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Abstract
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.
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
Aniridia is most commonly caused by haploinsufficiency of the PAX6 gene, characterised 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 eighty-six patients from sixty-two unrelated families with molecularly confirmed heterozygous PAX6 variants from a United Kingdom (UK)-based single-centre ocular genetics service. They were categorised into mutation groups and retrospective review of baseline to most recent clinical characteristics (ocular and systemic) were 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%, and foveal hypoplasia 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 varied amongst mutation groups. Overall, the missense mutation sub-group had the mildest phenotype, and surgically naïve eyes maintained better visual acuity. Systemic evaluation identified type 2 diabetes in 12.8%, which is twice the UK prevalence. This is the largest longitudinal study of aniridia in the United Kingdom, providing 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
Agonist CD40 antibodies are under clinical development in combination with chemotherapy as an approach to prime for anti-tumor 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 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 interleukin (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 anti-tumor 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
Antibody-mediated rejection (ABMR) continues to be a major problem undermining the success of kidney transplantation. Acute antibody-mediated rejection (ABMR) of kidney grafts is characterized by neutrophil and monocyte margination in the tubular capillaries and by graft transcripts indicating NK cell activation, but the myeloid cell mechanisms required for acute ABMR have remained unclear. Dysregulated donor-specific antibody (DSA) responses with high antibody titers are induced in B6.CCR5-/- mice transplanted with complete MHC mismatched A/J kidneys and are required for rejection of the grafts. This study tested the role of recipient myeloid cell production of myeloperoxidase (MPO) on the cellular and molecular components of acute ABMR. Despite induction of equivalent DSA titers, B6.CCR5-/- recipients rejected A/J kidneys between days 18-25 with acute ABMR whereas B6.CCR5-/-MPO-/- recipients rejected the grafts between days 46-54 with histopathological features of chronic graft injury. On day 15, myeloid cells infiltrating grafts from B6.CCR5-/- and B6.CCR5-/-MPO-/- recipients expressed marked phenotypic and functional transcript differences that correlated with development of acute vs. chronic allograft injury, respectively. Near the time of peak DSA titers, NK cell activation to proliferate and express CD107a was markedly decreased within allografts in B6.CCR5-/-MPO-/- recipients. Despite high titers of DSA, depletion of neutrophils reproduced the inhibition of NK cell activation and decreased macrophage infiltration but increased monocytes producing MPO. Overall, recipient myeloid cells producing MPO regulate graft-infiltrating monocyte/macrophage function and NK cell activation that are required for DSA-mediated acute kidney allograft injury and their absence switches DSA-mediated acute pathology and graft outcomes to chronic ABMR.
Authors
Satoshi Miyairi, Daisuke Ueda, Takafumi Yagisawa, Daigo Okada, Karen S. Keslar, Kazunari Tanabe, Nina Dvorina, Anna Valujskikh, William Baldwin 3rd, Stanley L. Hazen, Robert L. Fairchild
Abstract
Existing patient-derived-xenograft (PDX) mouse models of solid tumors lack a fully tumor-donor matched, syngeneic, and functional immune system. We developed such a model by engrafting lymphopenic recipient mice with a fresh, undisrupted piece of solid tumor, whereby tumor-infiltrating lymphocytes (TILs) persisted in the recipient mice for several weeks. Successful tumor engraftment was achieved in eighty-three to eighty-nine percent of tumor-infiltrating-lymphocytes-PDX (TIL-PDX) mice, and these were seen to harbor exhausted immuno-effector as well as functional immuno-regulatory cells persisting for at least six months post-engraftment. Combined treatment with interleukin-15 (IL-15) stimulation and immune checkpoint inhibition (ICI) resulted in complete or partial tumor response in this model. Further, depletion of Cytotoxic T-lymphocytes (CTLs) and/or Natural Killer (NK) cells before combined immunotherapy revealed that both cell types were required for maximal tumor regression. Our novel TIL-PDX model provides a valuable resource for powerful mechanistic and therapeutic studies in solid tumors.
Authors
Duy T. Le, Tridu R. Huynh, Bryan M. Burt, George Van Buren, Shawn A. Abeynaike, Cristina Zalfa, Rana Nikzad, Farrah Kheradmand, John J. Tyner, Silke Paust
Abstract
BACKGROUND. The role of humoral immunity in the coronavirus disease 2019 (COVID-19) is not fully understood owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome. METHODS. Using SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution. RESULTS. We identified linear epitopes from the Spike (S) and Nucleocapsid (N) protein and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S(811-825), S(881-895) and N(156-170) epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes. CONCLUSIONS. Epitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, it may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern (VOC) in both the peptide array and latex agglutination formats. FUNDING. Ontario Research Fund (ORF)-COVID-19 Rapid Research Fund, the Toronto COVID-19 Action Fund, Western University, the Lawson Health Research Institute, the London Health Sciences Foundation, and the AMOSO Innovation Fund.
Authors
Courtney Voss, Sally Esmail, Xuguang Liu, Michael J. Knauer, Suzanne Ackloo, Tomonori Kaneko, Lori E. Lowes, Peter J. Stogios, Almagul Seitova, Ashley Hutchinson, Farhad Yusifov, Tatiana Skarina, Elena Evdokimova, Peter Loppnau, Pegah Ghiabi, Taraneh Hajian, Shanshan Zhong, Husam Abdoh, Benjamin D. Hedley, Vipin Bhayana, Claudio M. Martin, Marat Slessarev, Benjamin Chin-Yee, Douglas D. Fraser, Ian Chin-Yee, Shawn S.C. Li
Abstract
Exposure to maternal obesity may promote metabolic dysfunction in offspring. We use infant mesenchymal stem cells (MSC) to experimentally examine cellular mechanisms of intergenerational health transmission. Our earlier reports show MSCs collected from infants of mothers with obesity had a dichotomous distribution in metabolic efficiency; they were either efficient (Ef-Ob) or inefficient (In-Ob) with respect to fatty acid oxidation (FAO). Here, we sought to determine if this was due to a primary defect in FAO. Accordingly, we measured FAO in myogenic differentiating MSCs under three conditions: 1) myogenesis alone, 2) excess fatty acid exposure, and 3) excess fatty acid exposure plus a chemical uncoupler to increase metabolic rate. Compared to NW and Ef-Ob MSCs, In-Ob displayed lower FAO in myogenesis alone and after fatty acid plus uncoupler, indicating In-Ob were less metabolically flexible after increasing lipid availability and metabolic rate, demonstrating a primary deficit in FAO. MSC FAO was negatively associated with fasting maternal glucose and insulin, and positively associated with fasting HDL-cholesterol. MSC FAO was negatively associated with infant fat mass. These data indicate a less favorable maternal metabolic milieu, independent of maternal BMI, reduces intrinsic MSC FAO and is linked to higher infant adiposity as early as birth.
Authors
Melissa L. Erickson, Zachary W. Patinkin, Allison M. Duensing, Dana Dabelea, Leanne M. Redman, Kristen E. Boyle
Abstract
The main mechanisms underlying sexually dimorphic outcomes in neonatal lung injury are unknown. We tested the hypothesis that hormonal- or sex chromosome-mediated mechanisms interact with hyperoxia exposure to impact injury and repair in the neonatal lung. To distinguish sex differences caused by gonadal hormones versus sex chromosome complement (XX versus XY), we used the four core genotypes (FCG) mice and exposed them to hyperoxia (95% FiO2, PND1-4: saccular stage) or room air. This model generates XX and XY mice that each have either testes (with Sry, XXM or XYM) or ovaries (without Sry, XXF or XYF). Lung alveolarization and vascular development were more severely impacted in XYM and XYF compared to XXF and XXM mice. Cell cycle-related pathways were enriched in the gonadal or chromosomal females, while muscle-related pathways were enriched in the gonadal males, and immune-response related pathways were enriched in chromosomal males. Female gene signatures showed a negative correlation with human patients that developed BPD or needed oxygen therapy at 28 days. These results demonstrate that chromosomal sex and not gonadal sex impacted the response to neonatal hyperoxia exposure. The female sex chromosomal complement was protective and could mediate sex-specific differences in neonatal lung injury.
Authors
Sandra L. Grimm, Xiaoyu Dong, Yuhao Zhang, Alexandre F. Carisey, Arthur P. Arnold, Bhagavatula Moorthy, Cristian Coarfa, Krithika Lingappan
Abstract
The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) has resulted in an unprecedented pandemic that has been accompanied by a global health crisis. Although the lungs are the main organs involved in COVID-19, systemic disease with a wide range of clinical manifestations also develops in SARS-CoV-2-infected patients. One of the major systems affected by this virus is the cardiovascular system. The presence of pre-existing cardiovascular disease increases mortality in COVID-19 patients, and cardiovascular injuries, including myocarditis, cardiac rhythm abnormalities, endothelial cell injury, thrombotic events, and myocardial interstitial fibrosis, are observed in some COVID-19 patients. The underlying pathophysiology of COVID-19-associated cardiovascular complications is not fully understood, although direct viral infection of myocardium and cytokine storm have been suggested as possible mechanisms of myocarditis. In this review, we summarize available data on SARS-CoV-2-related cardiac damage and discuss potential mechanisms of cardiovascular implications of this rapidly spreading virus.
Authors
Farnaz Farshidfar, Navid Koleini, Hossein Ardehali
Abstract
The skin lesion erythema migrans (EM) is an initial sign of the Ixodes-tick transmitted Borreliella spirochetal infection known as Lyme disease. T cells and innate immune cells have previously been shown to predominate the EM lesion and promote the reaction. Despite the established importance of B cells and antibodies in preventing infection, the role of B cells in the skin immune response to Borreliella is unknown. Here, we used single-cell RNA-Seq in conjunction with B cell receptor (BCR) sequencing to immunophenotype EM lesions and their associated B cells and BCR repertoires. We found that B cells were more abundant in EM in comparison to autologous uninvolved skin; many were clonally expanded and had circulating relatives. EM-associated B cells upregulated expression of MHC class II genes and exhibited preferential IgM isotype usage. A subset also exhibited low levels of somatic hypermutation despite a gene expression profile consistent with memory B cells. Our study demonstrates that single-cell gene expression with paired BCR sequencing can be used to interrogate the sparse B cell populations in human skin and reveals that B cells in the skin infection site in early Lyme disease express a phenotype consistent with local antigen presentation and antibody production.
Authors
Ruoyi Jiang, Hailong Meng, Khadir Raddassi, Ira Fleming, Kenneth B. Hoehn, Kenneth R. Dardick, Alexia A. Belperron, Ruth R. Montgomery, Alex K. Shalek, David A. Hafler, Steven H. Kleinstein, Linda K. Bockenstedt
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