STAT4 is expressed in neutrophils and promotes antimicrobial immunity
In this issue, Mehrpouya-Bahrami et al. uncover a key role for STAT4 in multiple neutrophil functions and innate immune responses. The cover art depicts the production of neutrophiI extracellular traps. Image credit: Cristina Risi.
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Research Articles
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Abstract
The acute respiratory distress syndrome (ARDS) is a highly lethal condition that impairs lung function and causes respiratory failure. Mechanical ventilation (MV) maintains gas exchange in patients with ARDS but exposes lung cells to physical forces that exacerbate injury. Our data demonstrate that mTOR complex 1 (mTORC1) is a mechanosensor in lung epithelial cells and that activation of this pathway during MV impairs lung function. We found that mTORC1 is activated in lung epithelial cells following volutrauma and atelectrauma in mice and humanized in vitro models of the lung microenvironment. mTORC1 is also activated in lung tissue of mechanically ventilated patients with ARDS. Deletion of Tsc2, a negative regulator of mTORC1, in epithelial cells impairs lung compliance during MV. Conversely, treatment with rapamycin at the time MV is initiated improves lung compliance without altering lung inflammation or barrier permeability. mTORC1 inhibition mitigates physiologic lung injury by preventing surfactant dysfunction during MV. Our data demonstrate that, in contrast to canonical mTORC1 activation under favorable growth conditions, activation of mTORC1 during MV exacerbates lung injury and inhibition of this pathway may be a novel therapeutic target to mitigate ventilator-induced lung injury during ARDS.
Authors
Hyunwook Lee, Qinqin Fei, Adam Streicher, Wenjuan Zhang, Colleen Isabelle, Pragi Patel, Hilaire C. Lam, Antonio Arciniegas-Rubio, Miguel Pinilla-Vera, Diana P. Amador-Munoz, Diana Barragan-Bradford, Angelica Higuera-Moreno, Rachel K. Putman, Lynette M. Sholl, Elizabeth P. Henske, Christopher M. Bobba, Natalia Higuita-Castro, Emily M. Shalosky, R. Duncan Hite, John W. Christman, Samir N. Ghadiali, Rebecca M. Baron, Joshua A. Englert
×Abstract
Cancer cells reprogram cellular metabolism to maintain adequate nutrient pools to sustain proliferation. Moreover, autophagy is a regulated mechanism to break down dysfunctional cellular components and recycle cellular nutrients. However, the requirement for autophagy and the integration in cancer cell metabolism is not clear in colon cancer. Here, we show a cell-autonomous dependency of autophagy for cell growth in colorectal cancer. Loss of epithelial autophagy inhibits tumor growth in both sporadic and colitis-associated cancer models. Genetic and pharmacological inhibition of autophagy inhibits cell growth in colon cancer–derived cell lines and patient-derived enteroid models. Importantly, normal colon epithelium and patient-derived normal enteroid growth were not decreased following autophagy inhibition. To couple the role of autophagy to cellular metabolism, a cell culture screen in conjunction with metabolomic analysis was performed. We identified a critical role of autophagy to maintain mitochondrial metabolites for growth. Loss of mitochondrial recycling through inhibition of mitophagy hinders colon cancer cell growth. These findings have revealed a cell-autonomous role of autophagy that plays a critical role in regulating nutrient pools in vivo and in cell models, and it provides therapeutic targets for colon cancer.
Authors
Samantha N. Devenport, Rashi Singhal, Megan D. Radyk, Joseph G. Taranto, Samuel A. Kerk, Brandon Chen, Joshua W. Goyert, Chesta Jain, Nupur K. Das, Katherine Oravecz-Wilson, Li Zhang, Joel K. Greenson, Y. Eugene Chen, Scott A. Soleimanpour, Pavan Reddy, Costas A. Lyssiotis, Yatrik M. Shah
×Abstract
BACKGROUND The fungal cell wall constituent 1,3-β-d-glucan (BDG) is a pathogen-associated molecular pattern that can stimulate innate immunity. We hypothesized that BDG from colonizing fungi in critically ill patients may translocate into the systemic circulation and be associated with host inflammation and outcomes.METHODS We enrolled 453 mechanically ventilated patients with acute respiratory failure (ARF) without invasive fungal infection and measured BDG, innate immunity, and epithelial permeability biomarkers in serially collected plasma samples.RESULTS Compared with healthy controls, patients with ARF had significantly higher BDG levels (median [IQR], 26 pg/mL [15–49 pg/mL], P < 0.001), whereas patients with ARF with high BDG levels (≥40 pg/mL, 31%) had higher odds for assignment to the prognostically adverse hyperinflammatory subphenotype (OR [CI], 2.88 [1.83–4.54], P < 0.001). Baseline BDG levels were predictive of fewer ventilator-free days and worse 30-day survival (adjusted P < 0.05). Integrative analyses of fungal colonization and epithelial barrier disruption suggested that BDG may translocate from either the lung or gut compartment. We validated the associations between plasma BDG and host inflammatory responses in 97 hospitalized patients with COVID-19.CONCLUSION BDG measurements offered prognostic information in critically ill patients without fungal infections. Further research in the mechanisms of translocation and innate immunity recognition and stimulation may offer new therapeutic opportunities in critical illness.FUNDING University of Pittsburgh Clinical and Translational Science Institute, COVID-19 Pilot Award and NIH grants (K23 HL139987, U01 HL098962, P01 HL114453, R01 HL097376, K24 HL123342, U01 HL137159, R01 LM012087, K08HK144820, F32 HL142172, K23 GM122069).
Authors
Georgios D. Kitsios, Daniel Kotok, Haopu Yang, Malcolm A. Finkelman, Yonglong Zhang, Noel Britton, Xiaoyun Li, Marina S. Levochkina, Amy K. Wagner, Caitlin Schaefer, John J. Villandre, Rui Guo, John W. Evankovich, William Bain, Faraaz Shah, Yingze Zhang, Barbara A. Methé, Panayiotis V. Benos, Bryan J. McVerry, Alison Morris
×Abstract
Signal transducer and activator of transcription 4 (STAT4) is expressed in hematopoietic cells and plays a key role in the differentiation of T helper 1 cells. Although STAT4 is required for immunity to intracellular pathogens, the T cell–independent protective mechanisms of STAT4 are not clearly defined. In this report, we demonstrate that STAT4-deficient mice were acutely sensitive to methicillin-resistant Staphylococcus aureus (MRSA) infection. We show that STAT4 was expressed in neutrophils and activated by IL-12 via a JAK2-dependent pathway. We demonstrate that STAT4 was required for multiple neutrophil functions, including IL-12–induced ROS production, chemotaxis, and production of the neutrophil extracellular traps. Importantly, myeloid-specific and neutrophil-specific deletion of STAT4 resulted in enhanced susceptibility to MRSA, demonstrating the key role of STAT4 in the in vivo function of these cells. Thus, these studies identify STAT4 as an essential regulator of neutrophil functions and a component of innate immune responses in vivo.
Authors
Pegah Mehrpouya-Bahrami, Alina K. Moriarty, Paulo De Melo, W. Coles Keeter, Nada S. Alakhras, Andrew S. Nelson, Madeline Hoover, Maria S. Barrios, Jerry L. Nadler, C. Henrique Serezani, Mark H. Kaplan, Elena V. Galkina
×Abstract
Mutations in the gene SFTPC, encoding surfactant protein C (SP-C), are associated with interstitial lung disease in children and adults. To assess the natural history of disease, we knocked in a familial, disease-associated SFTPC mutation, L188Q (L184Q [LQ] in mice), into the mouse Sftpc locus. Translation of the mutant proprotein, proSP-CLQ, exceeded that of proSP-CWT in neonatal alveolar type 2 epithelial cells (AT2 cells) and was associated with transient activation of oxidative stress and apoptosis, leading to impaired expansion of AT2 cells during postnatal alveolarization. Differentiation of AT2 to AT1 cells was also inhibited in ex vivo organoid culture of AT2 cells isolated from LQ mice; importantly, treatment with antioxidant promoted alveolar differentiation. Upon completion of alveolarization, SftpcLQ expression was downregulated, leading to resolution of chronic stress responses; however, the failure to restore AT2 cell numbers resulted in a permanent loss of AT2 cells that was linked to decreased regenerative capacity in the adult lung. Collectively, these data support the hypothesis that susceptibility to disease in adult LQ mice is established during postnatal lung development, and they provide a potential explanation for the delayed onset of disease in patients with familial pulmonary fibrosis.
Authors
Sneha Sitaraman, Emily P. Martin, Cheng-Lun Na, Shuyang Zhao, Jenna Green, Hitesh Deshmukh, Anne-Karina T. Perl, James P. Bridges, Yan Xu, Timothy E. Weaver
×Abstract
Estrogen-related receptor γ (Esrrg) is a murine lupus susceptibility gene associated with T cell activation. Here, we report that Esrrg controls Tregs through mitochondria homeostasis. Esrrg deficiency impaired the maintenance and function of Tregs, leading to global T cell activation and autoimmunity in aged mice. Further, Esrrg-deficient Tregs presented an impaired differentiation into follicular Tregs that enhanced follicular helper T cells’ responses. Mechanistically, Esrrg-deficient Tregs presented with dysregulated mitochondria with decreased oxygen consumption as well as ATP and NAD+ production. In addition, Esrrg-deficient Tregs exhibited decreased phosphatidylinositol and TGF-β signaling pathways and increased mTOR complex 1 activation. We found that the expression of human ESRRG, which is high in Tregs, was lower in CD4+ T cells from patients with lupus than in healthy controls. Finally, knocking down ESRRG in Jurkat T cells decreased their metabolism. Together, our results reveal a critical role of Esrrg in the maintenance and metabolism of Tregs, which may provide a genetic link between lupus pathogenesis and mitochondrial dysfunction in T cells.
Authors
Wei Li, Minghao Gong, Yuk Pheel Park, Ahmed S. Elshikha, Seung-Chul Choi, Josephine Brown, Nathalie Kanda, Wen-I Yeh, Leeana Peters, Anton A. Titov, Xiangyu Teng, Todd M. Brusko, Laurence Morel
×Abstract
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.
Authors
Kehong Chen, Joaquin Cagliani, Monowar Aziz, Chuyi Tan, Max Brenner, Ping Wang
×Abstract
Allogeneic hematopoietic stem cell transplantation is a viable treatment for multiple hematologic diseases, but its application is often limited by graft-versus-host disease (GVHD), where donor T cells attack host tissues in the skin, liver, and gastrointestinal tract. Here, we examined the role of the cellular energy sensor AMP kinase (AMPK) in alloreactive T cells during GVHD development. Early posttransplant, AMPK activity increased more than 15-fold in allogeneic T cells, and transplantation of T cells deficient in both AMPKα1 and AMPKα2 decreased GVHD severity in multiple disease models. Importantly, a lack of AMPK lessened GVHD without compromising antileukemia responses or impairing lymphopenia-driven immune reconstitution. Mechanistically, absence of AMPK decreased both CD4+ and CD8+ effector T cell numbers as early as day 3 posttransplant, while simultaneously increasing regulatory T cell (Treg) percentages. Improvements in GVHD resulted from cell-intrinsic perturbations in conventional effector T cells as depletion of donor Tregs had minimal impact on AMPK-related improvements. Together, these results highlight a specific role for AMPK in allogeneic effector T cells early posttransplant and suggest that AMPK inhibition may be an innovative approach to mitigate GVHD while preserving graft-versus-leukemia responses and maintaining robust immune reconstitution.
Authors
Darlene A. Monlish, Kevin J. Beezhold, Pailin Chiaranunt, Katelyn Paz, Nathan J. Moore, Andrea K. Dobbs, Rebecca A. Brown, John A. Ozolek, Bruce R. Blazar, Craig A. Byersdorfer
×Abstract
The blood-brain barrier (BBB) prevents antibodies from penetrating the CNS and limits conventional antibody-based approaches to brain tumors. We now show that ENT2, a transporter that regulates nucleoside flux at the BBB, may offer an unexpected path to circumventing this barrier to allow targeting of brain tumors with an anti-DNA autoantibody. Deoxymab-1 (DX1) is a DNA-damaging autoantibody that localizes to tumors and is synthetically lethal to cancer cells with defects in the DNA damage response. We found that DX1 penetrated brain endothelial cells and crossed the BBB, and mechanistic studies identify ENT2 as the key transporter. In efficacy studies, DX1 crosses the BBB to suppress orthotopic glioblastoma and breast cancer brain metastases. ENT2-linked transport of autoantibodies across the BBB has potential to be exploited in brain tumor immunotherapy, and its discovery raises hypotheses on actionable mechanisms of CNS penetration by neurotoxic autoantibodies in CNS lupus.
Authors
Zahra Rattray, Gang Deng, Shenqi Zhang, Anupama Shirali, Christopher K. May, Xiaoyong Chen, Benedette J. Cuffari, Jun Liu, Pan Zou, Nicholas J.W. Rattray, Caroline H. Johnson, Valentina Dubljevic, James A. Campbell, Anita Huttner, Joachim M. Baehring, Jiangbing Zhou, James E. Hansen
×Abstract
Agonist CD40 antibodies are under clinical development in combination with chemotherapy as an approach to prime for antitumor T cell immunity. However, treatment with anti-CD40 is commonly accompanied by both systemic cytokine release and liver transaminase elevations, which together account for the most common dose-limiting toxicities. Moreover, anti-CD40 treatment increases the potential for chemotherapy-induced hepatotoxicity. Here, we report a mechanistic link between cytokine release and hepatotoxicity induced by anti-CD40 when combined with chemotherapy and show that toxicity can be suppressed without impairing therapeutic efficacy. We demonstrate in mice and humans that anti-CD40 triggers transient hepatotoxicity marked by increased serum transaminase levels. In doing so, anti-CD40 sensitizes the liver to drug-induced toxicity. Unexpectedly, this biology is not blocked by the depletion of multiple myeloid cell subsets, including macrophages, inflammatory monocytes, and granulocytes. Transcriptional profiling of the liver after anti-CD40 revealed activation of multiple cytokine pathways including TNF and IL-6. Neutralization of TNF, but not IL-6, prevented sensitization of the liver to hepatotoxicity induced with anti-CD40 in combination with chemotherapy without impacting antitumor efficacy. Our findings reveal a clinically feasible approach to mitigate toxicity without impairing efficacy in the use of agonist CD40 antibodies for cancer immunotherapy.
Authors
Meredith L. Stone, Jesse Lee, Veronica M. Herrera, Kathleen Graham, Jae W. Lee, Austin Huffman, Heather Coho, Evan Tooker, Max I. Myers, Michael Giannone, Yan Li, Thomas H. Buckingham, Kristen B. Long, Gregory L. Beatty
×Abstract
Although CD8+ T cells recognize neoantigens that arise from somatic mutations in cancer, only a small fraction of nonsynonymous mutations give rise to clinically relevant neoantigens. In this study, HLA class I ligandomes of a panel of human colorectal cancer (CRC) and matched normal tissues were analyzed using mass spectrometry–based proteogenomic analysis. Neoantigen presentation was rare; however, the analysis detected a single neoantigen in a mismatch repair–deficient CRC (dMMR-CRC) tissue sample carrying 3967 nonsynonymous mutations, where abundant tumor-infiltrating lymphocytes (TILs) and inflamed gene expression status were observed in the tumor microenvironment (TME). Using the HLA class I ligandome data and gene expression profiles, a set of nonmutated tumor-associated antigen (TAA) candidates was concomitantly identified. Interestingly, CD8+ TILs predominantly recognized the detected neoantigen over the array of TAA candidates. Neoantigen-reactive CD8+ TILs showed PD-1 positivity and exhibited functional and specific responses. Moreover, T cell receptor (TCR) profiling identified the sequence of the neoantigen-reactive TCR clonotype and showed its expansion in the TME. Transduction of the sequenced TCR conferred neoantigen specificity and cytotoxicity to peripheral blood lymphocytes. The proteogenomic approach revealed the antigenic and reactive T cell landscape in dMMR-CRC, demonstrating the presence of an immunogenic neoantigen and its potential therapeutic applications.
Authors
Tomomi Hirama, Serina Tokita, Munehide Nakatsugawa, Kenji Murata, Yasuhito Nannya, Kazuhiko Matsuo, Hidetoshi Inoko, Yoshihiko Hirohashi, Shinichi Hashimoto, Seishi Ogawa, Ichiro Takemasa, Noriyuki Sato, Fumitake Hata, Takayuki Kanaseki, Toshihiko Torigoe
×Hematologic and systemic metabolic alterations due to Mediterranean class II G6PD deficiency in mice
Abstract
Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of WT stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RBCs) that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency, Med(-), was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs, and deficient mice had increased hemolytic sequelae to oxidant stress. Nonerythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD-deficient and WT mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and advance our basic understanding of altered metabolism in this setting.
Authors
Angelo D’Alessandro, Heather L. Howie, Ariel M. Hay, Karolina H. Dziewulska, Benjamin C. Brown, Matthew J. Wither, Matthew Karafin, Elizabeth F. Stone, Steven L. Spitalnik, Eldad A. Hod, Richard O. Francis, Xiaoyun Fu, Tiffany Thomas, James C. Zimring
×Abstract
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 nonlethal dose of lipopolysaccharide (LPS). SOCS3iEKO mice died 16–24 hours postinjection after severe kidney failure. Loss of SOCS3 led to an LPS-induced type I IFN–like program and high expression of prothrombotic and proadhesive genes. Consistently, we observed intraluminal leukocyte adhesion and neutrophil extracellular trap–osis (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 human umbilical vein endothelial cells, pulse-chase experiments showed that SOCS3 protein had a half-life less than 20 minutes. Inhibition of SOCS3 ubiquitination and proteasomal degradation led to protein accumulation and a stronger inhibition of IL-6 signaling and barrier function loss. Together, our data demonstrate that the regulation of SOCS3 protein levels is critical to inhibit IL-6–mediated endotheliopathy during shock and provide a promising therapeutic avenue to prevent multiorgan dysfunction through stabilization of endothelial SOCS3.
Authors
Nina Martino, Ramon Bossardi Ramos, Shuhan Lu, Kara Leyden, Lindsay Tomaszek, Sudeshna Sadhu, Gabrielle Fredman, Ariel Jaitovich, Peter A. Vincent, Alejandro P. Adam
×12-Lipoxygenase governs the innate immune pathogenesis of islet inflammation and autoimmune diabetes
Abstract
Macrophages and related myeloid cells are innate immune cells that participate in the early islet inflammation of type 1 diabetes (T1D). The enzyme 12-lipoxygenase (12-LOX) catalyzes the formation of proinflammatory eicosanoids, but its role and mechanisms in myeloid cells in the pathogenesis of islet inflammation have not been elucidated. Leveraging a model of islet inflammation in zebrafish, we show here that macrophages contribute significantly to the loss of β cells and the subsequent development of hyperglycemia. The depletion or inhibition of 12-LOX in this model resulted in reduced macrophage infiltration into islets and the preservation of β cell mass. In NOD mice, the deletion of the gene encoding 12-LOX in the myeloid lineage resulted in reduced insulitis with reductions in proinflammatory macrophages, a suppressed T cell response, preserved β cell mass, and almost complete protection from the development of T1D. 12-LOX depletion caused a defect in myeloid cell migration, a function required for immune surveillance and tissue injury responses. This effect on migration resulted from the loss of the chemokine receptor CXCR3. Transgenic expression of the gene encoding CXCR3 rescued the migratory defect in zebrafish 12-LOX morphants. Taken together, our results reveal a formative role for innate immune cells in the early pathogenesis of T1D and identify 12-LOX as an enzyme required to promote their prodiabetogenic phenotype in the context of autoimmunity.
Authors
Abhishek Kulkarni, Annie R. Pineros, Melissa A. Walsh, Isabel Casimiro, Sara Ibrahim, Marimar Hernandez-Perez, Kara S. Orr, Lindsey Glenn, Jerry L. Nadler, Margaret A. Morris, Sarah A. Tersey, Raghavendra G. Mirmira, Ryan M. Anderson
×Abstract
IFN-γ–driven responses to malaria have been shown to modulate the development and function of T follicular helper (TFH) cells and memory B cells (MBCs), with conflicting evidence of their involvement in the induction of antibody responses required to achieve clinical immunity and their association with disease outcomes. Using high-dimensional single-cell mass cytometry, we identified distinct populations of TH1-polarized CD4+ T cells and MBCs expressing the TH1-defining transcription factor T-bet, associated with either increased or reduced risk of Plasmodium vivax (P. vivax) malaria, demonstrating that inflammatory responses to malaria are not universally detrimental for infection. Furthermore, we found that, whereas class-switched but not IgM+ MBCs were associated with a reduced risk of symptomatic malaria, populations of TH1 cells with a stem central memory phenotype, TH17 cells, and T regulatory cells were associated with protection from asymptomatic infection, suggesting that activation of cell-mediated immunity might also be required to control persistent P. vivax infection with low parasite burden.
Authors
Lisa J. Ioannidis, Halina M. Pietrzak, Ann Ly, Retno A.S. Utami, Emily M. Eriksson, Stephanie I. Studniberg, Waruni Abeysekera, Connie S.N. Li-Wai-Suen, Dylan Sheerin, Julie Healer, Agatha M. Puspitasari, Dwi Apriyanti, Farah N. Coutrier, Jeanne R. Poespoprodjo, Enny Kenangalem, Benediktus Andries, Pak Prayoga, Novita Sariyanti, Gordon K. Smyth, Leily Trianty, Alan F. Cowman, Ric N. Price, Rintis Noviyanti, Diana S. Hansen
×Abstract
The small GTPase RhoA and its downstream effectors are critical regulators in the pathophysiological processes of asthma. The underlying mechanism, however, remains undetermined. Here, we generated an asthma mouse model with RhoA–conditional KO mice (Sftpc-cre;RhoAfl/fl) in type II alveolar epithelial cells (AT2) and demonstrated that AT2 cell–specific deletion of RhoA leads to exacerbation of allergen-induced airway hyperresponsiveness and airway inflammation with elevated Th2 cytokines in bronchoalveolar lavage fluid (BALF). Notably, Sftpc-cre;RhoAfl/fl mice showed a significant reduction in Tgf-β1 levels in BALF 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;RhoAfl/fl mice. Using RNA sequencing technology, we identified Slc26a4 (pendrin), a transmembrane anion exchange, as the most upregulated gene in RhoA-deficient AT2 cells. The upregulation of SLC26A4 was further confirmed in AT2 cells of asthmatic patients and mouse models 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 the percentage of forced expiratory volume in 1 second (FEV1%). Furthermore, SLC26A4 inhibition promoted epithelial TGF-β1 release and attenuated allergic airway inflammation. Our study reveals a RhoA/SLC26A4 axis in AT2 cells that functions as a protective mechanism against allergic airway inflammation.
Authors
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
×Abstract
TNFRSF13B encodes the transmembrane activator and CAML interactor (TACI) receptor, which drives plasma cell differentiation. Although TNFRSF13B supports host defense, dominant-negative TNFRSF13B alleles are common in humans and other species and only rarely associate with disease. We reasoned that the high frequency of disruptive TNFRSF13B alleles reflects balancing selection, the loss of function conferring advantage in some settings. Testing that concept, we investigated how a common human dominant-negative variant, TNFRSF13B A181E, imparts resistance to enteric pathogens. Mice engineered to express mono- or biallelic A144E variants of tnrsf13B, corresponding to A181E, exhibited a striking resistance to pathogenicity and transmission of Citrobacter rodentium, a murine pathogen that models enterohemorrhagic Escherichia coli, and resistance was principally owed to natural IgA deficiency in the intestine. In WT mice with gut IgA and in mutant mice reconstituted with enteric IgA obtained from WT mice, IgA induces LEE expression of encoded virulence genes, which confer pathogenicity and transmission. Taken together, our results show that C. rodentium and most likely other enteric organisms appropriated binding of otherwise protective antibodies to signal induction of the virulence program. Additionally, the high prevalence of TNFRSF13B dominant-negative variants reflects balancing selection.
Authors
Jeffrey L. Platt, Mayara Garcia de Mattos Barbosa, Daniel Huynh, Adam R. Lefferts, Juhi Katta, Cyra Kharas, Peter Freddolino, Christine M. Bassis, Christiane Wobus, Raif Geha, Richard Bram, Gabriel Nunez, Nobuhiko Kamada, Marilia Cascalho
×Abstract
Mutations in HNRNPA1 encoding heterogeneous nuclear ribonucleoprotein (hnRNP) A1 are a rare cause of amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP). hnRNPA1 is part of the group of RNA-binding proteins (RBPs) that assemble with RNA to form RNPs. hnRNPs are concentrated in the nucleus and function in pre-mRNA splicing, mRNA stability, and the regulation of transcription and translation. During stress, hnRNPs, mRNA, and other RBPs condense in the cytoplasm to form stress granules (SGs). SGs are implicated in the pathogenesis of (neuro-)degenerative diseases, including ALS and inclusion body myopathy (IBM). Mutations in RBPs that affect SG biology, including FUS, TDP-43, hnRNPA1, hnRNPA2B1, and TIA1, underlie ALS, IBM, and other neurodegenerative diseases. Here, we characterize 4 potentially novel HNRNPA1 mutations (yielding 3 protein variants: *321Eext*6, *321Qext*6, and G304Nfs*3) and 2 known HNRNPA1 mutations (P288A and D262V), previously connected to ALS and MSP, in a broad spectrum of patients with hereditary motor neuropathy, ALS, and myopathy. We establish that the mutations can have different effects on hnRNPA1 fibrillization, liquid-liquid phase separation, and SG dynamics. P288A accelerated fibrillization and decelerated SG disassembly, whereas *321Eext*6 had no effect on fibrillization but decelerated SG disassembly. By contrast, G304Nfs*3 decelerated fibrillization and impaired liquid phase separation. Our findings suggest different underlying pathomechanisms for HNRNPA1 mutations with a possible link to clinical phenotypes.
Authors
Danique Beijer, Hong Joo Kim, Lin Guo, Kevin O’Donovan, Inès Mademan, Tine Deconinck, Kristof Van Schil, Charlotte M. Fare, Lauren E. Drake, Alice F. Ford, Andrzej Kochański, Dagmara Kabzińska, Nicolas Dubuisson, Peter Van den Bergh, Nicol C. Voermans, Richard J.L.F. Lemmers, Silvère M. van der Maarel, Devon Bonner, Jacinda B. Sampson, Matthew T. Wheeler, Anahit Mehrabyan, Steven Palmer, Peter De Jonghe, James Shorter, J. Paul Taylor, Jonathan Baets
×Abstract
Aniridia is most commonly caused by haploinsufficiency of the PAX6 gene, characterized by variable iris and foveal hypoplasia, nystagmus, cataracts, glaucoma, and aniridia-related keratopathy (ARK). Genotype-phenotype correlations have previously been described; however, detailed longitudinal studies of aniridia are less commonly reported. We identified 86 patients from 62 unrelated families with molecularly confirmed heterozygous PAX6 variants from a UK-based single-center ocular genetics service. They were categorized into mutation groups, and a retrospective review of clinical characteristics (ocular and systemic) from baseline to most recent was recorded. One hundred and seventy-two eyes were evaluated, with a mean follow-up period of 16.3 ± 12.7 years. Nystagmus was recorded in 87.2% of the eyes, and foveal hypoplasia was found in 75%. Cataracts were diagnosed in 70.3%, glaucoma in 20.6%, and ARK in 68.6% of eyes. Prevalence, age of diagnosis and surgical intervention, and need for surgical intervention varied among mutation groups. Overall, the missense mutation subgroup had the mildest phenotype, and surgically naive eyes maintained better visual acuity. Systemic evaluation identified type 2 diabetes in 12.8% of the study group, which is twice the UK prevalence. This is the largest longitudinal study of aniridia in the UK, and as such, it can provide insights into prognostic indicators for patients and guiding clinical management of both ocular and systemic features.
Authors
Vivienne Kit, Dulce Lima Cunha, Ahmed M. Hagag, Mariya Moosajee
×Abstract
BACKGROUND Individuals recovering from COVID-19 frequently experience persistent respiratory ailments, which are key elements of postacute sequelae of SARS-CoV-2 infection (PASC); however, little is known about the underlying biological factors that may direct lung recovery and the extent to which these are affected by COVID-19 severity.METHODS We performed a prospective cohort study of individuals with persistent symptoms after acute COVID-19, collecting clinical data, pulmonary function tests, and plasma samples used for multiplex profiling of inflammatory, metabolic, angiogenic, and fibrotic factors.RESULTS Sixty-one participants were enrolled across 2 academic medical centers at a median of 9 weeks (interquartile range, 6–10 weeks) after COVID-19 illness: n = 13 participants (21%) had mild COVID-19 and were not hospitalized, n = 30 participants (49%) were hospitalized but were considered noncritical, and n = 18 participants (30%) were hospitalized and in the intensive care unit (ICU). Fifty-three participants (85%) had lingering symptoms, most commonly dyspnea (69%) and cough (58%). Forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), and diffusing capacity for carbon monoxide (DLCO) declined as COVID-19 severity increased (P < 0.05) but these values did not correlate with respiratory symptoms. Partial least-squares discriminant analysis of plasma biomarker profiles clustered participants by past COVID-19 severity. Lipocalin-2 (LCN2), MMP-7, and HGF identified by our analysis were significantly higher in the ICU group (P < 0.05), inversely correlated with FVC and DLCO (P < 0.05), and were confirmed in a separate validation cohort (n = 53).CONCLUSION Subjective respiratory symptoms are common after acute COVID-19 illness but do not correlate with COVID-19 severity or pulmonary function. Host response profiles reflecting neutrophil activation (LCN2), fibrosis signaling (MMP-7), and alveolar repair (HGF) track with lung impairment and may be novel therapeutic or prognostic targets.Funding National Heart, Lung, and Blood Institute (K08HL130557 and R01HL142818), American Heart Association (Transformational Project Award), the DeLuca Foundation Award, a donation from Jack Levin to the Benign Hematology Program at Yale University, and Duke University.
Authors
Hyung J. Chun, Elias Coutavas, Alexander B. Pine, Alfred I. Lee, Vanessa L. Yu, Marcus K. Shallow, Coral X. Giovacchini, Anne M. Mathews, Brian Stephenson, Loretta G. Que, Patty J. Lee, Bryan D. Kraft
×Abstract
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a pandemic. Severe disease is associated with dysfunction of multiple organs, but some infected cells do not express ACE2, the canonical entry receptor for SARS-CoV-2. Here, we report that the C-type lectin receptor L-SIGN interacted in a Ca2+-dependent manner with high-mannose–type N-glycans on the SARS-CoV-2 spike protein. We found that L-SIGN was highly expressed on human liver sinusoidal endothelial cells (LSECs) and lymph node lymphatic endothelial cells but not on blood endothelial cells. Using high-resolution confocal microscopy imaging, we detected SARS-CoV-2 viral proteins within the LSECs from liver autopsy samples from patients with COVID-19. We found that both pseudo-typed virus enveloped with SARS-CoV-2 spike protein and authentic SARS-CoV-2 virus infected L-SIGN–expressing cells relative to control cells. Moreover, blocking L-SIGN function reduced CoV-2–type infection. These results indicate that L-SIGN is a receptor for SARS-CoV-2 infection. LSECs are major sources of the clotting factors vWF and factor VIII (FVIII). LSECs from liver autopsy samples from patients with COVID-19 expressed substantially higher levels of vWF and FVIII than LSECs from uninfected liver samples. Our data demonstrate that L-SIGN is an endothelial cell receptor for SARS-CoV-2 that may contribute to COVID-19–associated coagulopathy.
Authors
Yuji Kondo, Jason L. Larabee, Liang Gao, Huiping Shi, Bojing Shao, Christopher M. Hoover, J. Michael McDaniel, Yen-Chun Ho, Robert Silasi-Mansat, Stephanie A. Archer-Hartmann, Parastoo Azadi, R. Sathish Srinivasan, Alireza R. Rezaie, Alain Borczuk, Jeffrey C. Laurence, Florea Lupu, Jasimuddin Ahamed, Rodger P. McEver, James F. Papin, Zhongxin Yu, Lijun Xia
×Abstract
Background Abdominal pain and constipation are 2 main symptoms in patients with constipation-predominant irritable bowel syndrome (IBS-C). This study aimed to investigate the effects and possible mechanisms of transcutaneous auricular vagal nerve stimulation (taVNS) in patients with IBS-C.Methods Forty-two patients with IBS-C were randomized into a 4-week sham-taVNS or taVNS treatment. The primary outcomes were complete spontaneous bowel movements per week (CSBMs/week) and visual analog scale (VAS) for abdominal pain. High-resolution anorectal manometry (HRAM) was performed to evaluate anorectal motor and sensory function. Cytokines and brain gut peptides were analyzed in blood samples. ECG was recorded for the assessment of autonomic function.Results Compared with sham-taVNS, (a) taVNS increased CSBMs/week (P = 0.001) and decreased VAS pain score (P = 0.001); (b) improved quality of life (P = 0.020) and decreased IBS symptom score (P = 0.001); (c) improved rectoanal inhibitory reflex (P = 0.014) and improved rectal sensation (P < 0.04); (d) decreased a number of proinflammatory cytokines and serotonin in circulation; and (e) enhanced vagal activity (P = 0.040). The vagal activity was weakly correlated with the CSBMs/week (r = 0.391; P = 0.010) and the VAS pain score (r = –0.347; P = 0.025).Conclusions Noninvasive taVNS improves both constipation and abdominal pain in patients with IBS-C. The improvement in IBS-C symptoms might be attributed to the integrative effects of taVNS on intestinal functions mediated via the autoimmune mechanisms.Trial registration www.chictr.org.cn, no. ChiCTR2000029644.Funding National Natural Science Foundation of China (grant no. 81970538 for FL).
Authors
Xiaodan Shi, Yedong Hu, Bo Zhang, Wenna Li, Jiande DZ Chen, Fei Liu
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Abstract
The NR4A family of orphan nuclear receptors (Nr4a1-3) plays redundant roles to establish and maintain Treg identity; deletion of multiple family members in the thymus results in Treg deficiency and a severe inflammatory disease. Consequently, it has been challenging to unmask redundant functions of the NR4A family in other immune cells. Here we use a competitive bone marrow chimera strategy, coupled with conditional genetic tools, to rescue Treg homeostasis and unmask such functions. Unexpectedly, chimeras harboring Nr4a1–/– Nr4a3–/– (DKO) bone marrow develop autoantibodies and a systemic inflammatory disease despite a replete Treg compartment of largely wild-type origin. This disease differs qualitatively from that seen with Treg-deficiency and is B cell-extrinsic. Negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO T cells with functional, phenotypic, and transcriptional features of anergy accumulate in chimeric mice. Nevertheless, we observe upregulation of genes encoding inflammatory mediators in anergic DKO T cells, and DKO T cells exhibit enhanced capacity for IL-2 production. These studies reveal cell-intrinsic roles for the NR4A family in both central and peripheral T cell tolerance, and demonstrate that each is essential to preserve immune homeostasis.
Authors
Ryosuke Hiwa, Hailyn V. Nielsen, James L. Mueller, Ravi Mandla, Julie Zikherman
Abstract
G protein-coupled receptors (GPCRs) are highly desirable drug targets for human disease. Although GPCR dysfunction drives development and progression of many tumors, including breast cancer (BC), targeting individual GPCRs has limited efficacy as a cancer therapy because numerous GPCRs are activated. Here, we sought a new way of blocking GPCR activation in HER2+-BC by targeting a subgroup of GPCRs that couple to Gi/o proteins (Gi/o-GPCRs). In mammary epithelial cells of transgenic mouse models, and BC cell lines, HER2 hyperactivation altered GPCR expression, particularly, Gi/o-GPCRs. Gi/o-GPCR stimulation transactivated EGFR and HER2 and activated the PI3K/AKT and Src pathways. If we uncoupled Gi/o-GPCRs from their cognate Gi/o proteins by pertussis toxin (PTx), then BC cell proliferation and migration was inhibited in vitro and HER2-driven tumor formation and metastasis suppressed in vivo. Moreover, targeting Gi/o-GPCR signaling via PTx, PI3K, or Src inhibitors enhanced HER2-targeted therapy. These results indicate that, in BC cells, HER2 hyperactivation drives aberrant Gi/o-GPCR signaling, and Gi/o-GPCR signals converge on PI3K/AKT and Src signaling pathways to promote cancer progression and resistance to HER2-targeted therapy. Our findings point to a new way to pharmacologically deactivate GPCR signaling to block tumor growth and enhance therapeutic efficacy.
Authors
Cancan Lyu, Yuanchao Ye, Maddison M. Lensing, Kay-Uwe Wagner, Ronald J. Weigel, Songhai Chen
Abstract
BACKGROUND. A previous Phase I study showed that the infusion of autologous Treg cells expanded ex-vivo into recent onset Type 1 Diabetes (T1D) patients had an excellent safety profile, however, the majority of the infused Tregs could no longer be detected in the peripheral blood three months post-infusion (NCT01210664-Treg-T1D Trial). Interleukin-2 (IL-2) has been shown to enhance human Treg cell survival and expansion at low doses in vivo. Therefore, we conducted a Phase 1 study (NCT02772679-TILT trial) combining polyclonal Treg and low-dose IL-2 and assessed the impact over time on Tregs populations and other immune cells. METHODS. T1D Patients were treated with a single infusion of autologous polyclonal Tregs, expanded ex vivo, followed by one or two 5-day courses of recombinant human low dose IL-2 (ld-IL-2) at 0.3 x 106 to 1 x 106 units/dose. Fluorescence-based flow cytometry, Cytometry by Time Of Flight (CyTOF) and 10X Genomics single cell RNAseq were used to follow the distinct immune cell populations phenotypes over time following immunotherapy. RESULTS. Multiparametric analysis revealed that the combination therapy led to an increase in the number of infused and endogenous Tregs but also resulted in a significant increase from baseline in a subset of activated NK, Mucosal-Associated Invariant T (MAIT) cells and clonal CD8+ T populations. CONCLUSIONS. These data support the hypothesis that Id-IL-2 expands exogenously administered Tregs but also can expand cytotoxic cells. These results have important implications for the use of a combination of ld-IL-2 and Tregs for the treatment of autoimmune diseases with pre-existing active immunity. TRIAL REGISTRATION. ClinicalTrials.gov NCT01210664 (Treg-T1D Trial), NCT02772679 (TILTtrial). FUNDING. Sean N. Parker Autoimmunity Research Laboratory Fund, National Center for Research Resources.
Authors
Shen Dong, Kamir J. Hiam-Galvez, Cody T. Mowery, Kevan C. Herold, Stephen E. Gitelman, Jonathan H. Esensten, Weihong Liu, Angela P. Lares, Ashley S. Leinbach, Michael Lee, Vinh Nguyen, Stanley J. Tamaki, Whitney Tamaki, Courtney M. Tamaki, Morvarid Mehdizadeh, Amy L. Putnam, Matthew H. Spitzer, C. Jimmie Ye, Qizhi Tang, Jeffrey A. Bluestone
Abstract
cGMP-dependent protein kinase 1α (PKG1α) promotes left ventricle (LV) compensation to pressure overload. PKG1-activating drugs improve heart failure (HF) outcomes but are limited by vasodilation-induced hypotension. Signaling molecules which mediate PKG1α cardiac therapeutic effects but do not promote PKG1α-induced hypotension could therefore represent improved therapeutic targets. We investigated roles of mixed lineage kinase 3 (MLK3) in mediating PKG1α effects on LV function after pressure overload, and in regulating blood pressure (BP). In a transaortic constriction HF model PKG activation with sildenafil preserved LV function in MLK3+/+, but not MLK3-/- littermates. MLK3 co-immunoprecipitated with PKG1α. MLK3-PKG1α co-interaction decreased in failing LVs. PKG1a phosphorylated MLK3 on Thr-277/Ser-281 sites required for kinase activation. MLK3-/- mice displayed hypertension and increased arterial stiffness, though PKG stimulation with sildenafil or the sGC stimulator BAY41-2272 still reduced BP in MLK3-/- mice. MLK3 kinase inhibition with URMC-099 did not affect BP, but induced LV dysfunction in mice. These data reveal MLK3 as a PKG1α substrate mediating PKG1α preservation of LV function but not acute PKG1α BP effects. Mechanistically, MLK3 kinase-dependent effects preserve LV function, while MLK3 kinase-independent signaling regulates BP. These findings suggest augmenting MLK3 kinase activity could preserve LV function in HF but avoid hypotension from PKG1α activation.
Authors
Timothy D. Calamaras, Suchita Pande, Robert A.U. Baumgartner, Seung Kyum Kim, Joseph C. McCarthy, Gregory L. Martin, Kelly Tam, Angela L. McLaughlin, Guang-rong Wang, Mark J. Aronovitz, Weiyu Lin, Jonathan I. Aguirre, Paulina Baca, Peiwen Liu, Daniel A. Richards, Roger J. Davis, Richard H. Karas, Iris Z. Jaffe, Robert M. Blanton
Abstract
Cardiac inflammation and fibrosis contribute significantly to hypertension-related adverse cardiac remodeling. IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been demonstrated as a key molecular link between inflammation and cardiovascular disease. However, the cell-specific contribution of IKKβ signaling towards adverse cardiac remodeling remains elusive. Cardiac fibroblasts are one of the most populous non-myocyte cell types in the heart that play a key role in mediating cardiac fibrosis and remodeling. To investigate the function of fibroblast IKKβ, we generated inducible fibroblast-specific IKKβ-deficient mice. Here we report an important role of IKKβ in the regulation of fibroblast functions and cardiac remodeling. Fibroblast-specific IKKβ deficient male mice were protected from angiotensin II (Ang II)-induced cardiac hypertrophy, fibrosis, and macrophage infiltration. Ablation of fibroblast IKKβ inhibited Ang II-stimulated fibroblast proinflammatory and profibrogenic responses, leading to ameliorated cardiac remodeling and improved cardiac function in IKKβ-deficient mice. Findings from this study establish fibroblast IKKβ as a key factor regulating cardiac fibrosis and function in hypertension-related cardiac remodeling.
Authors
Weiwei Lu, Zhaojie Meng, Rebecca Hernandez, Changcheng Zhou
