Previously we reported heightened expression of human endogenous retroviral protein HERV-K deoxyuridine triphosphate nucleotidohydrolase (dUTPase) in circulating monocytes and pulmonary arterial (PA) adventitial macrophages of PA hypertension (PAH) patients. Furthermore, recombinant HERV-K dUTPase increased IL6 in PA endothelial cells (PAECs) and caused pulmonary hypertension in rats. Here we show that monocytes overexpressing HERV-K dUTPase as opposed to GFP, can release HERV-K dUTPase in extracellular vesicles (EVs) that cause pulmonary hypertension in mice in association with endothelial mesenchymal transition (EndMT) related to induction of SNAIL/SLUG, and proinflammatory molecules IL6 as well as VCAM1. In PAECs, HERV-K dUTPase requires TLR4-myeloid differentiation primary response (MYD)-88 to increase IL6 and SNAIL/SLUG, and HERV-K dUTPase interaction with melanoma cell adhesion molecule (MCAM) is necessary to upregulate VCAM1. TLR4 engagement induces p-p38 activation of NF-κB in addition to pJNK-pSMAD3 required for SNAIL, and pSTAT1 for IL6. HERV-K dUTPase interaction with MCAM also induces p-p38 activation of NF-κB in addition to pERK1/2-activating transcription factor (ATF)-2 to increase VCAM1. Thus in PAH, monocytes or macrophages can release HERV-K dUTPase in EVs, and HERV-K dUTPase can engage dual receptors and signaling pathways to subvert PAEC transcriptional machinery to induce EndMT and associated pro-inflammatory molecules.
Shoichiro Otsuki, Toshie Saito, Shalina Taylor, Dan Li, Jan-Renier Moonen, David P. Marciano, Rebecca L. Harper, Aiqin Cao, Lingli Wang, Maria E. Ariza, Marlene Rabinovitch
Sepsis is a critical illness characterized by dysregulated inflammatory responses lacking counter-regulation. Specialized pro-resolving mediators are agonists for anti-inflammation and promoting resolution and are protective in preclinical sepsis models. Here, in human sepsis, we mapped resolution circuits for the specialized pro-resolving mediators resolvin D1 and resolvin D2 in peripheral blood neutrophils and monocytes, their regulation of leukocyte activation and function ex vivo, and their relationships to measures of clinical severity. Neutrophils and monocytes were isolated from healthy subjects and sepsis patients by inertial microfluidics and resolvin D1 and resolvin D2 receptor expression determined by flow cytometry. The impact of these resolvins on leukocyte activation was determined by isodielectric separation and leukocyte function by stimulated phagolysosome formation. Leukocyte pro-resolving receptor expression was significantly higher in sepsis. In nanomolar concentrations, resolvin D1 and resolvin D2 partially reversed sepsis-induced changes in leukocyte activation and function. Principal component analyses of leukocyte resolvin receptor expression and responses differentiated sepsis from health and were associated with measures of sepsis severity. These findings indicate that resolvin D1 and resolvin D2 signaling for anti-inflammation and resolution are uncoupled from leukocyte activation in early sepsis and suggest that indicators of diminished resolution signaling correlate with clinical disease severity.
Bakr Jundi, Do-Hyun Lee, Hyungkook Jeon, Melody G. Duvall, Julie Nijmeh, Raja-Elie E. Abdulnour, Mayra Pinilla-Vera, Rebecca M. Baron, Jongyoon Han, Joel Voldman, Bruce D. Levy
Mouse IgE and mast cell (MC) functions have been studied primarily using inbred strains. Here, we (a) identified effects of genetic background on mouse IgE and MC phenotypes, (b) defined the suitability of various strains for studying IgE and MC functions, and (c) began to study potentially novel genes involved in such functions. We screened 47 Collaborative Cross (CC) strains, as well as C57BL/6J and BALB/cJ mice, for strength of passive cutaneous anaphylaxis (PCA) and responses to the intestinal parasite Strongyloides venezuelensis (S.v.). CC mice exhibited a diversity in PCA strength and S.v. responses. Among strains tested, C57BL/6J and CC027 mice showed, respectively, moderate and uniquely potent MC activity. Quantitative trait locus analysis and RNA sequencing of BM-derived cultured MCs (BMCMCs) from CC027 mice suggested Sp140 as a candidate gene for MC activation. siRNA-mediated knock-down of Sp140 in BMCMCs decreased IgE-dependent histamine release and cytokine production. Our results demonstrated marked variations in IgE and MC activity in vivo, and in responses to S.v., across CC strains. C57BL/6J and CC027 represent useful models for studying MC functions. Additionally, we identified Sp140 as a gene that contributes to IgE-dependent MC activation.
Kazufumi Matsushita, Xin Li, Yuki Nakamura, Danyue Dong, Kaori Mukai, Mindy Tsai, Stephen B. Montgomery, Stephen J. Galli
The stimulator of interferon genes (STING) protein senses cyclic di-nucleotides released in response to double stranded DNA, and functions as an adaptor molecule for type I interferon (IFN-I) signaling by activating IFN-I stimulated genes (ISG). We found impaired T cell infiltration into the peritoneum in response to TNF-α in global and EC-specific STING-/- mice and discovered that T cell transendothelial migration (TEM) across mouse and human endothelial cells (EC) deficient in STING was strikingly reduced compared to control EC, whereas T cells adhesion was not impaired. STING-/- T cells showed no defect in TEM or adhesion to EC, or immobilized endothelial cell expressed molecules ICAM-1 and VCAM-1 compared to WT T cells. Mechanistically, CXCL10, an ISG and a chemoattractant for T cells, was dramatically reduced in TNF-α-stimulated STING-/- EC and genetic loss or pharmacologic antagonism of IFN-type I interferon receptor (IFNAR) pathway reduced T cell TEM. Our data demonstrate a central role for EC STING during T cell TEM that is dependent on the ISG CXCL10 and on IFN-I-IFNAR signaling.
Marina Anastasiou, Gail A. Newton, Kuljeet Kaur, Francisco J. Carrillo-Salinas, Sasha A. Smolgovsky, Abraham L. Bayer, Vladimir Ilyukha, Shruti Sharma, Alexander Poltorak, Francis W. Luscinskas, Pilar Alcaide
We explored the potential link between chronic inflammatory arthritis and COVID-19 pathogenic and resolving macrophage pathways and their role in COVID-19 pathogenesis. We found that BALF macrophage clusters FCN1pos and FCN1posSPP1pos predominant in severe COVID-19 were transcriptionally related to synovial tissue macrophage (STM) clusters CD48highS100A12pos and CD48posSPP1pos that drive Rheumatoid Arthritis (RA) synovitis. BALF macrophage cluster FABP4pos predominant in healthy lung was transcriptionally related to STM cluster TREM2pos that governs resolution of synovitis in RA remission. Plasma concentrations of SPP1 and S100A12 (key products of macrophage clusters shared with active RA) were high in severe COVID-19 and predicted the need for Intensive Care Unit transfer, and remained high in post-COVID-19 stage. High plasma levels of SPP1 were unique to severe COVID-19 when compared to other causes of severe pneumonia, and immunohistochemistry localized SPP1pos macrophages in the alveoli of COVID-19 lung. Investigation into SPP1 mechanisms of action revealed that it drives pro-inflammatory activation of CD14pos monocytes and development of PD-L1pos neutrophils, both hallmarks of severe COVID-19. In summary, COVID-19 pneumonitis appears driven by similar pathogenic myeloid cell pathways as those in RA, and their mediators such as SPP1 might be an upstream activator of the aberrant innate response in severe COVID-19 and predictive of disease trajectory including post-COVID-19 monitoring.
Lucy MacDonald, Stefano Alivernini, Barbara Tolusso, Aziza Elmesmari, Domenico Somma, Simone Perniola, Annamaria Paglionico, Luca Petricca, Silvia L. Bosello, Angelo Carfì, Michela Sali, Egidio Stigliano, Antonella Cingolani, Rita Murri, Vincenzo Arena, Massimo Fantoni, Massimo Antonelli, Francesco Landi, Francesco Franceschi, Maurizio Sanguinetti, Iain B. McInnes, Charles McSharry, Antonio Gasbarrini, Thomas D. Otto, Mariola Kurowska-Stolarska, Elisa Gremese
SOCS3 is the main inhibitor of the JAK/STAT3 pathway. This pathway is activated by interleukin 6 (IL-6), a major mediator of the cytokine storm during shock. To determine its role in the vascular response to shock, we challenged mice lacking SOCS3 in the adult endothelium (SOCS3iEKO) with a non-lethal dose of lipopolysaccharide (LPS). SOCS3iEKO mice died 16-24 hours post-injection after severe kidney failure. Loss of SOCS3 led to an LPS-induced type I interferon-like program, and high expression of pro-thrombotic and pro-adhesive genes. Consistently, we observed intraluminal leukocyte adhesion and NETosis, as well as retinal venular leukoembolization. Notably, heterozygous mice displayed an intermediate phenotype, suggesting a gene dose effect. In vitro studies were performed to study the role of SOCS3 protein levels in the regulation of the inflammatory response. In HUVEC, pulse-chase experiments showed that SOCS3 protein has a half-life below 20 minutes. Inhibition of SOCS3 ubiquitination and proteasomal degradation leads to protein accumulation and a stronger inhibition of IL-6 signaling and barrier function loss. Together, our data demonstrates that the regulation of SOCS3 protein levels is critical to inhibit IL-6-mediated endotheliopathy during shock and provides a promising new therapeutic avenue to prevent MODS though stabilization of endothelial SOCS3.
Nina Martino, Ramon Bossardi Ramos, Shuhan Lu, Kara Leyden, Lindsay Tomaszek, Sudeshna Sadhu, Gabrielle Fredman, Ariel Jaitovich, Peter A. Vincent, Alejandro P. Adam
TIGIT is a recently identified coinhibitory receptor that is upregulated in the setting of cancer and functionally contributes to the impairment of antitumor immunity. However, its role during sepsis is unknown. Because patients with cancer are 10 times more likely to die of sepsis than previously healthy (PH) patients with sepsis, we interrogated the role of TIGIT during sepsis in the context of preexistent malignancy. PH mice or cancer (CA) mice inoculated with lung carcinoma cells were made septic by cecal ligation and puncture (CLP). We found that sepsis induced TIGIT upregulation predominantly on Tregs and NK cells in both PH and CA mice. Anti-TIGIT Ab improved the 7-d survival of CA septic mice but not PH mice after CLP. Treatment of CA septic animals but not PH septic animals with anti-TIGIT mAb significantly reversed sepsis-induced loss of CD4+ T cells, CD8+ T cells, Foxp3+ Treg, and CD19+ B cells in the spleen, which was the result of decreased caspase-3+ apoptotic cells. In sum, we found that anti-TIGIT Ab reversed sepsis-induced T cell apoptosis in CA septic mice and led to a significant survival benefit, suggesting its use as a potential immunotherapy to improve outcomes in septic patients with cancer.
Wenxiao Zhang, Jerome C. Anyalebechi, Kimberly M. Ramonell, Ching-wen Chen, Jianfeng Xie, Zhe Liang, Deena B. Chihade, Shunsuke Otani, Craig M. Coopersmith, Mandy L. Ford
The omega-3 fatty acid docosahexaenoic acid (DHA) inversely relates to neurological impairments with aging; however, limited nondietary models manipulating brain DHA have hindered a direct linkage. We discovered that loss of long-chain acyl-CoA synthetase 6 in mice (Acsl6–/–) depletes brain membrane phospholipid DHA levels, independent of diet. Here, Acsl6–/– brains contained lower DHA compared with controls across the life span. The loss of DHA- and increased arachidonate-enriched phospholipids were visualized by MALDI imaging predominantly in neuron-rich regions where single-molecule RNA in situ hybridization localized Acsl6 to neurons. ACSL6 is also astrocytic; however, we found that astrocyte-specific ACSL6 depletion did not alter membrane DHA because astrocytes express a non–DHA-preferring ACSL6 variant. Across the life span, Acsl6–/– mice exhibited hyperlocomotion, impairments in working spatial memory, and increased cholesterol biosynthesis genes. Aging caused Acsl6–/– brains to decrease the expression of membrane, bioenergetic, ribosomal, and synaptic genes and increase the expression of immune response genes. With age, the Acsl6–/– cerebellum became inflamed and gliotic. Together, our findings suggest that ACSL6 promotes membrane DHA enrichment in neurons, but not in astrocytes, and is important for neuronal DHA levels across the life span. The loss of ACSL6 impacts motor function, memory, and age-related neuroinflammation, reflecting the importance of neuronal ACSL6-mediated lipid metabolism across the life span.
Regina F. Fernandez, Andrea S. Pereyra, Victoria Diaz, Emily S. Wilson, Karen A. Litwa, Jonatan Martínez-Gardeazabal, Shelley N. Jackson, J. Thomas Brenna, Brian P. Hermann, Jeffrey B. Eells, Jessica M. Ellis
The small GTPase RhoA and its downstream effectors are critical regulators in the pathophysiological pro¬cesses of asthma. The underlying mechanism, however, remains undetermined. Here, we generated asthma mouse model with RhoA conditional knockout mice (Sftpc-cre;RhoAf/f) in type II alveolar epithelial cells (AT2) and demonstrated that AT2 cell specific deletion of RhoA leads to exacerbation of allergen-induced airway hyper-responsiveness and airway inflammation with elevated Th2 cytokines in bronchoalveolar lavage fluid (BALF). Notably, Sftpc-cre;RhoAf/f mice showed a significant reduction in TGF-β1 levels in BALFs and lung tissues, and administration of recombinant TGF-β1 to the mice rescued TGF-β1 and alleviated the increased allergic airway inflammation observed in Sftpc-cre;RhoAf/f mice. Using RNA-seq technology, we identified Slc26a4 (pendrin), a transmembrane anion exchange, as the most up-regulated gene in RhoA-deficient AT2 cells. The up-regulation of SLC26A4 was further confirmed in AT2 cells of asthmatic patients and mouse model and in human airway epithelial cells expressing dominant- negative RhoA (RhoA-N19). SLA26A4 was also elevated in serum from asthmatic patients and negatively associated with FEV1%. Furthermore, SLC26A4 inhibitor promoted epithelial TGF-β1 release and attenuated allergic airway inflammation. Our study reveals a previously undefined RhoA-SLC26A4 axis in AT2 cells that functions as a protective mechanism against allergic airway inflammation.
Danh C. Do, Yan Zhang, Wei Tu, Xinyue Hu, Xiaojun Xiao, Jingsi Chen, Haiping Hao, Zhigang Liu, Jing Li, Shau-Ku Huang, Mei Wan, Peisong Gao
T cell receptor (TCR) stimulation leads to expression of the transcription factor TOX. Prolonged TCR signaling, such as encountered during chronic infections or in tumors, leads to sustained TOX expression, which is required for the induction of a state of exhaustion or dysfunction. While CD8 memory T cells (Tmem) in mice typically do not express TOX at steady state, some human Tmem express TOX, but appear fully functional. This seeming discrepancy between mouse and human T cells has led to the speculation that TOX is differentially regulated between these species, which could complicate the interpretation of pre-clinical mouse model studies. We report here that similarly to TCR-mediated signals, inflammatory cytokines are also sufficient to increase TOX expression in human and mouse Tmem. Thus, TOX expression is controlled by the environment, which provides an explanation for the different TOX expression patterns encountered in T cells isolated from specific pathogen free laboratory mice versus humans. Finally, we report that TOX is not necessary for cytokine-driven expression of PD-1. Overall, our data highlight that the mechanisms regulating TOX expression are conserved across species and indicate that TOX expression reflects a T cell’s activation state, and does not necessarily correlate with T cell dysfunction.
Nicholas J. Maurice, Jacqueline Berner, Alexis K. Taber, Dietmar Zehn, Martin Prlic
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