BACKGROUND. Responses of the metabolome to acute aerobic exercise may predict VO2max and longer-term outcomes, including the development of diabetes and its complications. METHODS. Serum samples were collected from overweight trained (OWT) and normal weight trained (NWT) runners prior to and immediately after a supervised 90-minute treadmill run at 60% VO2max (NWT = 14, OWT = 11) in a cross-sectional study. We applied a liquid chromatography high resolution-mass spectrometry based untargeted metabolomics platform to evaluate the effect of acute aerobic exercise on the serum metabolome. RESULTS. NWT and OWT metabolic profiles shared increased circulating acylcarnitines and free fatty acids (FFAs) with exercise while intermediates of adenine metabolism, inosine and hypoxanthine, were strongly correlated with body fat percentage and VO2max. Untargeted metabolomics-guided follow-up quantitative lipidomic analysis revealed that baseline levels of fatty acid esters of hydroxy fatty acids (FAHFAs) were generally diminished in the OWT group. FAHFAs negatively correlated with visceral fat mass and HOMA-IR. Strikingly, a 4-fold decrease in FAHFAs was provoked by acute aerobic running in NWT, an effect that negatively correlated with circulating IL-6, neither of which was observed in the OWT group. Machine learning models based on a pre-exercise metabolite profile that included FAHFAs, FFAs, and adenine intermediates predicted VO2max. CONCLUSION. These findings in overweight human participants and healthy controls indicate that exercise-provoked changes in FAHFAs distinguish normal weight from overweight individuals and could predict VO2max. These results support the notion that FAHFAs could modulate the inflammatory response, fuel utilization, and insulin resistance. TRIAL REGISTRATION. ClinicalTrials.gov NCT02150889. FUNDING. NIH DK091538, AG069781, DK098203, TR000114, UL1TR002494.
Alisa B. Nelson, Lisa S. Chow, David B. Stagg, Jacob R. Gillingham, Michael D. Evans, Meixia Pan, Curtis C. Hughey, Chad L. Myers, Xianlin Han, Peter A. Crawford, Patrycja Puchalska
NADPH deficiency exacerbates lupus in murine models and in patients, but the mechanisms remain unknown. One hypothesis is that NADPH oxidase suppresses autoimmunity by facilitating dead cell clearance via LC3-associated phagocytosis (LAP). The absence of LAP reportedly causes an autoinflammatory syndrome in aged, non-autoimmune mice. Prior work implicated cytochrome b-245, beta polypeptide (CYBB), a component of the NADPH oxidase complex, and RUN and cysteine-rich domain containing Beclin 1 interacting protein (RUBICON), as requisite for LAP. To test the hypothesis that NADPH oxidase deficiency exacerbates lupus via a defect in LAP, we deleted Rubicon in the B6.Sle1.Yaa and MRL.Faslpr lupus mouse models. Under this hypothesis, RUBICON deficiency should phenocopy NADPH oxidase deficiency, as both work in the same pathway. However, we observed the opposite—RUBICON deficiency resulted in reduced mortality, renal disease, and autoantibody titers to RNA-associated autoantigens. Given that our data contradicts the published role for LAP in autoimmunity, we assessed whether CYBB and RUBICON are requisite for LAP. We found instead that LAP is not dependent on either of these two pathways. Our data thus reveal RUBICON as a novel regulator of SLE, possibly by a B cell-intrinsic mechanism, but do not support a role for LAP in lupus.
Rachael A. Gordon, Christina C. Giannouli, Chirag Raparia, Sheldon I. Bastacky, Anthony Marinov, William Hawse, Richard T. Cattley, Jeremy S. Tilstra, Allison M. Campbell, Kevin M. Nickerson, Anne Davidson, Mark J. Shlomchik
Duration of protection from SARS-CoV-2 infection in people with HIV (PWH) following vaccination is unclear. In a sub-study of the phase 2/3 the COV002 trial (NCT04400838), 54 HIV positive male participants on antiretroviral therapy (undetectable viral loads, CD4+ T cells >350 cells/ul) received two doses of ChAdOx1 nCoV-19 (AZD1222) 4-6 weeks apart and were followed for 6 months. Responses to vaccination were determined by serology (IgG ELISA and MesoScale Discovery (MSD)), neutralisation, ACE-2 inhibition, gamma interferon ELISpot, activation-induced marker (AIM) assay and T cell proliferation. We show that 6 months after vaccination the majority of measurable immune responses were greater than pre-vaccination baseline, but with evidence of a decline in both humoral and cell mediated immunity. There was, however, no significant difference compared to a cohort of HIV-uninfected individuals vaccinated with the same regimen. Responses to the variants of concern were detectable, although were lower than wild type. Pre-existing cross-reactive T cell responses to SARS-CoV-2 spike were associated with greater post-vaccine immunity and correlated with prior exposure to beta coronaviruses. These data support the on-going policy to vaccinate PWH against SARS-CoV-2, and underpin the need for long-term monitoring of responses after vaccination.
Ane Ogbe, Matthew Pace, Mustapha Bittaye, Timothy Tipoe, Sandra Adele, Jasmini Alagaratnam, Parvinder K. Aley, M. Azim Ansari, Anna Bara, Samantha Broadhead, Anthony Brown, Helen Brown, Federica Cappuccini, Paola Cinardo, Wanwisa Dejnirattisai, Katie Ewer, Henry Fok, Pedro M. Folegatti, Jamie Fowler, Leila Godfrey, Anna L. Goodman, Bethany Jackson, Daniel Jenkin, Mathew Jones, Stephanie Longet, Rebecca A. Makinson, Natalie G. Marchevsky, Moncy Mathew, Andrea Mazzella, Yama F. Mujadidi, Lucia Parolini, Claire Petersen, Emma Plested, Katrina Pollock, Thurkka Rajeswaran, Maheshi N. Ramasamy, Sarah Rhead, Hannah Robinson, Nicola Robinson, Helen Sanders, Sonia Serrano Fandos, Tom Tipton, Anele Waters, Panagiota Zacharopoulou, Eleanor Barnes, Susanna Dunachie, Philip Goulder, Paul Klenerman, Gavin R. Screaton, Alan Winston, Adrian V.S. Hill, Sarah C. Gilbert, Miles Carroll, Andrew J. Pollard, Sarah Fidler, Julie Fox, Teresa Lambe, John Frater
Clinical outcomes in colorectal cancer (CRC) have been correlated with T cell infiltrates, but the specific populations of T cells, their functions, and how they influence clinical outcomes remains unclear. To comprehensively investigate the diverse phenotype and function of T cells in CRC, we profiled 37,931 single T cells from tumors and adjacent normal colon of 16 treatment-naïve CRC patients with respect to transcriptome, TCR sequence, and 23 cell surface markers. Our single-cell analysis identified phenotypically and functionally distinguishable effector CD4+ and CD8+ T cell-types within human tumors. We employed single-cell gene signatures from these T cell subsets to query the TCGA database to assess the prognostic significance of these subsets. Among CD8+ T-cell infiltrates, we found two distinct cytotoxic T cell types differentiated into clonally-expanded exhausted T cells. GZMK+ KLRG1+ cytotoxic T cells with a less dysfunctional phenotype were enriched in CRC patients with good outcomes. Strikingly, GNLY+ CD103+ cytotoxic T cells, including intraepithelial lymphocytes (IELs) with a more dysfunctional phenotype, were not associated with good clinical outcomes, despite high co-expression of CD39 and CD103, markers which denote tumor-reactivity. Together, this suggests that tumor-reactive cytotoxic T cells are effectively targeted to the tumor, yet their presence alone does not contribute to anti-tumor activity due to their impaired function, as reflected in clinical outcomes. Among CD4+ T cell-infiltrates, we found two distinct regulatory T cells (Treg) subtypes associated with opposite clinical outcomes. While total Tregs, predominantly Helios+ cells, were associated with good outcomes, Helios- CD38+ peripherally-induced Treg cells (pTregs) were strongly associated with bad outcomes independent of stage. CD38+ pTregs, which shared gene signatures with Th17 cells, possessed a highly suppressive phenotype, suggesting they are the elusive Treg population that inhibits anti-tumor immunity in CRC. These findings highlight the potential utility of these subpopulations in predicting clinical outcomes independent of stage. Furthermore, these observations support the potential for novel CRC therapies directed at CD38+ pTregs or CD8+ CD103+ T cells to augment existing T cell-targeted immunotherapies.
Kazuya Masuda, Adam Kornberg, Jonathan Miller, Sijie Lin, Nathan Suek, Theo Botella, Kerim A. Secener, Alyssa M. Baccarella, Liang Cheng, Matthew Ingham, Vilma Rosario, Ahmed M. Al-Mazrou, Steven A. Lee-Kong, Ravi P. Kiran, Marlon Stoeckius, Peter Smibert, Armando Del Portillo, Paul E. Oberstein, Peter A. Sims, Kelley S. Yan, Arnold Han
Mechanisms governing entry and exit of immune cells into, and out of, inflamed joints, remain poorly understood. We sought herein to identify the key molecular pathways regulating such migration. Using murine models of inflammation in conjunction with mice expressing a photoconvertible fluorescent protein we characterized the migration of cells from joints to draining lymph nodes (LN) and performed RNA-seq analysis on isolated cells, identifying genes associated with migration and retention. We further refined the gene list to those specific for joint inflammation. RNA-seq data revealed pathways and genes previously highlighted as characteristic of RA in patient studies, validating the methodology. Focusing on gene regulatory pathways associated with cell migration, adhesion and movement, we identified genes involved in the retention of immune cells in the inflamed joint, namely JAM-A, and identified a role for such molecules in T cell differentiation in vivo.Thus, using a combination of novel cell tracking approaches and murine models of inflammatory arthritis we have identified genes, pathways and anatomically specific tissue signatures regulating cell migration in a variety of inflamed sites. This unique skin and joint specific dataset will be an invaluable resource for the identification of novel therapeutic targets for arthritis and other inflammatory disorders.
Catriona T. Prendergast, Robert A. Benson, Hannah E. Scales, Caio S. Bonilha, John J. Cole, Iain McInnes, James M. Brewer, Paul Garside
Studying temporal gene expression shifts during disease progression provides important insights into the biological mechanisms that distinguish adaptive and maladaptive responses. Existing tools for the analysis of time course transcriptomic data are not designed to optimally identify distinct temporal patterns when analyzing dynamic differentially expressed genes (DDEGs). Moreover, there is a lack of methods to assess and visualize the temporal progression of biological pathways mapped from time course transcriptomic datasets. In this study, we developed an open-source R package TrendCatcher (https://github.com/jaleesr/TrendCatcher), which applies the smoothing spline ANOVA model and break point searching strategy to identify and visualize distinct dynamic transcriptional gene signatures and biological processes from longitudinal datasets. We used TrendCatcher to perform a systematic temporal analysis of COVID-19 peripheral blood transcriptomes, including bulk and single-cell RNA sequencing time course data. TrendCatcher uncovered the early and persistent activation of neutrophils and coagulation pathways as well as impaired type I interferon (IFN-I) signaling in circulating cells as a hallmark of patients who progressed to severe COVID-19, whereas no such patterns were identified in individuals receiving SARS-CoV-2 vaccinations or patients with mild COVID-19. These results underscore the importance of systematic temporal analysis to identify early biomarkers and possible pathogenic therapeutic targets.
Xinge Wang, Mark A. Sanborn, Yang Dai, Jalees Rehman
Dyslipidemia and autophagy have been implicated in the pathogenesis of blinding neovascular age-related macular degeneration (NV-AMD). Very low-density lipoprotein receptor (VLDLR), expressed in photoreceptors with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acids (FA). Since FA uptake is reduced in Vldlr-/- tissues, more remain in circulation, and the retina is fuel deficient, driving the formation in mice of neovascular lesions reminiscent of retinal angiomatous proliferation (RAP), a subtype of NV-AMD. Nutrient scarcity and energy failure are classically mitigated by increasing autophagy. We find that excess circulating lipids restrain retinal autophagy, which contributes to pathological angiogenesis in the Vldlr-/- RAP model. Triglyceride-derived FA sensed by free fatty acid receptor 1 (FFAR1) restricted autophagy and oxidative metabolism in photoreceptors. FFAR1 suppressed transcription factor EB (TFEB), a master regulator of autophagy and lipid metabolism. Reduced TFEB, in turn, decreased Sirtuin-3 expression and mitochondrial respiration. Metabolomic signatures of mouse RAP-like retinas were consistent with a role in promoting angiogenesis. This signature was also found in human NV-AMD vitreous. Restoring photoreceptor autophagy in Vldlr-/- retinas, either pharmacologically or by deleting Ffar1, enhanced metabolic efficiency and suppressed pathological angiogenesis. Dysregulated autophagy by circulating lipids might therefore contribute to the energy failure of photoreceptors driving neovascular eye diseases, and FFAR1 may be a target for intervention.
Emilie Heckel, Gael Cagnone, Tapan Agnihotri, Bertan Cakir, Ashim Das, Jin Sung Kim, Nicholas Kim, Geneviève Lavoie, Anu Situ, Sheetal Pundir, Ye Sun, Florian Wünnemann, Kerry A. Pierce, Courtney Dennis, Grant A. Mitchell, Sylvain Chemtob, Flavio A. Rezende, Gregor Andelfinger, Clary B. Clish, Philippe P. Roux, Przemyslaw Sapieha, Lois E.H. Smith, Jean-Sébastien Joyal
Altered islet architecture is associated with β cell dysfunction and Type 2 Diabetes (T2D) progression, but molecular effectors of islet spatial organization remain mostly unknown. Although Notch signaling is known to regulate pancreatic development, we observed “re-activated” β cell Notch activity in obese mouse models. To test the repercussions and reversibility of Notch effects, we generated doxycycline-dependent, β cell-specific Notch gain-of-function mice. As predicted, we found that Notch activation in post-natal β cells impaired glucose stimulated insulin secretion (GSIS) and glucose intolerance, but we observed a surprising remnant glucose intolerance after doxycycline withdrawal and cessation of Notch activity, associated with a marked disruption of normal islet architecture. Transcriptomic screening of Notch-active islets revealed increased Ephrin signaling. Commensurately, exposure to Ephrin ligands increased β cell repulsion, and impaired murine and human pseudo-islet formation. Consistent with our mouse data, Notch and Ephrin signaling are increased in metabolically-inflexible β cells in patients with T2D. These studies suggest than islet architecture can be permanently altered by β cell Notch/Ephrin signaling during a morphogenetic window in early life.
Alberto Bartolomé, Nina Suda, Junjie Yu, Changyu Zhu, Jinsook Son, Hongxu Ding, Andrea Califano, Domenico Accili, Utpal B. Pajvani
Standard-of-care treatment for advanced HER2+ breast cancers (BC) is comprised of two HER2-specific monoclonal antibodies (mAb), Trastuzumab (T) and Pertuzumab (P) with chemotherapy. While this combination (T+P) is highly effective, its synergistic mechanism of action (MOA) is not completely known. Initial studies had demonstrated that Pertuzumab suppressed HER2 hetero-dimerization as the potential therapeutic MOA, thus the improved outcome associated with the T+P combination MOA compared to Trastuzumab alone has been widely reported as being due to Pertuzumab-mediated suppression of HER2 signaling in combination with Trastuzumab-mediated induction of anti-tumor immunity. Unraveling this MOA may be critical to extend this combination strategy to other antigens or other cancers, as well as improving this current treatment modality. Using novel murine and human versions of Pertuzumab, we found it induced both Antibody-Dependent-Cellular-Phagocytosis (ADCP) by tumor-associated macrophages and suppression of HER2 oncogenic signaling. Most significantly, we identified that only T+P combination therapy, but not when either antibody used in isolation, allows for the activation of the classical complement pathway, resulting in both direct complement-dependent cytotoxicity (CDC) as well as complement-dependent cellular phagocytosis (CDCP) of HER2+ BC cells. Notably, we show that tumor expression of C1q was positively associated with survival outcome in HER2+ BC patients, whereas expression of complement regulators CD55 and CD59 were inversely correlated, suggesting the importance of complement activity in clinical outcomes. Accordingly, inhibition of C1 activity in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant anti-tumor MOA for T+P therapy that may be functionally enhanced to augment therapeutic efficacy in the clinic.
Li-Chung Tsao, Erika J. Crosby, Timothy N. Trotter, Junping Wei, Tao Wang, Xiao Yang, Amanda N. Summers, Gangjun Lei, Christopher A. Rabiola, Lewis A. Chodosh, William J. Muller, Herbert Kim Lyerly, Zachary C. Hartman
Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by the accumulation of myofibroblasts and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-seq on lung fibroblasts isolated from young and aged mice during the early resolution phase post-bleomycin injury. We discovered that relative to injured young fibroblasts, injured aged fibroblasts exhibited a pro-fibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified pro-viral integration site of Moloney murine leukemia virus 1 (PIM1) and its target Nuclear Factor of Activated T Cells-1 (NFATc1) as putative drivers of the sustained pro-fibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts in vitro potentiated their fibrogenic activation in a NFATc1-dependent manner. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Inhibition of PIM1 signaling in IPF lung explants ex vivo inhibited pro-survival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.
Tho X. Pham, Jisu Lee, Jiazhen Guan, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Steven K. Huang, Daniel J. Tschumperlin, Giovanni Ligresti
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