Acute rejection (AR) in renal transplantation is an established risk factor for reduced allograft survival. The incidence of AR is 10-20% despite standard of care immunosuppression, suggesting molecular pathways exist which are inadequately suppressed by current therapy. Molecules with regulatory control among these could serve as important targets for therapeutic manipulation to prevent rejection. Here, an integrative network-based computational strategy incorporating gene expression and genotype data of human renal allograft biopsy tissue was applied, to identify the master regulators- the key driver genes (KDGs)- within dyregulated AR pathways. A 982 meta-gene signature with differential expression in AR versus non-AR, was identified from a meta-analysis of microarray data from 735 human kidney allograft biopsy samples across seven data sets. Among the upregulated genes, enriched biologic processes include the immune response, leucocyte activation and antigen processing and presentation; where monocytes, macrophages and dendritic cells were identified as the major immune cell populations. Genomic key driver analysis of this signature predicted 14 KDGs. Expression of the KDGs provided risk stratification for subsequent graft loss with high prediction accuracy at 2 years post transplant (AUC=0.913) and 2 years post biopsy (AUC=0.889) in separate clinical cohorts. Interrogation of two drug-repositioning resources identified compounds with predicted efficacy against individual KDGs or a key driver-based gene set respectively, and therefore be repositioned for AR prevention. Minocycline, an FDA-approved tetracycline antibiotic, was chosen for experimental validation in a murine cardiac allograft model of AR. Minocycline alone attenuated the inflammatory profile of AR compared with controls, and when co-administered with immunosuppression prolonged graft survival. This study demonstrates the proof-of-concept that a network-based strategy, using gene expression and genotype data assists target prioritization for therapeutics in renal allograft rejection.
Zhengzi Yi, Karen L. Keung, Li Li, Min Hu, Bo Lu, Leigh Nicholson, Elvira Jimenez-Vera, Madhav C. Menon, Chengguo Wei, Stephen I. Alexander, Barbara Murphy, Philip J. O’Connell, Weijia Zhang
Critical illness is accompanied by the release of large amounts of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of neutrophils which is associated with adverse outcomes. The signalling pathways that mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor neutrophils exposed to purified C5a demonstrated a prolonged defect (7 hours) in phagocytosis of Staphylococcus aureus. Phosphoproteomic profiling of 2712 phosphoproteins identified persistent C5a signalling and selective impairment of phagosomal protein phosphorylation on exposure to S. aureus. Notable proteins included early endosomal marker ZFYVE16 and V-ATPase proton channel component ATPV1G1. A novel assay of phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification which was recapitulated in neutrophils from critically ill patients. Examination of the C5a-impaired protein phosphorylation indicated a role for the phosphatidylinositol 3-kinase VPS34 in phagosomal maturation. Inhibition of VPS34 impaired neutrophil phagosomal acidification and killing of S. aureus. This study provides a phosphoproteomic assessment of human neutrophil signalling in response to S. aureus and its disruption by C5a, identifying a defect in phagosomal maturation and new mechanisms of immune failure in critical illness.
Alexander J.T. Wood, Arlette M. Vassallo, Marie-Helene Ruchaud-Sparagano, Jonathan Scott, Carmelo Zinnato, Carmen Gonzalez-Tejedo, Kamal Kishore, Clive S. D’Santos, A. John Simpson, David K. Menon, Charlotte Summers, Edwin R. Chilvers, Klaus Okkenhaug, Andrew Conway Morris
Background: A treatment option for ADPKD has highlighted the need to identify rapidly progressive patients. Kidney size/age and genotype have predictive power for renal outcomes, but their relative and additive value, plus associated trajectories of disease progression, are not well defined. Methods: The value of genotypic and/or kidney imaging data (Mayo Imaging Class) to predict the time to functional (end stage kidney disease; ESKD, or decline in estimated glomerular filtration rate; eGFR) or structural (increase in height adjusted total kidney volume; htTKV) outcomes were evaluated in a Mayo Clinic PKD1/PKD2 population; and eGFR and htTKV trajectories from 20-65 years of age modeled and independently validated in similarly defined CRISP and HALT PKD patients. Results: Both genotypic and imaging groups strongly predicted ESKD and eGFR endpoints, with genotype improving the imaging predictions, and vice versa; a multivariate model had strong discriminatory power (C statistic = 0.845). However, imaging but not genotypic groups predicted htTKV growth, although more severe genotypic and imaging groups had larger kidneys at a young age. The trajectory of eGFR decline was linear from baseline in the most severe genotypic and imaging groups, but curvilinear in milder groups. Imaging class trajectories differentiated htTKV growth rates; severe classes had rapid early growth and large kidneys but growth later slowed. Conclusions: The value of imaging, genotypic, and combined data to identify rapidly progressive patients was demonstrated, and reference values for clinical trials provided. Our data indicates that differences in kidney growth rates before adulthood significantly define patients with severe disease. Funding: NIDDK grants: Mayo DK058816, DK090728; CRISP DK056943, DK056956, DK056957, DK056961; HALT PKD DK062410, DK062408, DK062402, DK082230, DK062411, DK062401.
Sravanthi Lavu, Lisa E. Vaughan, Sarah R. Senum, Timothy L. Kline, Arlene B. Chapman, Ronald D. Perrone, Michal Mrug, William E. Braun, Theodore I. Steinman, Frederic F. Rahbari-Oskoui, Godela M. Brosnahan, Kyongtae T. Bae, Douglas Landsittel, Fouad T. Chebib, Alan S. L. Yu, Vicente E. Torres, Peter C. Harris
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a relative paucity of cancer cells that are surrounded by an abundance of non-tumor cells and extracellular matrix, known as stroma. The interaction between stroma and cancer cells contributes to poor outcome, but how proteins from these individual compartments drive aggressive tumor behavior is not known. Here, we report the proteomic analysis of laser-capture microdissected (LCM) PDAC samples. We isolated stroma, tumor, and bulk samples from a cohort with long- and short-term survivors. Compartment-specific proteins were measured by mass spectrometry, yielding the largest PDAC proteome landscape to date. These analyses revealed that in bulk analysis, tumor-derived proteins were typically masked and that LCM was required to reveal biology and novel prognostic markers. We validated tumor CALB2 and stromal COL11A1 expression as compartment-specific prognostic markers. We identified and functionally addressed the contributions of the tumor cell receptor EPHA2 to tumor cell viability and motility, underscoring the value of compartment-specific protein analysis in PDAC.
Tessa Y.S. Le Large, G. Mantini, Laura L. Meijer, T.V. Pham, N. Funel, Nicole C.T. van Grieken, B. Kok, Jaco C. Knol, H.W.M. van Laarhoven, S.R. Piersma, C.R. Jimenez, G. Kazemier, E. Giovannetti, M.F. Bijlsma
Triple-negative breast cancers (TNBCs) are highly heterogeneous and aggressive, with high mortality rates. Although TNBC is typically more responsive to chemotherapy than other breast cancer subtypes, many patients develop chemo-resistance. The molecular processes contributing to chemo-resistance, and the roles of tumor cell-stromal crosstalk in establishing chemo-resistance are complex and largely unclear. Here we report molecular studies of paired TNBC patient-derived xenografts (PDX) established from patient biopsies before and after the development of chemo-resistance. Interestingly, the chemo-resistant model acquired a distinct KRASQ61R mutation that activates K-Ras. The chemo-resistant KRAS-mutant model showed gene expression and proteomic changes indicative of altered tumor cell metabolism. Specifically, KRAS-mutant PDXs exhibit increased redox ratios and decreased activation of AMPK, a protein involved in responding to metabolic homeostasis. Additionally, the chemo-resistant model exhibited increased immunosuppression including expression of CXCL1 and CXCL2, cytokines responsible for recruiting immunosuppressive leukocytes to tumors. Notably, chemo-resistant KRAS-mutant tumors harbored increased numbers of granulocytic myeloid-derived suppressor cells (gMDSCs). Interestingly, previously established gene expression signatures of Ras/MAPK activity correlated with myeloid/neutrophil-recruiting CXCL1/2 expression and negatively with T-cell recruiting chemokines (CXCL9/10/11) across TNBC patients, even in the absence of KRAS mutations. Importantly, MEK inhibition induced tumor suppression in mice while simultaneously reversing metabolic and immunosuppressive phenotypes including chemokine production and gMDSC tumor recruitment in the chemo-resistant KRAS mutant tumors. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemo-resistance.
Derek A. Franklin, Joe T. Sharick, Paula I. Gonzalez-Ericsson, Violeta Sanchez, Phillip Dean, Susan R. Opalenik, Stefano Cairo, Jean-Gabriel Judde, Michael T. Lewis, Jenny C. Chang, Melinda E. Sanders, Rebecca S. Cook, Melissa C. Skala, Jennifer Bordeaux, Jehovana Orozco Bender, Christine A. Vaupel, Gary Geiss, Douglas Hinerfeld, Justin M. Balko
Arrestin domain containing 3 (ARRDC3) represents a newly discovered α-arrestin involved in obesity, inflammation and cancer. Here we demonstrated a pro-inflammation role of ARRDC3 in H. pylori-associated gastritis. Increased ARRDC3 was detected in gastric mucosa of patients and mice infected with H. pylori. ARRDC3 in gastric epithelial cells (GECs) was induced by H. pylori, regulated by ERK and PI3K-AKT pathways in a cagA-dependent manner. Human gastric ARRDC3 correlated with the severity of gastritis, and mouse ARRDC3 from non-BM-derived cells promoted gastric inflammation. This inflammation was characterized by the CXCR2-dependent influx of CD45+CD11b+Ly6C-Ly6G+ neutrophils, whose migration was induced via the ARRDC3-dependent production of CXCL2 by GECs. Importantly, gastric inflammation was attenuated in ARRDC3-/- mice but increased in protease-activated receptor 1 (PAR1)-/- mice. Mechanistically, ARRDC3 in GECs directly interacted with PAR1 and negatively regulated PAR1 via ARRDC3-mediated lysosomal degradation, which abrogated the suppression of CXCL2 production and following neutrophil chemotaxis by PAR1, thereby contributing to the development of H. pylori-associated gastritis. This study identifies a novel regulatory network involving H. pylori, GECs, ARRDC3, PAR1, and neutrophils, which collectively exert a pro-inflammatory effect within gastric microenvironment. Efforts to inhibit this ARRDC3-dependent pathway may prove valuable strategies in treating of H. pylori-associated gastritis.
Yu-gang Liu, Yong-sheng Teng, Zhi-guo Shan, Ping Cheng, Chuan-jie Hao, Yi-pin Lv, Fang-yuan Mao, Shi-ming Yang, Weisan Chen, Yong-Liang Zhao, Nan You, Quan-ming Zou, Yuan Zhuang
Kidney disease is one of the most devastating complications of diabetes, and tubular atrophy predicts diabetic kidney disease (DKD) progression to end stage renal disease. We have proposed that fatty acids bound to albumin contribute to tubular atrophy by inducing lipotoxicity, following filtration across damaged glomeruli, and subsequent proximal tubule reabsorption by a fatty acid transport protein-2 (FATP2)-dependent mechanism. To address this possibility, genetic (Leprdb/db eNOS-/-) and induced (high fat diet plus low dose streptozotocin) mouse models of obesity and DKD, were bred with global FATP2 gene (Slc27a2)-deleted mice, and then phenotyped. DKD-prone mice with the Slc27a2-/- genotype demonstrated normalization of glomerular filtration rate, reduced albuminuria, improved kidney histopathology, and longer lifespan compared to diabetic Slc27a2+/+ mice. Genetic and induced DKD-prone Slc27a2-/- mice also exhibited markedly reduced fasting plasma glucose, with mean values approaching euglycemia, despite increased obesity and decreased physical activity. Glucose lowering in DKD-prone Slc27a2-/- mice was accompanied by beta-cell hyperplasia and sustained insulin secretion. Together, our data indicate that FATP2 uniquely regulates DKD pathogenesis by a combined lipotoxicity and glucotoxicity (glucolipotoxicity) mechanism.
Shenaz Khan, Robert J. Gaivin, Caroline Abramovich, Michael Boylan, Jorge Calles, Jeffrey R. Schelling
M3 muscarinic acetylcholine receptor (M3R) is one of the autoantigens associated with Sjögren’s syndrome (SS) and is localized in exocrine glands where disease specific inflammation occurs. The inflammatory lesion is characterized by infiltration of CD4+ T cells, including clonally expanded Th17 cells. We undertook this study to identify circulating M3R specific Th17 cells, and to determine functional properties of those cells. Using ELISpot method, we identified M3R reactive Th17 cells in the peripheral blood of patients with primary SS (pSS). Among examined 10 pSS, 10 healthy subjects (HS), and 5 IgG4-related disease (IgG4-RD) patients, M3R reactive IL-17 secreting cells were significantly increased in five pSS patients specifically. The commonest T cell epitope, which was analyzed and confirmed by co-culture of isolated CD4+ T cells with antigen presenting cells plus M3R peptides in vitro, was peptide 83-95 of M3R. Peptide recognition was partly in HLA DR restricted manner, confirmed by blocking assay. M3R reactive Th17 cells positivity correlated with higher titers of anti-M3R antibodies, whose systemic disease activity score tended to be higher. Our studies highlight the role of tissue specific autoantigen derived circulating Th17 cells in pSS, for which further work might lead to antigen specific targeted therapy.
Saori Abe, Hiroto Tsuboi, Hanae Kudo, Hiromitsu Asashima, Yuko Ono, Fumika Honda, Hiroyuki Takahashi, Mizuki Yagishita, Shinya Hagiwara, Yuya Kondo, Isao Matsumoto, Takayuki Sumida
One of the major challenges in using pancreatic cancer patient-derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for biopsy specimen with limited tumor cell yield, typical characteristics of biopsies from endoscopic ultrasound-guided fine needle aspirations (EUS-FNAs).Here, we tested conditioned media of individual PDOs for cell-free tumor DNA (cfDNA) to detect driver mutations already early on during the expansion process in order to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72h after biopsy, recapitulate the mutational profile of the primary tumor indicating suitability of this approach to subject PDOs to drug testing in a reduced timeframe. In addition, we demonstrate that this workflow is practicable even in patients of whom the amount of tumor material was not sufficient for molecular characterization by established means.Our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach this can be achieved in a rapid and feasible fashion with broad implications in clinical practice.
Zahra Dantes, Hsi-Yu Yen, Nicole Pfarr, Christof Winter, Katja Steiger, Alexander Muckenhuber, Alexander Hennig, Sebastian Lange, Thomas Engleitner, Rupert Öllinger, Roman Maresch, Felix Orben, Irina Heid, Georgios A. Kaissis, Kuangyu Shi, Geoffrey J. Topping, Fabian Stögbauer, Matthias Wirth, Katja Peschke, Aristeidis Papargyriou, Massoud Rezaee-Oghazi, Karin Feldmann, Arlett P. G. Schäfer, Raphela Ranjan, Clara Lubeseder-Martellato, Daniel E. Stange, Thilo Welsch, Marc E. Martignoni, Güralp Onur Ceyhan, Helmut Friess, Alexander Herner, Lucia Liotta, Matthias Treiber, Guido von Figura, Mohamed Abdelhafez, Peter Klare, Christoph Schlag, Hana Algül, Jens T. Siveke, Rickmer F. Braren, Gregor Weirich, Wilko Weichert, Dieter Saur, Roland Rad, Roland Schmid, Günter Schneider, Maximilian Reichert
The bromodomain and extraterminal (BET) family of epigenetic reader proteins are key regulators of inflammatory and hypertrophic gene expression in the heart. We previously identified the activation of pro-inflammatory gene networks as a key early driver of dilated cardiomyopathy (DCM) in transgenic mice expressing a mutant form of phospholamban (PLNR9C) – a genetic cause of DCM in humans. We hypothesized that BETs coactivate this inflammatory process, representing a critical node in the progression of DCM. To test this hypothesis, PLNR9C or age-matched wild type mice were treated longitudinally with the small molecule BET bromodomain inhibitor JQ1 or vehicle. BET inhibition abrogated adverse cardiac remodeling, reduced cardiac fibrosis, and prolonged survival in PLNR9C mice by inhibiting expression of pro-inflammatory gene networks at all stages of disease. Specifically, JQ1 had profound effects on pro-inflammatory gene network expression in cardiac fibroblasts, while having little effect on gene expression in cardiomyocytes. Cardiac fibroblast proliferation was also substantially reduced by JQ1. Mechanistically, we demonstrated that BRD4 serves as a direct and essential regulator of NFkB-mediated pro-inflammatory gene expression in cardiac fibroblasts. Interdicting pro-inflammatory gene expression via BET bromodomain inhibition could be a novel therapeutic strategy for chronic DCM in humans.
Andrew Antolic, Hiroko Wakimoto, Zhe Jiao, Joshua M. Gorham, Steven R. DePalma, Madeleine E. Lemieux, David A. Conner, Da Young Lee, Jun Qi, Jonathan G. Seidman, James E. Bradner, Jonathan D. Brown, Saptarsi M. Haldar, Christine E. Seidman, Michael A. Burke
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