Clostridioides difficile is a leading cause of nosocomial infection responsible for significant morbidity and mortality with limited options for therapy. Secreted C. difficile toxin B (TcdB) is a major contributor to disease pathology and select TcdB-specific Abs may protect against disease recurrence. However, the high frequency of recurrence suggests that the memory B cell response, essential for new Ab production following C. difficile re-exposure, is insufficient. We therefore isolated TcdB-specific memory B cells from individuals with a history of C. difficile infection and performed single-cell deep sequencing of their Ab genes. Herein, we report that TcdB-specific memory B cell-encoded antibodies showed somatic hypermutation but displayed limited isotype class switch. Memory B cell-encoded monoclonal antibodies generated from the gene sequences revealed low to moderate affinity for TcdB and a limited ability to neutralize TcdB. These findings indicate that memory B cells are an important factor in C. difficile disease recurrence.
Hemangi B. Shah, Kenneth Smith, Edgar J. Scott, II, Jason L. Larabee, Judith A. James, Jimmy D. Ballard, Mark L. Lang
Post-prandial triglycerides (TGs) are elevated in people with type 2 diabetes (T2D) and glucoregulatory agents such as Glucagon-like-peptide-1 (GLP-1) receptor agonists and Dipeptidyl Peptidase-4 (DPP-4) inhibitors simultaneously reduce post-prandial TG excursion. Although the glucose-lowering mechanisms of DPP-4 have been extensively studied, how the reduction of DPP-4 activity improves lipid tolerance remains unclear. Here we demonstrate that gut-selective and systemic inhibition of DPP-4 activity reduces post-prandial TG excursion in young mice. Genetic inactivation of Dpp4 simultaneously within endothelial cells (ECs) and hematopoietic cells using Tie2-Cre reduces intestinal lipoprotein secretion under regular chow (RC) diet conditions. Bone marrow transplantation revealed a key role for hematopoietic cells in modulation of lipid responses arising from genetic reduction of DPP-4 activity. Unexpectedly, deletion of Dpp4 in enterocytes increases TG excursion in high fat diet (HFD)-fed mice. Moreover, chemical reduction of DPP-4 activity and increased levels of GLP-1 are uncoupled from triglyceride excursion in older or HFD-fed mice, yet lipid tolerance remains improved in older Dpp4-/- and Dpp4EC-/- mice. Taken together, this study defines new roles for specific DPP-4 compartments, age, and diet as modifiers of DPP-4 activity linked to control of gut lipid metabolism.
Elodie M. Varin, Antonio Hanson, Jacqueline L. Beaudry, My-Anh Nguyen, Xiemin Cao, Laurie Baggio, Erin E. Mulvihill, Daniel J. Drucker
Recent evidence shows that the naïve heart harbors a population of intravascular recirculating B cells that make close contact with the microvascular endothelium of the heart and arrest their transit as they pass through the heart. However, the timing of their appearance and their organ specificity remain unknown. To address this knowledge gap, we performed a systematic analysis of B cells isolated from the myocardium and other organs, from embryonic life to early adulthood. We found that B cells are present in the developing heart by day E13.5. The phenotype of myocardial B cells changed dynamically during development. While neonatal heart B cells were mostly CD11b+ and CD11b-CD21-CD23-, adult B cells were predominantly CD11b-CD21+CD23+. Histological analysis and intravital microscopy of lung and liver showed that organ-associated B cells in contact with the microvascular endothelium were not specific to the heart. Flow cytometric analysis of perfused hearts, livers, lungs and spleen at different developmental stages showed that the dynamic changes in B cell subpopulations observed in the heart during development mirrored changes observed in the spleen, peripheral blood and other organs. Single cell RNAseq analysis of B cells showed that myocardial-associated B cells were part of a larger population of organ-associated B cells that had a distinct gene expression profile. These findings broaden our understanding of the biology of myocardial-associated B cells and suggest that current models of the dynamics of naïve B cell during development are incomplete.
Cibele Rocha-Resende, Wei Yang, Wenjun Li, Daniel Kreisel, Luigi Adamo, Douglas Mann
Skin lesions in dermatomyositis (DM) patients are common, frequently refractory, and have prognostic significance. Histologically, DM lesions appear like cutaneous lupus erythematosus (CLE) lesions and frequently cannot be differentiated. We thus undertook to examine the transcriptional profile of DM biopsies and compared them to CLE lesions in order to identify unique features. Type I interferon (IFN) signaling, including upregulation of IFN kappa, was a common pathway in both DM and CLE, but CLE also exhibited other inflammatory pathways. Importantly, DM lesions could be distinguished from CLE by a 5-gene biomarker panel that included an upregulation of IL18. Using single-cell RNA-sequencing, we further identified keratinocytes as the main source of increased IL-18 in DM skin. The novel molecular signature identified in this study has significant clinical implications for differentiating DM from CLE lesions, and we have highlighted the potential role for IL-18 in the pathophysiology of DM skin disease.
Lam Tsoi, Mehrnaz Gharaee-Kermani, Celine C. Berthier, Tori Nault, Grace Hile, Shannon N. Estadt, Matthew T. Patrick, Rachael Wasikowski, Allison C. Billi, Lori Lowe, Tamra J. Reed, Johann Gudjonsson, J. Michelle Kahlenberg
Evidence has mounted that insulin can be synthesized in various brain regions including the hypothalamus. However, the distribution and functions of insulin-expressing cells in the hypothalamus remain elusive. Herein, we show that in the mouse hypothalamus, the perikarya of insulin-positive neurons are located in the paraventricular nucleus (PVN) and their axons project to the median eminence; these findings define parvocellular neurosecretory PVN insulin neurons. Contrary to corticotrophin-releasing hormone expression, insulin expression in the PVN was inhibited by restraint stress (RS) in both adult and young mice. Acute RS–induced inhibition of PVN insulin expression in adult mice decreased both pituitary growth hormone (GH) mRNA level and serum GH concentration, which were attenuated by overexpression of PVN insulin. Notably, PVN insulin knockdown or chronic RS in young mice hindered normal growth via the down-regulation of GH gene expression and secretion, whereas PVN insulin overexpression in young mice prevented chronic RS–induced growth retardation by elevating GH production. Our results suggest that in both normal and stressful conditions, insulin synthesized in the parvocellular PVN neurons plays an important role in the regulation of pituitary GH production and body length, unveiling a physiological function of brain-derived insulin.
Jaemeun Lee, Kyungchan Kim, Jae Hyun Cho, Jin Young Bae, Timothy P. O’Leary, James D. Johnson, Yong Chul Bae, Eun-Kyoung Kim
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
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
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