Chronic obstructive pulmonary disease (COPD) is mainly caused by cigarette smoking, characterized by chronic inflammation in vulnerable individuals. However, it is unknown how genetic factors may shape chronic inflammation in COPD. To understand how hedgehog interacting protein, encoded by HHIP gene identified in the genome-wide association study in COPD, plays a role in inflammation, we utilized Hhip+/- mice that resemble persistent inflammation and emphysema observed in human COPD upon aging. By performing single cell RNA sequencing of the whole lung from mice at different ages, we found that Hhip+/- mice develop a cytotoxic immune response with a specific increase in Klrg1+CD8+T cells with upregulated Ifnγ expression recapitulating human COPD. Hhip expression was restricted to a lung fibroblast subpopulation that has increased interaction with CD8+T lymphocytes in Hhip+/- compared to Hhip+/+ during aging. Hhip-expressing lung fibroblasts have upregulated IL-18 pathway genes in Hhip+/- lung fibroblasts, which was sufficient to drive increased levels of IFNγ in CD8+T cells ex vivo. Our finding provides insight into how a common genetic variation contributes to the amplified lymphocytic inflammation in COPD.
Jeong H. Yun, ChangHee Lee, Tao Liu, Siqi Liu, Edy Y. Kim, Shuang Xu, Jeffrey L. Curtis, Luca Pinello, Russell P. Bowler, Edwin K. Silverman, Craig P. Hersh, Xiaobo Zhou
Cell lines are the mainstay in understanding the biology of COVID-19 infection, but do not recapitulate many of the complexities of human infection. The use of human lung tissue is one solution for the study of such novel respiratory pathogens. We hypothesized that a cryopreserved bank of human lung tissue allows for the ex vivo study of the inter-individual heterogeneity of host response to SARS-CoV-2 infection, thus providing a bridge between studies with cell lines and studies in animal models. We generated a cryobank of tissues from 21 donors, many of whom had clinical risk factors for severe COVID-19. Cryopreserved tissues preserved 90% cell viability and contained heterogenous populations of metabolically active epithelial, endothelial, and immune cell subsets of the human lung. Samples were readily infectable with HCoV-OC43 and SARS-CoV-2 coronaviruses, and demonstrated comparable susceptibility to infection. In contrast, we observed a marked donor-dependent heterogeneity in the expression of IL6, CXCL8 and IFNB1 in response to SARS-CoV2 infection. Treatment of tissues with dexamethasone and the experimental drug, n-hydroxycytidine, suppressed viral growth in all samples, whereas chloroquine and remdesivir had no detectable effect. Metformin and sirolimus, molecules with predicted but unproven antiviral activity, each suppressed viral replication in tissues from a subset of donors. In summary, we developed a novel system for the ex vivo study of human SARS-CoV- 2 infection using primary human lung tissue from a library of donor tissues. This model may be useful for drug screening and for understanding basic mechanisms of COVID-19 pathogenesis.
Matthew A. Schaller, Yamini Sharma, Zadia Dupee, Duy T. Nguyen, Juan M. Urueña, Ryan A. Smolchek, Julia C. Loeb, Tiago N. Machuca, John A. Lednicky, David J. Odde, Robert F. Campbell, W. Gregory Sawyer, Borna Mehrad
Innate immunity and chronic inflammation are involved in atherosclerosis and atherothrombosis leading to target organ damage in essential hypertension (EH). However, the role of neutrophils in EH is still elusive. We investigated the association between angiotensin II (Ang II) and neutrophil extracellular traps (NETs) in pathogenesis of EH. Plasma samples, kidney biopsies and surgical specimens of abdominal aortic aneurysms (AAA) from EH patients were used. Cell-based assays, NETs/human aortic endothelial cells co-cultures and in situ studies were performed. Increased plasma levels of NETs and tissue factor (TF) activity were detected in untreated, newly-diagnosed, EH patients. Stimulation of control neutrophils with plasma from untreated EH patients generated TF-enriched NETs promoting endothelial collagen production. Ang II induced NETosis in vitro via a reactive oxygen species (ROS)/peptidylarginine deiminase type 4 and autophagy-dependent pathway. Circulating NETs and thrombin generation levels were reduced significantly in EH patients starting treatment with Ang II receptor blockers, whereas their plasma was unable to trigger procoagulant NETs. Moreover, TF-bearing NETotic neutrophils/remnants were accumulated in sites of interstitial renal fibrosis and in the subendothelial layer of AAA. These data reveal the important pathogenic role of Ang II/ROS/NETs/TF axis in EH, linking thromboinflammation with endothelial dysfunction and fibrosis.
Akrivi Chrysanthopoulou, Eugenia Gkaliagkousi, Antonios Lazaridis, Stella Arelaki, Panagiotis Pateinakis, Maria Ntinopoulou, Alexandros Mitsios, Christina Antoniadou, Christos Argyriou, George S. Georgiadis, Vasileios Papadopoulos, Alexandra Giatromanolaki, Konstantinos Ritis, Panagiotis Skendros
Macrophage activation syndrome (MAS) is a life-threatening cytokine storm complicating systemic juvenile idiopathic arthritis (SJIA) driven by IFNγ. SJIA and MAS are associated with an unexplained emerging lung disease (SJIA-LD), with our recent work supporting pulmonary activation of IFNγ pathways pathologically linking SJIA-LD and MAS. Our objective was to mechanistically define the novel observation of pulmonary inflammation in the TLR9 mouse model of MAS. In acute MAS, lungs exhibit mild but diffuse CD4-predominant, perivascular interstitial inflammation with elevated IFNγ, IFN-induced chemokines, and AMΦ expression of IFNγ-induced genes. Single-cell RNA-sequencing confirmed IFN-driven transcriptional changes across lung cell types with myeloid expansion and detection of MAS-specific macrophage populations. Systemic MAS resolution was associated with increased AMΦ and interstitial lymphocytic infiltration. AMΦ transcriptomic analysis confirmed IFNγ-induced proinflammatory polarization during acute MAS, which switches towards an anti-inflammatory phenotype after systemic MAS resolution. Interestingly, recurrent MAS led to increased alveolar inflammation and lung injury, and reset AMΦ polarization towards a proinflammatory state. Furthermore, in mice bearing macrophages insensitive to IFNγ, both systemic feature of MAS and pulmonary inflammation were attenuated. These findings demonstrate that experimental MAS induces IFNγ-driven pulmonary inflammation replicating key features of SJIA-LD, and provides a model system for testing novel treatments directed towards SJIA-LD.
Denny K. Gao, Nathan Salomonis, Maggie Henderlight, Christopher Woods, Kairavee Thakkar, Alexei A. Grom, Sherry Thornton, Michael B. Jordan, Kathryn A. Wikenheiser-Brokamp, Grant S. Schulert
Hyperstimulation of the cholecystokinin receptor (CCK1R), a Gq-protein coupled receptor (GPCR), in pancreatic acinar cells is commonly used to induce pancreatitis in rodents. Human pancreatic acinar cells lack CCK1R but express cholinergic receptor muscarinic 3 (M3R), another GPCR. To test whether M3R activation is involved in pancreatitis, a mutant M3R was conditionally expressed in pancreatic acinar cells in mice. This mutant receptor loses responsiveness to its native ligand acetylcholine but can be activated by an inert small molecule, clozapine-N-oxide (CNO). Intracellular calcium and amylase were elicited by CNO in pancreatic acinar cells isolated from mutant M3R mice but not WT mice. Similarly, acute pancreatitis (AP) could be induced by a single injection of CNO in the transgenic mice but not WT mice. Compared with the cerulein-induced AP, CNO caused more widespread acinar cell death and inflammation. Furthermore, chronic pancreatitis developed at 4 weeks after 3 episodes of CNO-induced AP. In contrast, in mice with three recurrent episodes of cerulein-included AP, pancreas histology was restored in 4 weeks. Furthermore, the M3R antagonist ameliorated the severity of cerulein-induced AP in WT mice. We conclude that M3R activation can cause the pathogenesis of pancreatitis. This model may provide an alternative approach for pancreatitis research.
Jianhua Wan, Jiale Wang, Larry E. Wagner II, Oliver H. Wang, Fu Gui, Jiaxiang Chen, Xiaohui Zhu, Ashley N. Haddock, Brandy H. Edenfield, Brian Haight, Debabrata Mukhopadhyay, Ying Wang, David I. Yule, Yan Bi, Baoan Ji
Stimulator of IFN genes (STING) activates TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3) to produce type I IFNs. Extracellular cold-inducible RNA-binding protein (eCIRP) is released from cells during hemorrhagic shock (HS). We hypothesized that eCIRP activates STING to induce inflammation and acute lung injury (ALI) after HS. WT and STING–/– mice underwent controlled hemorrhage by bleeding, followed by fluid resuscitation. Blood and lungs were collected at 4 hours after resuscitation. Serum ALT, AST, LDH, IL-6, and IFN-β were significantly decreased in STING–/– mice compared with WT mice after HS. In STING–/– mice, the levels of pTBK1 and pIRF3, and expression of TNF-α, IL-6, and IL-1β mRNAs and proteins in the lungs, were significantly decreased compared with WT HS mice. The 10-day mortality rate in STING–/– mice was significantly reduced. I.v. injection of recombinant mouse CIRP (rmCIRP) in STING–/– mice showed a significant decrease in pTBK1 and pIRF3 and in IFN-α and IFN-β mRNAs and proteins in the lungs compared with rmCIRP-treated WT mice. Treatment of TLR4–/–, MyD88–/–, and TRIF–/– macrophages with rmCIRP significantly decreased pTBK1 and pIRF3 levels and IFN-α and IFN-β mRNAs and proteins compared with WT macrophages. HS increases eCIRP levels, which activate STING through TLR4/MyD88/TRIF pathways to exacerbate inflammation.
Kehong Chen, Joaquin Cagliani, Monowar Aziz, Chuyi Tan, Max Brenner, Ping Wang
BACKGROUND. Epicardial adipose tissue (EAT) directly overlies the myocardium with changes in its morphology and volume associated with myriad cardiovascular and metabolic diseases. However, EAT’s immune structure and cellular characterization remain incompletely described. This study aimed to define the immune phenotype of EAT in humans, and compare such profiles across lean, obese and diabetic patients. METHODS. A total of 152 adult patients undergoing open chest coronary artery bypass grafting (CABG), valve repair/replacement (VR) surgery or combined CABG/valve surgery were recruited to the study. Patients’ clinical and biochemical data alongside epicardial adipose tissue (EAT), subcutaneous adipose tissue (SAT) and pre-operative blood samples were collected. Immune cell profiling was evaluated by flow cytometry and complemented by gene expression studies of immune mediators. Bulk RNA-seq was performed in EAT across different metabolic profiles to assess whole transcriptome changes observed in these groups. RESULTS. Flow cytometry analysis demonstrated that EAT is highly enriched in adaptive immune (T and B) cells. Whilst overweight/obese and diabetic patients had similar EAT cellular profiles to lean control patients, the EAT exhibited significantly (P≤.01) raised expression of immune mediators including: interleukin1 (IL1), IL6, tumour necrosis factorα (TNFα) and interferonγ (IFNγ). These changes were not observed in either SAT or blood. Neither underlying coronary artery disease nor the presence of hypertension significantly altered the immune profiles observed. Bulk RNA-seq demonstrated significant alterations in metabolic and inflammatory pathways in the EAT of overweight/obese patients compared with lean controls. CONCLUSIONS. Adaptive immune cells are the predominant immune cell constituent in human EAT and SAT. The presence of underlying cardiometabolic conditions, specifically obesity and diabetes, rather than cardiac disease phenotype appears to alter the inflammatory profile of EAT. Obese states markedly alter EAT metabolic and inflammatory signalling genes, underlining the impact of obesity on the EAT transcriptome profile.
Vishal Vyas, Hazel Blythe, Elizabeth G. Wood, Balraj Sandhar, Shah-Jalal Sarker, Damian Balmforth, Shirish G. Ambekar, John Yap, Stephen J. Edmondson, Carmelo Di Salvo, Kit Wong, Neil Roberts, Rakesh Uppal, Ben Adams, Alex Shipolini, Aung Y. Oo, David Lawrence, Shyam Kolvekar, Kulvinder S. Lall, Malcolm C. Finlay, M. Paula Longhi
Host genes define the severity of inflammation and immunity but specific loci doing so are unknown. Here we show that TNFRSF13B variants which enhance defense against certain pathogens, also control immune-mediated injury of transplants, by regulating innate B cells’ functions. Analysis of TNFRSF13B in human kidney transplant recipients revealed that 33% of the subjects with antibody-mediated rejection (AMR) but less than 6% of those with stable graft function had TNFRSF13B missense mutations. To explore mechanisms underlying aggressive immune responses we investigated allo-immunity and rejection in mice. Cardiac allografts in Tnfrsf13b-mutant mice underwent early and severe AMR. The dominance and precocity of AMR in Tnfrsf13b-deficient mice was not caused by increased alloantibodies. Rather, Tnfrsf13b mutations decreased “natural” IgM and compromised complement regulation leading to complement deposition in allografted hearts and autogenous kidneys. Thus, wild type TNFRSF13B and Tnfrsf13b support innate B cell functions that limit complement-associated inflammation; in contrast, common variants of these genes, intensify inflammatory responses that help clear microbial infections but allow inadvertent tissue injury to ensue. The wide variation in inflammatory reactions associated with TNFRSF13B diversity suggests polymorphisms could underlie variation in host defense and explosive inflammatory responses that sometimes enhances morbidity associated with immune responses.
Mayara Garcia de Mattos Barbosa, Adam R. Lefferts, Daniel Huynh, Hui Liu, Yu Zhang, Beverly Fu, Jenna Barnes, Milagros Samaniego, Richard J. Bram, Raif Geha, Ariella Shikanov, Eline T. Luning Prak, Evan A. Farkash, Jeffrey L. Platt, Marilia Cascalho
Neutrophil-mediated activation and injury of the endothelium play a role in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap/NET-derived histones) with polyanionic compounds has been suggested as a potential strategy for protecting the endothelium from such insults. Here, we report that the FDA-approved polyanionic agent defibrotide (a pleotropic mixture of oligonucleotides) directly engages histones and thereby blocks their pathological effects on endothelium. In vitro, defibrotide counteracted endothelial cell activation and pyroptosis-mediated cell death, whether triggered by purified NETs or recombinant histone H4. In vivo, defibrotide stabilized the endothelium and protected against histone-accelerated inferior vena cava thrombosis in mice. Mechanistically, defibrotide demonstrated direct and tight binding to histone H4 as detected by both electrophoretic mobility shift assay and surface plasmon resonance. Taken together, these data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.
Hui Shi, Alex A. Gandhi, Stephanie A. Smith, Qiuyu Wang, Diane Chiang, Srilakshmi Yalavarthi, Ramadan A. Ali, Chao Liu, Gautam Sule, Pei-Suen Tsou, Yu Zuo, Yogendra Kanthi, Evan A. Farkash, Jiandie D. Lin, James H. Morrissey, Jason S. Knight
Antibody-mediated glomerulonephritis (AGN) is a clinical manifestation of many autoimmune kidney diseases for which few effective treatments exist. Chronic inflammatory circuits in renal glomerular and tubular cells lead to tissue damage in AGN. These cells are targeted by the cytokine IL-17, which has recently been shown to be a central driver of the pathogenesis of AGN. However, surprisingly little is known about the regulation of pathogenic IL-17 signaling in the kidney. Here, using a well-characterized mouse model of AGN, we show that IL-17 signaling in renal tubular epithelial cells (RTECs) is necessary for AGN development. We also show that Regnase-1, an RNA binding protein with endoribonuclease activity, is a negative regulator of IL-17 signaling in RTECs. Accordingly, mice with a selective Regnase-1 deficiency in RTECs exhibited exacerbated kidney dysfunction in AGN. Mechanistically, Regnase-1 inhibits IL-17–driven expression of the transcription factor IκBξ and, consequently, its downstream gene targets, including Il6 and Lcn2. Moreover, deletion of Regnase-1 in human RTECs reduced inflammatory gene expression in a IκBξ-dependent manner. Overall, these data identify an IL-17–driven inflammatory circuit in RTECs during AGN that is constrained by Regnase-1.
De-Dong Li, Rami Bechara, Kritika Ramani, Chetan V. Jawale, Yang Li, Jay K. Kolls, Sarah L. Gaffen, Partha S. Biswas
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