Preeclampsia is a serious pregnancy disorder that lacks effective treatments other than delivery. Improper sensing of oxygen changes during placentation by prolyl hydroxylases (PHD), specifically PHD2, causes placental Hypoxia-Inducible Factor-1 (HIF1) buildup and abnormal downstream signaling in early-onset preeclampsia; yet therapeutic targeting of HIF1 has never been attempted. Here we generated a conditional (placenta-specific) knockout of Phd2 in mice (Phd2-/- cKO) to reproduce HIF1 excess and to assess anti-HIF therapy. Conditional deletion of Phd2 in the junctional zone (JZ) during pregnancy increased placental HIF1 content, resulting in abnormal placentation, impaired remodeling of the uterine spiral arteries, and fetal growth restriction. Pregnant dams developed new-onset hypertension at mid-gestation (E9.5) in addition to proteinuria and renal and cardiac pathology, hallmarks of severe preeclampsia in humans. Daily injection of acriflavine, a small-molecule inhibitor of HIF1, to pregnant Phd2-/- cKO mice from E7.5 (prior to hypertension) or E10.5 (after hypertension has been established) to E14.5 corrected placental dysmorphologies and improved fetal growth. Moreover, it reduced maternal blood pressure and reverted renal and myocardial pathology. Thus, therapeutic targeting of the HIF pathway may improve placental development and function, as well as maternal and fetal health, in preeclampsia.
Julien Sallais, Chanho Park, Sruthi Alahari, Tyler Porter, Ruizhe Liu, Merve Kurt, Abby Farrell, Martin Post, Isabella Caniggia
Acute graft-versus-host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT) inflicted by alloreactive T cells primed in secondary lymphoid organs (SLOs) and subsequent damage to aGvHD target tissues. In recent years, regulatory T cell (Treg) transfer and/or expansion has emerged as a promising therapy to modulate aGvHD. However, cellular niches essential for fostering Tregs to prevent aGvHD have not been explored, yet. Here, we tested whether and to what extent MHC class II (MHCII) expressed on Ccl19+ fibroblastic reticular cells (FRCs) shape the donor CD4+ T cell response during aGvHD. Animals lacking MHCII expression on Ccl19-Cre-expressing FRCs (MHCIIΔCcl19) showed aberrant CD4+ T cells activation in the effector phase resulting in exacerbated aGvHD that was associated with significantly reduced expansion of Foxp3+ Tregs and invariant natural killer T (iNKT) cells. Skewed Treg maintenance in MHCIIΔCcl19 mice resulted in loss of protection from aGvHD provided by adoptively transferred donor Tregs. In contrast, although FRCs upregulated co-stimulatory surface receptors, degraded and processed exogenous antigens after myeloablative irradiation, FRCs were dispensable to activate alloreactive CD4+ T cells in two mouse models of aGvHD. In sum, these data reveal an immunoprotective, MHCII-mediated function of FRC niches in secondary lymphoid organs (SLOs) after allo-HCT and highlights a hitherto unknown framework of cellular and molecular interactions that regulate CD4+ T cell alloimmunity.
Haroon Shaikh, Joern Pezoldt, Zeinab Mokhtari, Juan Gamboa Vargas, Duc-Dung Le, Josefina Peña Mosca, Estibaliz Arellano-Viera, Michael A.G. Kern, Caroline Graf, Niklas Beyersdorf, Manfred B. Lutz, Angela Riedel, Maike Büttner-Herold, Alma Zernecke, Hermann Einsele, Antoine-Emmanuel Saliba, Burkhard Ludewig, Jochen Huehn, Andreas Beilhack
The DNA methyltransferase inhibitor decitabine has classically been used to reactivate silenced genes and as a pre-treatment for anti-cancer therapies. In a new variation of this idea, this study explores the concept of adding low-dose decitabine following administration of chemotherapy to bolster therapeutic efficacy. We find that addition of decitabine following treatment with the chemotherapy gemcitabine improves survival and slows tumor growth in a mouse model of high-grade sarcoma. Unlike prior studies in epithelial tumor models, low-dose decitabine did not induce a robust anti-tumor T cell response in sarcoma. Furthermore, low-dose decitabine synergizes with gemcitabine independently of the immune system. Mechanistic analyses demonstrate that the combination therapy induces bi-phasic cell cycle arrest and apoptosis. Therapeutic efficacy was found to be sequence dependent, with gemcitabine priming cells for treatment with decitabine through inhibition of ribonucleotide reductase. This study identifies a unique application of low-dose decitabine to augment the cytotoxic effects of conventional chemotherapy in an immune-independent manner. The concepts explored in this study represent a promising new paradigm for cancer treatment by augmenting chemotherapy through addition of low-dose decitabine to increase tolerability and improve patient response. These findings have widespread implications for the treatment of sarcomas and other aggressive malignancies.
Wade R. Gutierrez, Amanda Scherer, Jeffrey D. Rytlewski, Emily A. Laverty, Alexa P. Sheehan, Gavin R. McGivney, Qierra R. Brockman, Vickie Knepper-Adrian, Grace A. Roughton, Dawn E. Quelle, David J. Gordon, Varun Monga, Rebecca D. Dodd
The fluid covering the surface of airway epithelia represents a first barrier against pathogens. The chemical and physical properties of the airway surface fluid are controlled by the activity of ion channels and transporters. In cystic fibrosis (CF), loss of CFTR chloride channel function causes airway surface dehydration, bacterial infection, and inflammation. We investigated the effects of IL-17A plus TNF-α, two cytokines with a relevant role in CF and other chronic lung diseases. Transcriptome analysis revealed a profound change with upregulation of several genes involved in ion transport, anti-bacterial defense, and neutrophil recruitment. At the functional level, bronchial epithelia treated in vitro with the cytokine combination showed upregulation of ENaC sodium channel, ATP12A proton pump, ADRB2 beta-adrenergic receptor, and SLC26A4 anion exchanger. The overall result of IL-17A/TNF-α treatment was hyperviscosity of the airway surface as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Importantly, stimulation with a beta-adrenergic agonist switched airway surface to a low viscosity state in non-CF but not in CF epithelia. Our study suggests that CF lung disease is sustained by a vicious cycle in which epithelia cannot exit from the hyperviscous state thus perpetuating the proinflammatory airway surface condition.
Daniela Guidone, Martina Buccirossi, Paolo Scudieri, Michele Genovese, Sergio Sarnataro, Rossella De Cegli, Federico Cresta, Vito Terlizzi, Gabrielle Planelles, Gilles Crambert, Isabelle Sermet-Gaudelus, Luis J.V. Galietta
Acquired aplastic anemia (AA) is caused by autoreactive T-cell-mediated destruction of early hematopoietic cells. Somatic loss of human leukocyte antigen (HLA) Class I alleles was identified as a mechanism of immune escape in surviving hematopoietic cells of some AA patients. However, pathogenicity, structural characteristics and clinical impact of specific HLA alleles in AA remain poorly understood. Here, we evaluated somatic HLA loss in 505 AA patients from two multi-institutional cohorts. Using a combination of HLA mutation frequencies, peptide-binding structures, and association with AA in an independent cohort of 6,323 patients from the National Marrow Donor Program, we identified 19 AA risk alleles and 12 non-risk alleles and established a novel AA HLA pathogenicity stratification. Our results define pathogenicity for the majority of common HLA-A/B alleles across diverse populations. Our study demonstrates that HLA alleles confer different risks of developing AA, but once AA develops, specific alleles are not associated with response to immunosuppression or trans-plant outcomes. However, higher pathogenicity alleles, particularly HLA-B*14:02, are associated with higher rates of clonal evolution in adult AA patients. Our study provides novel insights into the immune pathogenesis of AA, opening the door to future autoantigen identification and improved under-standing of clonal evolution in AA.
Timothy S. Olson, Benjamin F. Frost, Jamie L. Duke, Marian Dribus, Hongbo M. Xie, Zachary D. Prudowsky, Elissa Furutani, Jonas Gudera, Yash B. Shah, Deborah Ferriola, Amalia Dinou, Ioanna Pagkrati, Soyoung Kim, Yixi Xu, Meilun He, Shannon Zheng, Sally Nijim, Ping Lin, Chong Xu, Taizo Nakano, Joseph H. Oved, Beatriz M. Carreno, Yung-Tsi Bolon, Shahinaz M. Gadalla, Steven G.E. Marsh, Sophie Paczesny, Stephanie J. Lee, Dimitrios S. Monos, Akiko Shimamura, Alison A. Bertuch, Loren Gragert, Stephen Spellman, Daria V. Babushok
Rosacea is a chronic skin disorder characterized by abnormal neurovascular and inflammatory conditions on the central face. Despite increasing evidence suggests that rosacea is associated with metabolic disorders, the role of metabolism in rosacea pathogenesis remains unknown. Here, via targeted metabolomics approach, we characterized significantly altered metabolic signatures in rosacea patients, especially for amino acid-related metabolic pathways. Among these, glutamic acid and aspartic acid are highlighted and positively correlated with the disease severity in rosacea patients. We further demonstrated that glutamic acid and aspartic acid can facilitate the development of erythema and telangiectasia, typical features of rosacea, in the skin of mice. Mechanistically, glutamic acid and aspartic acid stimulate the production of vasodilation-related neuropeptides from peripheral neuron and keratinocytes, and induce the release of nitric oxide from endothelial cells and keratinocytes. Interestingly, we provided evidence showing that doxycycline can improve the symptoms of rosacea patients possibly by targeting amino acid metabolic pathway. These findings reveal that abnormal amino acid metabolism promotes neurovascular reactivity in rosacea, and raise the possibility of targeting dysregulated metabolism as a promising strategy for clinical treatment.
Tangxiele Liu, Wenqin Xiao, Mengting Chen, Rui Mao, San Xu, Qinqin Peng, Zhixiang Zhao, Qian Wang, Hongfu Xie, Zhili Deng, Ji Li
Expression of the transcription factor Interferon Regulatory Factor 4 (IRF4) is required for the development of lung conventional dendritic cells type 2 (cDC2s) that elicit Th2 responses, yet how IRF4 functions in lung cDC2s throughout the acute and memory allergic response is not clear. Here, we use a novel mouse model that loses IRF4 expression after lung cDC2 development to demonstrate that mice with IRF4-deficient DCs display impaired memory responses to allergen. This defect in the memory response is a direct result of ineffective Th2 induction and impaired recruitment of activated effector T cells to the lung after sensitization. IRF4-deficient DCs demonstrate defects in their migration to the draining lymph node and in T cell priming. Finally, T cells primed by IRF4-competent DCs mediate potent memory responses independently of IRF4-expressing DCs, demonstrating that IRF4-expressing DCs are not necessary during the memory response. Thus, IRF4 controls a program in mature DCs governing Th2 priming and effector responses, but IRF4-expressing DCs are dispensable during tissue resident-memory T cell (TRM cell)-dependent memory responses.
Daniel F. Camacho, Tania E. Velez, Maile K. Hollinger, Esther Wang, Chanie L. Howard, Eli P. Darnell, Domenick E. Kennedy, Paulette A. Krishack, Cara L. Hrusch, Marcus R. Clark, James J. Moon, Anne I. Sperling
Pseudomonas aeruginosa undergoes diversification during infection of the cystic fibrosis (CF) lung. Understanding these changes requires model systems that capture the complexity of the CF lung environment. We previously identified loss-of-function mutations in the two-component regulatory system sensor kinase gene pmrB, in P. aeruginosa from CF and from experimental infection of mice. Here, we demonstrate that whilst such mutations lower in vitro MICs for multiple antimicrobial classes, this is not reflected in increased antibiotic susceptibility in vivo. Loss of PmrB impairs aminoarabinose modification of lipopolysaccharide, increasing the negative charge of the outer membrane and promoting uptake of cationic antimicrobials. However, in vivo, this can be offset by increased membrane binding of other positively charged molecules present in lungs. The polyamine spermidine readily coats the surface of PmrB-deficient P. aeruginosa, reducing susceptibility to antibiotics that rely on charge differences to bind the outer membrane and increasing biofilm formation. Spermidine is elevated in lungs during P. aeruginosa infection in mice and during episodes of antimicrobial treatment in people with CF. These findings highlight the need to study antimicrobial resistance under clinically relevant environmental conditions. Microbial mutations carrying fitness costs in vitro may be advantageous during infection, where host resources can be utilised.
Chowdhury M. Hasan, Sian Pottenger, Angharad E. Green, Adrienne A. Cox, Jack S. White, Trevor Jones, Craig Winstanley, Aras Kadioglu, Megan H. Wright, Daniel R. Neill, Joanne L. Fothergill
The individual contribution of specific myeloid subsets such as CD1c+ conventional dendritic cells (cDC) to perpetuation of Rheumatoid Arthritis (RA) pathology remains unclear. In addition, the specific innate sensors driving pathogenic activation of CD1c+ cDCs in RA patients and their functional implications have not been characterized. Here, we assessed phenotypical, transcriptional and functional characteristics of CD1c+ and CD141+ cDCs and monocytes from the blood and synovial fluid of RA patients. Increased levels of CCR2 and the IgG receptor CD64 on circulating CD1c+ cDC associated with the presence of this DC subset in the synovial membrane in RA patients. Moreover, synovial CD1c+ cDCs are characterized by increased expression of proinflammatory cytokines and high abilities to induce pathogenic IFNγ+IL-17+ CD4+ T cells in vitro. Finally, we identified the crosstalk between Fcγ Receptors and NLRC4 as a new potential molecular mechanism mediating pathogenic activation, CD64 upregulation and functional specialization of CD1c+ cDCs in response to dsDNA-IgG in RA patients.
Cristina Delgado-Arévalo, Marta Calvet-Mirabent, Ana Triguero-Martinez, Enrique Vázquez de Luis, Alberto Benguría-Filippini, Raquel Largo, Diego Calzada-Fraile, Olga Popova, Ildefonso Sánchez-Cerrillo, Ilya Tsukalov, Roberto Moreno-Vellisca, Hortensia de la Fuente, Gabriel Herrero-Beaumont, Almudena R. Ramiro, Francisco Sánchez-Madrid, Santos Castañeda, Ana Dopazo, Isidoro González-Álvaro, Enrique Martin-Gayo
No disease-modifying drug exists for osteoarthritis (OA). Despite success in animal models, candidate drugs continue to fail in clinical trials due to the unmapped interpatient heterogeneity and disease complexity. We have utilized a single-cell cytometry-by-time-of-flight (cyTOF) based platform to precisely outline the effects of candidate drugs on human OA chondrocytes. OA chondrocytes harvested from patients undergoing total knee arthroplasty were treated with two drugs, an NF-κB pathway inhibitor, BMS-345541, and a chondroinductive small molecule, Kartogenin, that showed preclinical success in animal models for OA. cyTOF conducted with 30 metal isotope-labeled antibodies parsed the effects of the drugs on inflammatory, senescent, and chondroprogenitor populations. The NF-κB pathway inhibition decreased the expression of NF-κB, HIF2A and iNOS in multiple chondrocyte clusters and significantly depleted four p16ink4a expressing senescent populations including NOTCH1+STRO1+ chondroprogenitors. While Kartogenin also affected select p16ink4a expressing senescent clusters, there was a less discernible effect on chondroprogenitor populations. Overall, BMS-345541 elicited a uniform drug response in all patients while only a few responded to Kartogenin. These studies demonstrate that a single-cell cyTOF-based drug screening platform can provide insights into patient response assessment and their stratification.
Neety Sahu, Fiorella C. Grandi, Nidhi Bhutani
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