Research Article
Abstract
Severe lung damage resulting from COVID-19 involves complex interactions between diverse populations of immune and stromal cells. In this study, we used a spatial transcriptomics approach to delineate the cells, pathways, and genes present across the spectrum of histopathological damage in COVID-19–affected lung tissue. We applied correlation network–based approaches to deconvolve gene expression data from 46 areas of interest covering more than 62,000 cells within well-preserved lung samples from 3 patients. Despite substantial interpatient heterogeneity, we discovered evidence for a common immune-cell signaling circuit in areas of severe tissue that involves crosstalk between cytotoxic lymphocytes and pro-inflammatory macrophages. Expression of IFNG by cytotoxic lymphocytes was associated with induction of chemokines, including CXCL9, CXCL10, and CXCL11, which are known to promote the recruitment of CXCR3+ immune cells. The TNF superfamily members BAFF (TNFSF13B) and TRAIL (TNFSF10) were consistently upregulated in the areas with severe tissue damage. We used published spatial and single-cell SARS-CoV-2 data sets to validate our findings in the lung tissue from additional cohorts of patients with COVID-19. The resulting model of severe COVID-19 immune-mediated tissue pathology may inform future therapeutic strategies.
Authors
Amy R. Cross, Carlos E. de Andrea, María Villalba-Esparza, Manuel F. Landecho, Lucia Cerundolo, Praveen Weeratunga, Rachel E. Etherington, Laura Denney, Graham Ogg, Ling-Pei Ho, Ian S.D. Roberts, Joanna Hester, Paul Klenerman, Ignacio Melero, Stephen N. Sansom, Fadi Issa
Abstract
SMA with respiratory distress type 1 (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S) are results of mutations in immunoglobulin mu DNA binding protein 2 (IGHMBP2). IGHMBP2 is a UPF1-like helicase with proposed roles in several cellular processes, including translation. This study examines activator of basal transcription 1 (ABT1), a modifier of SMARD1-nmd disease pathology. Microscale thermophoresis and dynamic light scattering demonstrate that IGHMBP2 and ABT1 proteins directly interact with high affinity. The association of ABT1 with IGHMBP2 significantly increases the ATPase and helicase activity as well as the processivity of IGHMBP2. The IGHMBP2/ABT1 complex interacts with the 47S pre-rRNA 5′ external transcribed spacer and U3 small nucleolar RNA (snoRNA), suggesting that the IGHMBP2/ABT1 complex is important for pre-rRNA processing. Intracerebroventricular injection of scAAV9-Abt1 decreases FVB-Ighmbp2nmd/nmd disease pathology, significantly increases lifespan, and substantially decreases neuromuscular junction denervation. To our knowledge, ABT1 is the first disease-modifying gene identified for SMARD1. We provide a mechanism proposing that ABT1 decreases disease pathology in FVB-Ighmbp2nmd/nmd mutants by optimizing IGHMBP2 biochemical activity (ATPase and helicase activity). Our studies provide insight into SMARD1 pathogenesis, suggesting that ABT1 modifies IGHMBP2 activity as a means to regulate pre-rRNA processing.
Authors
Gangadhar P. Vadla, Sara M. Ricardez Hernandez, Jiude Mao, Mona O. Garro-Kacher, Zachary C. Lorson, Ronin P. Rice, Sarah A. Hansen, Christian L. Lorson, Kamal Singh, Monique A. Lorson
Abstract
CD4+ T cells play a critical role in antitumor immunity via recognition of peptide antigens presented on MHC class II (MHC-II). Although some solid cancers can be induced to express MHC-II, the extent to which this enables direct recognition by tumor-specific CD4+ T cells is unclear. We isolated and characterized T cell antigen receptors (TCRs) from naturally primed CD4+ T cells specific for 2 oncoproteins, HPV-16 E6 and the activating KRASG12V mutation, from patients with head and neck squamous cell carcinoma and pancreatic ductal adenocarcinoma, respectively, and determined their ability to recognize autologous or human leukocyte antigen–matched antigen-expressing tumor cells. We found in both cases that the TCRs were capable of recognizing peptide-loaded target cells expressing the relevant MHC-II or B cell antigen-presenting cells (APCs) when the antigens were endogenously expressed and directed to the endosomal pathway but failed to recognize tumor cells expressing the source protein even after induction of surface MHC-II expression by IFN-γ or transduction with CIITA. These results suggest that priming and functional recognition of both a nuclear (E6) and a membrane-associated (KRAS) oncoprotein are predominantly confined to crosspresenting APCs rather than via direct recognition of tumor cells induced to express MHC-II.
Authors
Spencer E. Brightman, Martin S. Naradikian, Rukman R. Thota, Angelica Becker, Leslie Montero, Milad Bahmanof, Ashmitaa Logandha Ramamoorthy Premlal, Jason A. Greenbaum, Bjoern Peters, Ezra E.W. Cohen, Aaron M. Miller, Stephen P. Schoenberger
Abstract
Organic anion transporter 1 (OAT1/SLC22A6, NKT) is a multispecific drug transporter in the kidney with numerous substrates, including pharmaceuticals, endogenous metabolites, natural products, and uremic toxins. Here, we show that OAT1 regulates levels of gut microbiome–derived metabolites. We depleted the gut microbiome of Oat1-KO and WT mice and performed metabolomics to analyze the effects of genotype (KO versus WT) and microbiome depletion. OAT1 is an in vivo intermediary between the host and the microbes, with 40 of the 162 metabolites dependent on the gut microbiome also impacted by loss of Oat1. Chemoinformatic analysis revealed that the altered metabolites (e.g., indoxyl sulfate, p-cresol sulfate, deoxycholate) had more ring structures and sulfate groups. This indicates a pathway from gut microbes to liver phase II metabolism, to renal OAT1–mediated transport. The idea that multiple gut-derived metabolites directly interact with OAT1 was confirmed by in vitro transport and magnetic bead binding assays. We show that gut microbiome–derived metabolites dependent on OAT1 are impacted in a chronic kidney disease (CKD) model and human drug-metabolite interactions. Consistent with the Remote Sensing and Signaling Theory, our results support the view that drug transporters (e.g., OAT1, OAT3, OATP1B1, OATP1B3, MRP2, MRP4, ABCG2) play a central role in regulating gut microbe–dependent metabolism, as well as interorganismal communication between the host and microbiome.
Authors
Jeffry C. Granados, Vladimir Ermakov, Koustav Maity, David R. Vera, Geoffrey Chang, Sanjay K. Nigam
Abstract
The expression of indoleamine 2,3-dioxygenase (IDO), a robust immunosuppressant, is significantly induced in macaque tuberculosis (TB) granulomas, where it is expressed on IFN-responsive macrophages and myeloid-derived suppressor cells. IDO expression is also highly induced in human TB granulomas, and products of its activity are detected in patients with TB. In vivo blockade of IDO activity resulted in the reorganization of the granuloma with substantially greater T cells being recruited to the core of the lesions. This correlated with better immune control of TB and reduced lung M. tuberculosis burdens. To study if the IDO blockade strategy can be translated to a bona fide host-directed therapy in the clinical setting of TB, we studied the effect of IDO inhibitor 1-methyl-d-tryptophan adjunctive to suboptimal anti-TB chemotherapy. While two-thirds of controls and one-third of chemotherapy-treated animals progressed to active TB, inhibition of IDO adjunctive to the same therapy protected macaques from TB, as measured by clinical, radiological, and microbiological attributes. Although chemotherapy improved proliferative T cell responses, adjunctive inhibition of IDO further enhanced the recruitment of effector T cells to the lung. These results strongly suggest the possibility that IDO inhibition can be attempted adjunctive to anti-TB chemotherapy in clinical trials.
Authors
Bindu Singh, Chivonne Moodley, Dhiraj K. Singh, Ruby A. Escobedo, Riti Sharan, Garima Arora, Shashank R. Ganatra, Vinay Shivanna, Olga Gonzalez, Shannan Hall-Ursone, Edward J. Dick Jr., Deepak Kaushal, Xavier Alvarez, Smriti Mehra
Abstract
The G protein–coupled receptor melanocortin-4 receptor (MC4R) and its associated protein melanocortin receptor–associated protein 2 (MRAP2) are essential for the regulation of food intake and body weight in humans. MC4R localizes and functions at the neuronal primary cilium, a microtubule-based organelle that senses and relays extracellular signals. Here, we demonstrate that MRAP2 is critical for the weight-regulating function of MC4R neurons and the ciliary localization of MC4R. More generally, our study also reveals that GPCR localization to primary cilia can require specific accessory proteins that may not be present in heterologous cell culture systems. Our findings further demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggest that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.
Authors
Adelaide Bernard, Irene Ojeda Naharros, Xinyu Yue, Francois Mifsud, Abbey Blake, Florence Bourgain-Guglielmetti, Jordi Ciprin, Sumei Zhang, Erin McDaid, Kellan Kim, Maxence V. Nachury, Jeremy F. Reiter, Christian Vaisse
Abstract
Modifications to vaccine delivery that increase serum antibody longevity are of great interest for maximizing efficacy. We have previously shown that a delayed fractional (DFx) dosing schedule (0-1-6 month) — using AS01B-adjuvanted RH5.1 malaria antigen — substantially improves serum IgG durability as compared with monthly dosing (0-1-2 month; NCT02927145). However, the underlying mechanism and whether there are wider immunological changes with DFx dosing were unclear. Here, PfRH5-specific Ig and B cell responses were analyzed in depth through standardized ELISAs, flow cytometry, systems serology, and single-cell RNA-Seq (scRNA-Seq). Data indicate that DFx dosing increases the magnitude and durability of circulating PfRH5-specific B cells and serum IgG1. At the peak antibody magnitude, DFx dosing was distinguished by a systems serology feature set comprising increased FcRn binding, IgG avidity, and proportion of G2B and G2S2F IgG Fc glycans, alongside decreased IgG3, antibody-dependent complement deposition, and proportion of G1S1F IgG Fc glycan. Concomitantly, scRNA-Seq data show a higher CDR3 percentage of mutation from germline and decreased plasma cell gene expression in circulating PfRH5-specific B cells. Our data, therefore, reveal a profound impact of DFx dosing on the humoral response and suggest plausible mechanisms that could enhance antibody longevity, including improved FcRn binding by serum Ig and a potential shift in the underlying cellular response from circulating short-lived plasma cells to nonperipheral long-lived plasma cells.
Authors
Carolyn M. Nielsen, Jordan R. Barrett, Christine Davis, Jonathan K. Fallon, Cyndi Goh, Ashlin R. Michell, Catherine Griffin, Andrew Kwok, Carolin Loos, Samuel Darko, Farida Laboune, Mehmet Tekman, Ababacar Diouf, Kazutoyo Miura, Joseph R. Francica, Amy Ransier, Carole A. Long, Sarah E. Silk, Ruth O. Payne, Angela M. Minassian, Douglas A. Lauffenburger, Robert A. Seder, Daniel C. Douek, Galit Alter, Simon J. Draper
Abstract
The central physiological role of the bone marrow renders bone marrow stromal cells (BMSCs) particularly sensitive to aging. With bone aging, BMSCs acquire a differentiation potential bias in favor of adipogenesis over osteogenesis, and the underlying molecular mechanisms remain unclear. Herein, we investigated the factors underlying age-related changes in the bone marrow and their roles in BMSCs’ differentiation. Antibody array revealed that CC chemokine ligand 3 (CCL3) accumulation occurred in the serum of naturally aged mice along with bone aging phenotypes, including bone loss, bone marrow adiposity, and imbalanced BMSC differentiation. In vivo Ccl3 deletion could rescue these phenotypes in aged mice. CCL3 improved the adipogenic differentiation potential of BMSCs, with a positive feedback loop between CCL3 and C/EBPα. CCL3 activated C/EBPα expression via STAT3, while C/EBPα activated CCL3 expression through direct promoter binding, facilitated by DNA hypomethylation. Moreover, CCL3 inhibited BMSCs’ osteogenic differentiation potential by blocking β-catenin activity mediated by ERK-activated Dickkopf-related protein 1 upregulation. Blocking CCL3 in vivo via neutralizing antibodies ameliorated trabecular bone loss and bone marrow adiposity in aged mice. This study provides insights regarding age-related bone loss and bone marrow adiposity pathogenesis and lays a foundation for the identification of new targets for senile osteoporosis treatment.
Authors
Degang Yu, Shuhong Zhang, Chao Ma, Sen Huang, Long Xu, Jun Liang, Huiwu Li, Qiming Fan, Guangwang Liu, Zanjing Zhai
Abstract
Chronic inflammation is associated with lung tumorigenesis, in which NF-κB–mediated epigenetic regulation plays a critical role. Lung tumor suppressor G protein–coupled receptor, family C, member 5A (GPRC5A), is repressed in most non–small cell lung cancer (NSCLC); however, the mechanisms remain unclear. Here, we show that NF-κB acts as a transcriptional repressor in suppression of GPRC5A. NF-κB induced GPRC5A repression both in vitro and in vivo. Intriguingly, transactivation of NF-κB downstream targets was not required, but the transactivation domain of RelA/p65 was required for GPRC5A repression. NF-κB did not bind to any potential cis-element in the GPRC5A promoter. Instead, p65 was complexed with retinoic acid receptor α/β (RARα/β) and recruited to the RA response element site at the GPRC5A promoter, resulting in disrupted RNA polymerase II complexing and suppressed transcription. Notably, phosphorylation on serine 276 of p65 was required for interaction with RARα/β and repression of GPRC5A. Moreover, NF-κB–mediated epigenetic repression was through suppression of acetylated histone H3K9 (H3K9ac), but not DNA methylation of the CpG islands, at the GPRC5A promoter. Consistently, a histone deacetylase inhibitor, but not DNA methylation inhibitor, restored GPRC5A expression in NSCLC cells. Thus, NF-κB induces transcriptional repression of GPRC5A via a complex with RARα/β and mediates epigenetic repression via suppression of H3K9ac.
Authors
Hongyong Song, Xiaofeng Ye, Yueling Liao, Siwei Zhang, Dongliang Xu, Shuangshuang Zhong, Bo Jing, Tong Wang, Beibei Sun, Jianhua Xu, Wenzheng Guo, Kaimi Li, Min Hu, Yanbin Kuang, Jing Ling, Tuo Zhang, Yadi Wu, Jing Du, Feng Yao, Y. Eugene Chin, Qi Wang, Binhua P. Zhou, Jiong Deng
Abstract
Pulmonary fibrosis is characterized by stiffening of the extracellular matrix. Fibroblasts migrate in the direction of greater stiffness, a phenomenon termed durotaxis. The mechanically guided fibroblast migration could be a crucial step in the progression of lung fibrosis. In this study, we found primary human lung fibroblasts sense increasing matrix stiffness with a change of mitochondrial dynamics in favor of mitochondrial fission and increased production of ATP. Mitochondria polarize in the direction of a physiologically relevant stiffness gradient, with conspicuous localization to the leading edge, primarily lamellipodia and filopodia, of migrating lung fibroblasts. Matrix stiffness–regulated mitochondrial fission and durotactic lung fibroblast migration are mediated by a dynamin-related protein 1/mitochondrial fission factor–dependent (DRP1/MFF-dependent) pathway. Importantly, we found that the DRP1/MFF pathway is activated in fibrotic lung myofibroblasts in both human IPF and bleomycin-induced mouse lung fibrosis. These findings suggest that energy-producing mitochondria need to be sectioned via fission and repositioned in durotactic lung fibroblasts to meet the higher energy demand. This represents a potentially new mechanism through which mitochondria may contribute to the progression of fibrotic lung diseases. Inhibition of durotactic migration of lung fibroblasts may play an important role in preventing the progression of human idiopathic pulmonary fibrosis.
Authors
Ting Guo, Chun-sun Jiang, Shan-Zhong Yang, Yi Zhu, Chao He, A. Brent Carter, Veena B. Antony, Hong Peng, Yong Zhou
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