Research
Abstract
Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare but serious disease with poorly understood mechanisms. Here we report that patients with EGPA have elevated levels of TSLP, IL-25, and sST2, well characterized cytokine “alarmins” that activate or modulate type 2 innate lymphoid cells (ILC2s). Patients with active EGPA have a concurrent reduction in circulating ILC2s, suggesting a role for ILC2s in the pathogenesis of this disease. To explore the mechanism of these findings in patients, we established a model of EGPA in which active vasculitis and pulmonary hemorrhage are induced by IL-33 administration in predisposed, hypereosinophilic mice. In this model, induction of pulmonary hemorrhage and vasculitis is dependent on ILC2s and signaling through IL4Ra. In the absence of IL4Ra or STAT6, IL-33-treated mice have less vascular leak and pulmonary edema, less endothelial activation, and reduced eotaxin production, cumulatively leading to a reduction of pathologic eosinophil migration into the lung parenchyma. These results offer a mouse model for use in future mechanistic studies of EGPA, and suggest that IL-33, ILC2s and IL4Ra signaling may be potential targets for further study and therapeutic targeting in patients with EGPA.
Authors
Maya E. Kotas, Jérémie Dion, Steven Van Dyken, Roberto R. Ricardo-Gonzalez, Claire J. Danel, Camille Taillé, Luc Mouthon, Richard M. Locksley, Benjamin Terrier
Natural killer cells associate with amyotrophic lateral sclersois in a sex- and age-dependent manner
Abstract
NK cells are innate immune cells implicated in ALS; whether NK cells impact ALS in a sex- and age-specific manner was investigated. In mice, NK cells were depleted in male and female SOD1G93A ALS mice, survival and neuroinflammation were assessed, and data were stratified by sex. NK cell depletion extended survival in female but not male ALS mice with sex-specific effects on spinal cord microglia. In humans, NK cell numbers, NK cell subpopulations, and NK cell surface markers were examined in prospectively collected blood from ALS and control subjects; longitudinal changes in these metrics were correlated to Revised ALS Functional Rating Scale (ALSFRS-R) slope and stratified by sex and age. Expression of NK cell trafficking and cytotoxicity markers were elevated in ALS subjects, and changes in CXCR3+ NK cells and seven trafficking and cytotoxicity markers (CD11a, CD11b, CD38, CX3CR1, NKG2D, NKp30, NKp46) correlated with disease progression. Age impacted the associations between ALSFRS-R and markers NKG2D and NKp46, while sex impacted the NKp30 association. Collectively, these findings suggest that NK cells contribute to ALS progression in a sex- and age-specific manner and demonstrate that age and sex are critical variables when designing and assessing ALS immunotherapy.
Authors
Benjamin J. Murdock, Joshua P. Famie, Caroline E. Piecuch, Kristen D. Raue, Faye E. Mendelson, Cole H. Pieroni, Sebastian D. Iniguez, Lili Zhao, Stephen A. Goutman, Eva L. Feldman
Abstract
Right ventricular (RV) fibrosis is a key feature of maladaptive RV hypertrophy and dysfunction and is associated with poor outcomes in pulmonary hypertension (PH). However, mechanisms and therapeutic strategies to mitigate RV fibrosis remain unrealized. Previously, we identified that cardiac fibroblast α7 nicotinic acetylcholine receptor (α7 nAChR) drives smoking induced RV fibrosis. Here we sought to define the role of α7 nAChR in RV dysfunction and fibrosis in the settings of RV pressure overload as seen in PH. We show that RV tissue from PH patients has increased collagen content and ACh expression. Using experimental rat model of PH, we demonstrate that RV fibrosis and dysfunction are associated with increases in ACh and α7 nAChR expression in the RV but not in the LV. In vitro studies show that α7 nAChR activation leads to an increase in adult ventricular fibroblast proliferation and collagen content mediated by a Ca2+/ epidermal growth factor receptor (EGFR) signaling mechanism. Pharmacological antagonism of nAChR decreases RV collagen content and improves RV function in the PH model. Further, mice lacking α7 nAChR exhibit improved RV diastolic function and have lower RV collagen content in response to persistently increased RV afterload, compared to wild-type controls. These finding indicate that enhanced α7 nAChR signaling is an important mechanism underlying RV fibrosis and dysfunction, and targeted inhibition of α7 nAChR is a novel therapeutic strategy in the setting of increased RV afterload.
Authors
Alexander Vang, Denielli da Silva Gonçalves Bos, Ana Fernandez-Nicolas, Peng Zhang, Alan R. Morrison, Thomas J. Mancini, Richard T. Clements, Iuliia Polina, Michael W. Cypress, Bong Sook Jhun, Edward Hawrot, Ulrike Mende, Jin O-Uchi, Gaurav Choudhary
Abstract
The hypothalamus is a critical regulator of glucose metabolism and is capable of correcting diabetes conditions independently of an effect on energy balance. The small GTPase Rap1 in the forebrain is implicated in high-fat diet (HFD)-induced obesity and glucose imbalance. Here, we report that increasing Rap1 activity selectively in the medial hypothalamus elevated blood glucose without increasing the body weight of HFD-fed mice. In contrast, decreasing hypothalamic Rap1 activity protected mice from diet-induced hyperglycemia but did not prevent weight gain. The remarkable glycemic effect of Rap1 was reproduced when Rap1 was specifically deleted in SF1-positive neurons in the ventromedial hypothalamic nucleus (VMH) known to regulate glucose metabolism. While having no effect on body weight regardless of sex, diet, and age, Rap1 deficiency in the VMH SF1 neurons markedly lowered blood glucose and insulin levels, improved glucose and insulin tolerance, and protected mice against HFD-induced neural leptin resistance and peripheral insulin resistance at the cellular and whole-body levels. Lastly, acute pharmacological inhibition of brain Epac2, a direct activator of Rap1, corrected glucose imbalance in obese mouse models. Our findings uncover the primary role of VMH Rap1 in glycemic control and implicate Rap1 signaling as a potential target for therapeutic intervention in diabetes.
Authors
Kentaro Kaneko, Hsiao-Yun Lin, Yukiko Fu, Pradip K. Saha, Ana B. De la Puente-Gomez, Yong Xu, Kousaku Ohinata, Peter Chen, Alexei Morozov, Makoto Fukuda
Abstract
Mice are normally unaffected by SARS-CoV-2 infection since the virus does not bind effectively to the murine version of the ACE2 receptor molecule. Here we report that induced mild pulmonary morbidities render SARS-CoV-2 refractive CD-1 mice to be susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low-doses of the acute-lung-injury stimulants bleomycin or ricin caused a severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates of >50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart and serum of low-dose-ricin pretreated, as compared to non-pretreated mice. Furthermore, lung extracts prepared 2-3 days after viral infection contained subgenomic RNA and virus particles capable of replication, only when derived from the pretreated mice. The deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against SARS-CoV-2 RBD. Thus, viral cell entry in the sensitized mice seems to depend on viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. This unique mode of viral entry, observed over a mildly injured tissue background, may contribute to the exacerbation of COVID-19 pathologies in patients with preexisting morbidities.
Authors
Reut Falach, Liat Bar-On, Shlomi Lazar, Tamar Kadar, Ohad Mazor, Moshe Aftalion, David Gur, Yentl Evgy, Ohad Shifman, Tamar Aminov, Ofir Israeli, Inbar Cohen-Gihon, Galia Zaide, Hila Gutman, Yaron Vagima, Efi Makdasi, Dana Stein, Ronit Rosenfeld, Ron Alcalay, Eran Zahavy, Haim Levy, Itai Glinert, Amir Ben-Shmuel, Tomer Israely, Sharon Melamed, Boaz Politi, Hagit Achdout, Shmuel Yitzhaki, Chanoch Kronman, Tamar Sabo
Abstract
BACKGROUND. The chemokine system of ligands and receptors is implicated in the progression of Alcohol-associated hepatitis (AH). Finding upstream regulators could lead to novel therapies. MOTHODS. The coordinated expression of chemokines in livers of healthy controls (HC) and patients with AH in two distinct cohorts of patients with various chronic liver diseases. Studies in cultured hepatocytes and in tissue-specific knockouts were used for mechanistic insight into a potential upstream regulator of chemokine expression in AH. RESULTS. Selected C-X-C chemokine members of the Interleukin-8 (IL-8) chemokine family and C-C chemokine CCl20 were highly associated with AH compared to HC, but not in patients with liver diseases of other etiologies (NAFLD or HCV). Our previous studies implicate Macrophage migration inhibitory factor (MIF) as a pleiotropic cytokine/chemokine with the potential to coordinately regulate chemokine expression in AH. LPS-stimulated expression of multiple chemokines in cultured hepatocytes was dependent on MIF. Gao-binge ethanol feeding to mice induced a similar coordinated chemokine expression in livers of wild-type mice; this was prevented in hepatocyte-specific Mif knockout (MifΔHep) mice. CONCLUSIONS. This study demonstrates that patients with AH exhibit a specific, coordinately expressed chemokine signature and hepatocyte-derived MIF might drive this inflammatory response.
Authors
Kyle L. Poulsen, Xiude D. Fan, Christopher D. Kibler, Emily Huang, Xiaoqin Wu, Megan R. McMullen, Lin Leng, Richard Bucala, Meritxell Ventura-Cots, Josepmaria Argemi, Ramon Bataller, Laura E. Nagy
Abstract
Idiopathic Pulmonary Fibrosis (IPF) is characterized by aberrant repair that diminishes lung function via mechanisms that remain poorly understood. C-C chemokine receptor (CCR10) and its ligand, CCL28, were both elevated in IPF compared with normal donors. CCR10 was highly expressed by various cells from IPF lungs, most notably stage-specific embryonic antigen (SSEA)-4+ mesenchymal progenitor cells (MPCs). In vitro, CCL28 promoted the proliferation of CCR10+ MPCs while CRISPR-Cas9-mediated targeting of CCR10 resulted in the death of MPCs. Following the intravenous injection of various cells from IPF lungs into immunodeficient (NSG) mice, human CCR10+ cells initiated and maintained fibrosis in NSG mice. Eph receptor A3 (EphA3) was among the highest expressed receptor tyrosine kinases detected on IPF CCR10+ cells. Ifabotuzumab-targeted killing of EphA3+ cells significantly reduced the numbers of CCR10+ cells and ameliorated pulmonary fibrosis in humanized NSG mice. Thus, human CCR10+ cells promote pulmonary fibrosis and EphA3 mAb-directed elimination of these cells inhibits lung fibrosis.
Authors
Miriam S. Hohmann, David M. Habiel, Milena S. Espindola, Guanling Huang, Isabelle Jones, Rohan Narayanan, Ana Lucia Coelho, Justin M. Oldham, Imre Noth, Shwu-Fan Ma, Adrianne Kurkciyan, Jonathan L. McQualter, Gianni Carraro, Barry Stripp, Peter Chen, Dianhua Jiang, Paul W. Noble, William Parks, John Woronicz, Geoffrey Yarranton, Lynne A. Murray, Cory M. Hogaboam
Abstract
Long noncoding RNAs (lncRNAs) are increasingly implicated in the pathology of diabetic complications. Here we examined the role of lncRNAs in monocyte dysfunction and inflammation associated with human type 2 diabetes mellitus (T2D). RNA-seq analysis of CD14+ monocytes from patients with T2D versus healthy controls revealed downregulation of anti-inflammatory and anti-proliferative genes along with several lncRNAs, including a novel divergent lncRNA DRAIR (Diabetes Regulated anti-inflammatory RNA) and its nearby gene CPEB2. High glucose and palmitic acid downregulated DRAIR in cultured CD14+ monocytes, whereas anti-inflammatory cytokines and monocyte-to-macrophage differentiation upregulated DRAIR via KLF4 transcription factor. DRAIR overexpression increased anti-inflammatory and macrophage differentiation genes but inhibited pro-inflammatory genes. Conversely, DRAIR knockdown attenuated anti-inflammatory genes, promoted inflammatory responses, and inhibited phagocytosis. DRAIR regulated target gene expression through interaction with chromatin, and inhibition of the repressive epigenetic mark H3K9me2 and its corresponding methyltransferase G9a. Mouse orthologous Drair and Cpeb2 were also downregulated in peritoneal macrophages from T2D db/db mice, and Drair knockdown in non-diabetic mice enhanced pro-inflammatory genes in macrophages. Thus, DRAIR modulates inflammatory phenotype of monocytes/macrophages via epigenetic mechanisms, and its downregulation in T2D may promote chronic inflammation. Augmentation of endogenous lncRNAs like DRAIR could serve as novel anti-inflammatory therapies for diabetic complications.
Authors
Marpadga A. Reddy, Vishnu Amaram, Sadhan Das, Vinay Singh Tanwar, Rituparna Ganguly, Mei Wang, Linda Lanting, Linxiao Zhang, Maryam Abdollahi, Zhuo Chen, Xiwei Wu, Sridevi Devaraj, Rama Natarajan
Abstract
Clinical phenotyping of term and preterm labor is imprecise, and disagreement persists on categorization relative to underlying pathobiology, which remains poorly understood. We performed RNA sequencing (RNA-seq) of 31 specimens of human uterine myometrium from 10 term and 21 preterm cesarean deliveries with rich clinical context information. A molecular signature of 4,814 transcripts stratified myometrial samples into quiescent (Q) and non-quiescent (NQ) phenotypes, independent of gestational age and incision site. Similar stratifications were achieved using expressed genes in Ca2+ signaling and TGF-β pathways. For maximal parsimony, we evaluated the expression of just two Ca2+ transporter genes, ATP2B4 (encoding PMCA4) and ATP2A2 (coding for SERCA2), and found that their ratio reliably distinguished NQ and Q specimens in the current study, and also in two publically available RNA-seq datasets (GSE50599 and GSE80172), with an overall AUC of 0.94. Cross-validation of the ATP2B4/ATP2A2 ratio by qPCR in an expanded cohort (by 11 additional specimens) achieved complete separation (AUC=1.00) of NQ vs. Q specimens. While providing additional insight into the associations between clinical features of term and preterm labor and myometrial gene expression, our study also offers a practical algorithm for unbiased classification of myometrial biopsies by their overall contractile program.
Authors
William E. Ackerman IV, Catalin S. Buhimschi, Ali Snedden, Taryn L. Summerfield, Guomao Zhao, Irina A. Buhimschi
Abstract
The epithelial cell-derived cytokines IL-25, IL-33 and TSLP initiate type 2 inflammation in allergic diseases including asthma. However, the signaling pathway regulating these cytokines expression remains elusive. Since microRNAs are pivotal regulators of gene expression, we profiled microRNA expression in bronchial epithelial brushings from type 2-low and type 2-high asthma patients. MiR-206 was the most highly expressed epithelial microRNA in type 2-high asthma relative to type 2-low asthma but was downregulated in both subsets compared with healthy controls. CD39, an ectonucleotidase degrading ATP, was a target of miR-206 and upregulated in asthma. Allergen-induced acute extracellular ATP accumulation led to miR-206 downregulation and CD39 upregulation in human bronchial epithelial cells, forming a feedback loop to eliminate excessive ATP. Airway ATP levels were markedly elevated and strongly correlated with IL-25 and TSLP expression in asthma patients. Intriguingly, airway miR-206 antagonism increased Cd39 expression, reduced ATP accumulation, suppressed Il-25, Il-33, Tslp expression and group 2 innate lymphoid cell expansion, and alleviated type 2 inflammation in a mouse model of allergic airway inflammation. In contrast, airway miR-206 overexpression had opposite effects. Overall, epithelial miR-206 upregulates airway IL-25, TSLP expression by targeting CD39-extracellular ATP axis, which represents a novel therapeutic target in type 2-high asthma.
Authors
Kan Zhang, Yuchen Feng, Yuxia Liang, Wenliang Wu, Chenli Chang, Dian Chen, Shengchong Chen, Jiali Gao, Gongqi Chen, Lingling Yi, Dan Cheng, Guohua Zhen
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