact on exercise tolerance and metabolic profile in CKD patients. METHODS. We conducted a randomized placebo-controlled, double blind, cross-over trial comparing CoQ10, NR, and placebo in 25 patients with eGFR of <60ml/min/1.73m2. Subjects received NR (1000 mg/day), CoQ10 (1200 mg/day), or placebo for 6 weeks each. Primary outcomes were aerobic capacity (VO2 peak) and work efficiency measured using graded cycle ergometry testing. We performed semi-targeted plasma metabolomics and lipidomics. RESUTS. Participant mean age was 61.0 ± 11.6 years and mean eGFR was 36.9±9.2 ml/min/1.73m2. Compared to placebo, we found no differences in VO2 peak (P=0.30, 0.17), total work (P=0.47, 0.77), and total work efficiency (P=0.46, 0.55) post NR or CoQ10 supplementation. NR decreased submaximal VO2 at 30W (P=0.03) and VO2 at 60W (P=0.07) compared to placebo. No changes in eGFR were observed post-NR or CoQ10 (P=0.14, 0.88). CoQ10 increased free fatty acids and decreased complex medium/long chain triglycerides. NR supplementation significantly altered TCA cycle intermediates and glutamate that are involved in reactions that exclusively use NAD+ and NADP+ as cofactors. NR decreased a broad range of lipid groups including triglycerides and ceramides. CONCLUSIONS. Six-weeks of treatment with NR or CoQ10 improved markers of systemic mitochondrial metabolism and lipid profiles but did not improve VO2 peak or total work efficiency. TRIAL REGISTRATION. ClinicalTrials.gov (NCT03579693) FUNDING. This study was supported by National Institutes of Diabetes and Digestive Kidney Diseases R01 DK101509 (to BK), R03 DK114502 (to BR), R01 DK125794 (to BR), R01 DK101509 (to JG), Dialysis Clinics Incorporated C-4112 (to BR), Northern California VA Health Care System (BR).
Armin Ahmadi, Gwenaelle Begue, Ana P. Valencia, Jennifer E. Norman, Benjamin Lidgard, Brian J. Bennett, Matthew P. Van Doren, David J. Marcinek, Sili Fan, David K. Prince, Jorge L. Gamboa, Jonathan Himmelfarb, Ian H. de Boer, Bryan R. Kestenbaum, Baback Roshanravan
Trigeminal neuralgia (TN) is a classical neuralgic pain condition with distinct clinical characteristics. Modeling TN in rodents proves challenging. Recently, we found that a foramen in rodent skull base, the foramen lacerum, provides direct access to the trigeminal nerve root. Using this access, we developed FLIT (Foramen Lacerum Impingement of Trigeminal nerve root) model and observed distinct pain-like behaviors in rodents, including paroxysmal asymmetric facial grimaces, head tilt when eating, avoidance of solid chew, lack of wood chewing, etc. The FLIT model recapitulated key clinical features of TN, including lancinating pain-like behavior, and dental pain-like behavior. Importantly, when compared with a trigeminal neuropathic pain model (infraorbital nerve chronic constriction injury, IoN-CCI), the FLIT model was associated with significantly higher numbers of c-Fos positive cells in the primary somatosensory cortex (S1), unraveling robust cortical activation in the FLIT model. Using intravital two-photon calcium imaging, synchronized S1 neural dynamics were only present in the FLIT but not the IoN-CCI model, revealing differential implication of cortical activation in different pain models. Taken together, FLIT is a clinically relevant rodent model of TN which could facilitate pain research and therapeutics development.
Weihua Ding, Liuyue Yang, Qian Chen, Kun Hu, Yan Liu, Eric Bao, Changning Wang, Jianren Mao, Shiqian Shen
Cephalic tetanus (CT) is a severe form of tetanus that follows head wounds and the intoxication of cranial nerves by tetanus neurotoxin (TeNT). Hallmarks of CT are cerebral palsy, which anticipates the typical spastic paralysis of tetanus, and rapid evolution of cardiorespiratory deficit even without generalized tetanus. How TeNT causes this unexpected flaccid paralysis, and how the canonical spasticity then rapidly evolves into cardiorespiratory defects remain unresolved aspects of CT pathophysiology. Using electrophysiology and immunohistochemistry, we demonstrate that TeNT cleaves its substrate VAMP within facial neuromuscular junctions and causes a botulism-like paralysis overshadowing its canonical spasticity. Meanwhile, TeNT spreads among brainstem neuronal nuclei and, as shown by an assay to monitor the ventilation ability of CT mice, it harms essential functions like respiration. A partial axotomy of the facial nerve revealed a still-unknown ability of TeNT to undergo intra-brainstem diffusion, which allows the toxin to spread onto brainstem nuclei devoid of direct peripheral efferents. Other showing a mechanism possibly involved in the transition from local to generalized tetanus, these findings suggest that patients with idiopathic facial nerve palsy should be immediately considered for CT and treated with antisera to block the potential progression of a life-threatening form of tetanus.
Federico Fabris, Stefano Varani, Marika Tonellato, Ivica Matak, Petra Šoštarić, Patrik Meglić, Matteo Caleo, Aram Megighian, Ornella Rossetto, Cesare Montecucco, Marco Pirazzini
Respiratory syncytial virus (RSV) infection causes significant morbidity and mortality in infants, immunocompromised, and older individuals. There is an urgent need for effective antivirals and vaccines for high risk individuals. We used two complementary in vivo models to analyze RSV-associated human lung pathology and human immune correlates of protection. RSV infection resulted in widespread human lung epithelial damage, a pro-inflammatory innate immune response, and elicited a natural adaptive human immune response that conferred protective immunity. We demonstrated a key role for human T cells in controlling RSV infection. Specifically, primed human CD8+ T cells or CD4+ T cells effectively and independently control RSV replication in human lung tissue in the absence of an RSV-specific antibody response. These preclinical data support the development of RSV vaccines which also elicit effective T cell responses to improve RSV vaccine efficacy.
Chandrav De, Raymond J. Pickles, Wenbo Yao, Baolin Liao, Allison E. Boone, Mingyu Choi, Diana M. Battaglia, Frederic B. Askin, Jason K. Whitmire, Guido Silvestri, J. Victor Garcia, Angela Wahl
Respiration can positively impact cerebrospinal fluid (CSF) flow in the brain, yet its effects on central nervous system (CNS) fluid homeostasis including waste clearance function via the glymphatic and meningeal lymphatic systems remain unclear. Here, we investigated the effect of supporting respiratory function via continuous positive airway pressure (CPAP) on glymphatic-lymphatic function in spontaneously breathing anesthetized rodents. To do this, we used a systems approach combining engineering, magnetic resonance imaging, computational fluid dynamics analysis, and physiological testing. We first designed a nasal CPAP device for use in the rat and demonstrated that it functioned similar to clinical devices as evidenced by its ability to open the upper airway, augment end-expiratory lung volume, and improve arterial oxygenation. We further showed that CPAP increased CSF flow speed at the skull base and augmented glymphatic transport regionally. The CPAP-induced augmented CSF flow speed was associated with an increase in intracranial pressure (ICP), including the ICP waveform pulse amplitude. We suggest that the augmented pulse amplitude with CPAP underlies the increase in CSF bulk flow and glymphatic transport. Our results provide new insights into the functional crosstalk at the pulmonary-CSF interface and suggest that CPAP might have therapeutic benefit for sustaining glymphatic-lymphatic function.
Burhan Ozturk, Sunil Koundal, Ehab Al Bizri, Xinan Chen, Zachary H. Gursky, Feng Dai, Andrew S. Lim, Paul Heerdt, Jonathan Kipnis, Allen Tannenbaum, Hedok Lee, Helene Benveniste
BACKGROUND. There is considerable heterogeneity in the effect of weight loss on metabolic function in people with obesity. METHODS. We evaluated muscle and liver insulin sensitivity, body composition, and circulating factors associated with insulin action before and after ~20% weight loss in women identified as “Responders” (n=11) or “Non-responders” (n=11), defined as the top (>75% increase) and bottom (<5% increase) quartiles of the weight loss-induced increase in glucose disposal rate (GDR) during a hyperinsulinemic-euglycemic clamp procedure, among 43 women with obesity (BMI: 44.1±7.9 kg/m2). RESULTS. At baseline, GDR, which provides an index of muscle insulin sensitivity, and the hepatic insulin sensitivity index were >50% lower in Responders than Non-Responders, but both increased much more after weight loss in Responders than Non-responders, which eliminated the differences between groups. Weight loss also caused greater decreases in intrahepatic triglyceride content and plasma adiponectin and PAI-1 concentrations in Responders than Non-Responders and greater insulin-mediated suppression of plasma free fatty acids, branched-chain amino acids and C3/C5 acylcarnitines in Non-Responders than Responders, so that differences between groups at baseline were no longer present after weight loss. The effect of weight loss on total body fat mass, intra-abdominal adipose tissue volume, adipocyte size, and circulating inflammatory markers were not different between groups. CONCLUSION. The results from our study demonstrate the heterogeneity in the effects of marked weight loss on muscle and hepatic insulin sensitivity in people with obesity is determined by baseline insulin action, and reaches a ceiling when “normal” insulin action is achieved. CLINICAL TRIAL REGISTRATION. NCT00981500, NCT01299519, NCT02207777 FUNDING. This study was supported by National Institutes of Health grants P30 DK056341 (Washington University Nutrition and Obesity Research Center), P30 DK020579 (Washington University Diabetes Research Center), P30 DK052574 (Washington University Digestive Disease Research Center), and UL1 TR002345 (Washington University Institute of Clinical and Translational Sciences), T32 HL130357 (Obesity and Cardiovascular Disease Postdoctoral Training Program), grants from the American Diabetes Association (1-18-ICTS-119), the Longer Life Foundation (2019-011), and the Atkins Philanthropic Trust.
Bettina Mittendorfer, Brandon D. Kayser, Mihoko Yoshino, Jun Yoshino, Jeramie D. Watrous, Mohit Jain, J. Christopher Eagon, Bruce W. Patterson, Samuel Klein
Laryngotracheal stenosis (LTS) is pathologic fibrotic narrowing of the larynx and trachea characterized by hypermetabolic fibroblasts and CD4-mediated inflammation. However, the role of CD4 T-cells in promoting LTS fibrosis is unknown. The mechanistic target of rapamycin (mTOR) signaling pathways have been shown to regulate T-cell phenotype. Here we sought to investigate the influence of mTOR signaling in CD4 T-cells on LTS pathogenesis. In this study, human LTS specimens revealed an increased population of CD4-cells expressing the activated isoform of mTOR. In a murine LTS model, targeting mTOR with systemic sirolimus and a sirolimus-eluting airway stent reduced fibrosis and TH17-cells. Selective deletion of mTOR in CD4+-cells reduced TH17-cells and attenuated fibrosis, demonstrating CD4-cells’ pathologic role in LTS. Multispectral immunofluorescence of human LTS revealed increased TH17-cells. In-vitro, TH17-cells increased collagen-1 production by LTS fibroblasts, which was prevented with sirolimus pretreatment of TH17-cells. Collectively, mTOR signaling drives pathologic CD4 T-cell phenotypes in LTS, and targeting mTOR with sirolimus is effective at treating LTS through inhibition of pro-fibrotic TH17-cells. Finally, sirolimus may be delivered locally with a drug-eluting stent, transforming clinical therapy for LTS.
Kevin M. Motz, Ioan A. Lina, Idris Samad, Michael K. Murphy, Madhavi Duvvuri, Ruth J. Davis, Alexander Gelbard, Liam Chung, Yee Chan-Li, Samuel Collins, Jonathan D. Powell, Jennifer H. Elisseeff, Maureen R. Horton, Alexander T. Hillel
Immune responses in people with multiple sclerosis (pwMS) on disease-modifying therapies (DMTs) have been of significant interest throughout the COVID-19 pandemic. Lymphocyte-targeting immunotherapies including anti-CD20 treatments and sphingosine-1-phosphate receptor (S1PR) modulators attenuate antibody responses after vaccination. Evaluation of cellular responses after vaccination is therefore of particular importance in these populations. In this study, we analysed CD4 and CD8 T cell functional responses to SARS-CoV-2 spike peptides in healthy controls and pwMS on five different DMTs by flow cytometry. Although pwMS on rituximab and fingolimod therapies had low antibody responses after both two and three vaccine doses, T cell responses in pwMS on rituximab were preserved after a third vaccination, even when an additional dose of rituximab was administered between vaccine doses two and three. PwMS taking fingolimod had low detectable T cell responses in peripheral blood. CD4 and CD8 T cell responses to SARS-CoV-2 variants of concern Delta and Omicron were lower than to the ancestral Wuhan-Hu-1 variant. Our results indicate the importance of assessing both cellular and humoral responses after vaccination and suggest that even in the absence of robust antibody responses vaccination can generate immune responses in pwMS.
Asia-Sophia Wolf, Anthony Ravussin, Marton König, Mathias H. Øverås, Guri Solum, Ingrid Fadum Kjønstad, Adity Chopra, Trygve Holmøy, Hanne F. Harbo, Silje Watterdal Syversen, Kristin Kaasen Jørgensen, Einar A. Høgestøl, John T. Vaage, Elisabeth G. Celius, Fridtjof Lund-Johansen, Ludvig A. Munthe, Gro Owren Nygaard, Siri Mjaaland
Altered mitochondrial function without a well-defined cause has been documented in the patients with ulcerative colitis (UC). In our efforts to understand UC pathogenesis, we observed reduced expression of clustered mitochondrial homologue, CLUH, only in the active UC tissues compared to the unaffected areas from the same patient and healthy controls. Stimulation with bacterial toll like receptor (TLR) ligands similarly reduced CLUH expression in the human primary macrophages. Further, CLUH negatively regulated secretion of pro-inflammatory cytokines IL6, TNF-α and rendered a pro-inflammatory niche in the TLR stimulated macrophage. CLUH was further found to bind to mitochondrial fission protein DRP-1 and also regulated DRP-1 transcription in the human macrophages. In the TLR ligand stimulated macrophages, absence of CLUH led to enhanced DRP-1 availability for mitochondrial fission and smaller dysfunctional mitochondrial pool was observed. Mechanistically, this fissioned mitochondrial pool in turn enhanced mitochondrial ROS production, reduced mitophagy and lysosomal function in the CLUH knockout macrophages. Remarkably, our studies in the mice model of colitis with CLUH knockdown displayed exacerbated disease pathology. Taken together, this is the first report signifying the role of CLUH in UC pathogenesis, by means of regulating inflammation via maintaining mitochondrial-lysosomal functions in the human macrophages and intestinal mucosa.
Shaziya Khan, Desh Raj, Shikha Sahu, Anam Naseer, Nishakumari C. Singh, Sunaina Kumari, Sharmeen Ishteyaque, Jyotsna Sharma, Promila Lakra, Madhav Nilakanth Mugale, Arun Kumar Trivedi, Mrigank Srivastava, Tulika Chandra, Vivek Bhosale, Manoj Kumar Barthwal, Shashi Kumar Gupta, Kalyan Mitra, Aamir Nazir, Uday C. Ghoshal, Amit Lahiri
Obesity-associated metabolic inflammation drives the development of insulin resistance and type 2 diabetes, notably through modulating innate and adaptive immune cells in metabolic organs. The nutrient sensor liver kinase B1 (LKB1) has recently been shown to control cellular metabolism and T cell priming functions of dendritic cells (DCs). Here, we report that hepatic DCs from high-fat diet (HFD)-fed obese mice display increased LKB1 phosphorylation and that LKB1 deficiency in DCs (CD11cΔLKB1) worsened HFD-driven hepatic steatosis and impaired glucose homeostasis. Loss of LKB1 in DCs was associated with increased expression of T helper 17-polarizing cytokines and accumulation of hepatic IL-17A+ T helper cells in HFD-fed mice. Importantly, IL-17A neutralization rescued metabolic perturbations in HFD-fed CD11cΔLKB1 mice. Mechanistically, deficiency of the canonical LKB1 target AMPK in HFD-fed CD11cΔAMPKα1 mice recapitulated neither the hepatic Th17 phenotype nor the disrupted metabolic homeostasis, suggesting the involvement of other and/or additional LKB1 downstream effectors. We indeed provide evidence that the control of Th17 responses by DCs via LKB1 is actually dependent on both AMPKalpha1 and AMPK-related salt-inducible kinase(s) signaling. Altogether, our data reveal a key role for LKB1 signaling in DCs in protection against obesity-induced metabolic dysfunctions by limiting hepatic Th17 responses.
Hendrik J.P. van der Zande, Eline C. Brombacher, Joost M. Lambooij, Leonard R. Pelgrom, Anna Zawistowska-Deniziak, Thiago A. Patente, Graham A. Heieis, Frank Otto, Arifa Ozir-Fazalalikhan, Maria Yazdanbakhsh, Bart Everts, Bruno Guigas
Elevation of glucagon levels and increase in alpha cell proliferation is associated with states of hyperglycemia in diabetes. A better understanding of the molecular mechanisms governing glucagon secretion could have major implications in understanding abnormal responses to hypoglycemia in diabetes patients and provide novel avenues for diabetes management. Our previous studies have highlighted the role of nutrient signaling via mTOR complex 1 (mTORC1) regulation that controls glucagon secretion and alpha cell mass and that hyperglucagonemia can improve glucose homeostasis by diminishing glucagon action in the liver. However, it is unclear if short-term effects of mTORC1 activation are sufficient to induce glucagon secretion without changes in alpha cell mass and whether short-term hyperglucagonemia reduces liver glucagon action in a reversible manner. Using mice with inducible induction of the regulator of the mTORC1 complex (Rheb) in alpha cells (αRhebTg), we showed that short-term activation of mTORC1 signaling is sufficient to induce hyperglucagonemia as a result of increased glucagon secretion. Hyperglucagonemia in the αRhebTg was also associated with an increase in alpha cell size and mass expansion. This model allowed us to identify the effects of chronic and short-term hyperglucagonemia on glucose homeostasis by regulating glucagon signaling in the liver. Short-term hyperglucagonemia impaired glucose tolerance, which was reversible over time. Decrease in liver glucagon effects in αRhebTg mice was associated with reduced expression of the glucagon receptor (GCGR) and genes involved in gluconeogenesis, amino acid metabolism, and urea production. However, only genes regulating gluconeogenesis returned to baseline upon improvement of glycemia. Overall, these studies demonstrate that hyperglucagonemia exerts a biphasic response on glucose metabolism: short-term hyperglucagonemia leads to glucose intolerance, whereas chronic exposure to glucagon generates decrease on hepatic glucagon action along with improved glucose tolerance.
Camila Lubaczeuski, Nadejda Bozadjieva-Kramer, Ruy A. Louzada, George K. Gittes, Gil Leibowitz, Ernesto Bernal-Mizrachi
Elevated blood glucose levels, or hyperglycemia, can increase brain excitability and amyloid-beta (Aβ) release offering a mechanistic link between type-2-diabetes and Alzheimer’s disease (AD). Since the cellular mechanisms governing this relationship are poorly understood, we explored whether ATP-sensitive potassium (KATP) channels, which couple changes in energy availability with cellular excitability, play a role in AD pathogenesis. First, we demonstrate that KATP channel subunits, Kir6.2/KCNJ11 and SUR1/ABCC8 are expressed on excitatory and inhibitory neurons in the human brain and cortical expression of KCNJ11 and ABCC8 changes with Alzheimer’s pathology in humans and mice. Next, we explored whether eliminating neuronal KATP channel activity uncoupled the relationship between metabolism, excitability, and Aβ pathology in a novel mouse model of cerebral amyloidosis and neuronal KATP channel ablation (e.g. APP/PS1, Kir6.2-/- mouse). Using both acute and chronic paradigms, we demonstrate that Kir6.2-KATP channels are metabolic sensors that regulate hyperglycemic-dependent increases in interstitial fluid levels of Aβ, amyloidogenic processing of APP, and amyloid plaque formation, which may be dependent on lactate release. These studies identify a new role for Kir6.2-KATP channels in Alzheimer’s disease and suggest that pharmacological manipulation of Kir6.2-KATP channels holds therapeutic promise in reducing Aβ pathology in diabetic or prediabetic patients.
John Grizzanti, William R. Moritz, Morgan C. Pait, Molly Stanley, Sarah D. Kaye, Caitlin M. Carroll, Nicholas J. Constantino, Lily J. Deitelzweig, James A. Snipes, Derek Kellar, Emily E. Caesar, Ryan J. Pettit-Mee, Stephen M. Day, Jonathon P. Sens, Noelle I. Nicol, Jasmeen Dhillon, Maria S. Remedi, Drew D. Kiraly, Celeste M. Karch, Colin G. Nichols, David M. Holtzman, Shannon L. Macauley
Alloreactivity can drive autoimmune syndromes. After allogeneic hematopoietic stem cell transplantation (allo-HCT) chronic graft-versus-host disease (cGVHD), a B cell-mediated autoimmune-like syndrome, commonly occurs. Because donor-derived B cells continually develop under selective pressure from host alloantigens, aberrant B Cell Receptor (BCR)-activation and IgG production can emerge and contribute to cGVHD pathobiology. To better understand molecular programing of B cells under selective pressure of alloantigens, we performed scRNA-Seq analysis on high numbers of purified B cells from allo-HCT patients. An unsupervised analysis revealed 10 clusters, distinguishable by signature genes for maturation, activation and memory. We found striking transcriptional differences in the memory B cell compartment after allo-HCT compared to healthy or infected individuals. To identify intrinsic properties when B-cell tolerance is lost after allo-HCT, we then assessed clusters for differentially expressed genes (DEGs) between patients with vs. without autoimmune-like manifestations (Active cGVHD vs. No cGVHD, respectively). DEGs were found in Active cGVHD in both naive and BCR-activated clusters, suggesting functional diversity. Some DEGs were also differentially expressed across most clusters, suggesting common molecular programs that may promote B cell plasticity. Our study of human allo-HCT and cGVHD provides new understanding of B-cell memory in the face of chronic alloantigen stimulation.
Jonathan C. Poe, Jiyuan Fang, Dadong Zhang, Marissa R. Lee, Rachel A. DiCioccio, Hsuan Su, Xiaodi Qin, Jennifer Y. Zhang, Jonathan Visentin, Sonali J. Bracken, Vincent T. Ho, Kathy S. Wang, Jeremy J. Rose, Steven Z. Pavletic, Frances T. Hakim, Wei Jia, Amy N. Suthers, Itaevia M. Curry-Chisolm, Mitchell E. Horwitz, David A. Rizzieri, William C. McManigle, Nelson J. Chao, Adela R. Cardones, Jichun Xie, Kouros Owzar, Stefanie Sarantopoulos
BACKGROUND. The Omicron BA.5 subvariant of SARS-CoV-2 markedly escapes neutralizing antibodies induced by vaccination due to mutations in the Spike (S) protein. Solid organ transplant recipients (SOTRs) suffer high COVID-19 morbidity and demonstrate poor Omicron strain recognition after COVID-19 vaccination. T cell responses may provide a crucial second line of defense. Therefore, it is critical to understand which vaccine regimens induce robust, conserved T cell responses. METHODS. We evaluated anti-S IgG titers, subvariant pseudo-neutralization, and S-specific CD4+ and CD8+ T cell responses from SOTRs in a national, prospective observational trial (n=75). Participants were selected if they received 3 doses of mRNA (homologous boosting) or two doses of mRNA followed by Ad26.COV2.S (heterologous boosting). RESULTS. Homologous boosting with three mRNA doses induced the highest anti-S IgG titers. However, antibodies induced by both vaccine regimens demonstrated significantly lower pseudo-neutralization against BA.5 compared to the ancestral strain. In contrast, vaccine-induced S-specific T cells maintained cross-reactivity against BA.5 compared to ancestral recognition. Homologous boosting induced higher frequencies of activated polyfunctional CD4+ T cell responses, with polyfunctional IL-21+ peripheral T follicular helper cells increased in mRNA-1273 compared to BNT¬¬162b2. IL-21+ cells robustly correlated with antibody titers. Heterologous boosting with Ad26.COV2.S did not increase CD8+ responses compared to homologous boosting. CONCLUSIONS. These data demonstrate that boosting with the ancestral strain can induce cross-reactive T cell responses against emerging variants of concern in SOTRs, but alterative vaccine strategies are required to induce robust CD8+ T cell responses. TRIAL REGISTRATION. IRB00248540 FUNDING. U01AI138897, U54CA260492, Emory COVID-19 research repository
Elizabeth A. Thompson, Wabathi Ngecu, Laila Stoddart, T. Scott Johnston, Amy Chang, Katherine Cascino, Jennifer L. Alejo, Aura T. Abedon, Hady Samaha, Nadine Rouphael, Aaron A.R. Tobian, Dorry L. Segev, William A. Werbel, Andrew H. Karaba, Joel N. Blankson, Andrea L. Cox
DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.
Amjad Horani, Deepesh Gupta, Jian Xu, Huihui Xu, Lis del C. Puga Molina, Celia M. Santi, Sruthi Ramagiri, Steven K. Brennan, Jiehong Pan, Jeffrey R. Koenitzer, Tao Huang, Rachael M. Hyland, Sean P. Gunsten, Shin-Cheng Tzeng, Jennifer M. Strahle, Pleasantine Mill, Moe R. Mahjoub, Susan K. Dutcher, Steven L. Brody
The development and progression of endometriotic lesions are poorly understood, but immune cell dysfunction and inflammation are closely associated with the pathophysiology of endometriosis. There is a need for 3D in vitro models to permit the study of interactions between cell types and the microenvironment. To address this, we developed endometriotic spheroids (ES) to explore the role of epithelial-stromal interactions and model peritoneal invasion associated with lesion development. Using a non-adherent microwell culture system, spheroids were generated with immortalized endometriotic epithelial cells(12Z) combined with endometriotic stromal (iEc-ESC) or uterine stromal (iHUF) cell lines. Transcriptomic analysis found 4,522 differentially expressed genes in ES compared to spheroids containing uterine stromal cells. The top increased gene sets were inflammation-related pathways, and an overlap with baboon endometriotic lesions was highly significant. Finally, to mimic invasion of endometrial tissue into the peritoneum, a model was developed with human peritoneal mesothelial cells in an extracellular matrix. Invasion was increased in presence of estradiol or proinflammatory macrophages and suppressed by a progestin. Taken together, our results strongly support the concept that ES are an appropriate model for dissecting mechanisms that contribute to endometriotic lesion development.
Yong Song, Gregory W. Burns, Niraj R. Joshi, Ripla Arora, Ji-Yong Julie Kim, Asgerally T. Fazleabas