Monocytes play an important role in the regulation of alloimmune responses after heart transplantation (HTx). Recent studies have highlighted the importance of immunometabolism in the differentiation and function of myeloid cells. While the importance of glucose metabolism in monocyte differentiation and function has been reported, a role for fatty acid β-oxidation (FAO) has not been explored. Heterotopic HTx was performed using hearts from Balb/c donor mice implanted into C57Bl/6 recipient mice and treated with etomoxir (eto), an irreversible inhibitor of carnitine palmitoyltransferase 1 (Cpt1), a rate-limiting step of FAO, or vehicle control. FAO inhibition prolonged HTx survival, reduced early T cell infiltration/activation and reduced dendritic cell (DC) and macrophage infiltration to heart allografts of eto-treated recipients. ELISPOT demonstrated eto-treated HTx were less reactive to activated donor antigen presenting cells. FAO inhibition reduced monocyte-to-DC and monocyte-to-macrophage differentiation in vitro and in vivo. FAO inhibition did not alter the survival of heart allografts when transplanted into Ccr2-deficient recipients, suggesting the effects of FAO inhibition were dependent on monocyte mobilization. Finally, we confirmed the importance of FAO on monocyte differentiation in vivo using conditional deletion of Cpt1a. Our findings demonstrate that targeting FAO attenuates alloimmunity after HTx, in part through impairing monocyte differentiation.
Yuehui Zhu, Hao Dun, Li Ye, Yuriko Terada, Leah P. Shriver, Gary J. Patti, Daniel Kreisel, Andrew E. Gelman, Brian W. Wong
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines pose as the most effective approach for mitigating COVID-19 pandemic. High-degree efficacy of SARS-CoV-2 vaccines in clinical trials indicates that vaccination invariably induces an adaptive immune response in vaccine recipients. However, the emergence of breakthrough infections in vaccinated individuals suggest that the breadth and magnitude vaccine-induced adaptive immune response may varies. We assessed vaccine-induced SARS-CoV-2 T-cell response in twenty-one vaccinated individuals and found that SARS-CoV-2 specific T-cells were invariably detected in all individuals. However, the magnitude and breadth of SARS-CoV-2 specific T-cell response varied. Vaccination induced mainly a CD4+ T-cell dominant SARS-CoV-2 specific immune response and the frequencies of SARS-CoV-2 specific T-cell varied across vaccinated individuals. To gain insights into whether SARS-CoV-2 vaccines can induce a long-lived T-cell immune response we investigated differentiation states and cytokine profiles to identify immune features associated with superior recall function and longevity. We identified distinct hierarchically organized differentiation states and cytokine expression patterns. SARS-CoV-2 specific CD4+ T-cells were polyfunctional and produced high levels of IL-2, which could be associated with superior longevity. Stratifying the vaccinated individuals based on the breadth and magnitude of vaccine-induced SARS-CoV-2 response identified two distinct response groups: individuals with high abundance vs low abundance of SARS-CoV-2 T-cells. The fractions of TNF-a and IL-2 producing SARS-CoV-2 T-cells were the main determinants distinguishing high vs low responders. Lastly, we identified the majority of vaccine-induced SARS-CoV-2 T-cells were reactive against conserved regions of mutant S-protein, suggesting that vaccine-induced SARS-CoV-2 T-cells could provide continued protection against emerging variants-of-concern.
Li Li, Muharrem Muftuoglu, Shaoheng Liang, Mahesh Basyal, Jiangxing LV, Mehmet E. Akdogan, Ken Chen, Michael Andreeff, Simrit Parmar
The importance of healthy mitochondrial function is implicated in the prevention of chronic/diabetic kidney diseases (CKD/DKD). Sex differences also play an important role in DKD. Our previous studies revealed that mitochondrial substrate overload (modeled by homozygous deletion of carnitine acetyl-transferase – CrAT) in proximal tubules causes renal injury. Here we demonstrate the importance of intact mitochondrial substrate efflux by titrating the amount of overload through the generation of a heterozygous CrAT knockout model (“PT-CrATHET” mouse). Intriguingly, these animals developed renal injury similarly to their homozygous counterparts. Mitochondria were structurally and functionally impaired in both sexes. Transcriptomic analyses, however, revealed striking sex differences. Male mice shut down fatty acid oxidation and several other metabolism-related pathways. Females had a significantly weaker transcriptional response in metabolism but activation of inflammatory pathways was prominent. Proximal tubular cells from PT-CrATHET mice of both sexes exhibited a shift towards a more glycolytic phenotype, but females were still able to oxidize fatty acid-based substrates. Our results demonstrate that maintaining mitochondrial substrate metabolism balance is crucial to satisfy proximal tubular energy demand. Our findings have potentially broad implications as both the glycolytic shift and the sexual dimorphisms discovered herein offer new modalities for future interventions for treating kidney disease.
Allison McCrimmon, Kerin M. Cahill, Claudia Kruger, Margaret E. Mangelli, Emily Bouffard, Timothy Dobroski, Kelly N. Michanczyk, Susan J. Burke, Robert C. Noland, Daria V. Ilatovskaya, Krisztian Stadler
Kidney fibrosis is the final common pathway of progressive kidney diseases, the underlying mechanisms of which is not fully understood. The purpose of the current study is to investigate a role of Piezo1, a mechanosensitive nonselective cation channel, in kidney fibrosis. In human fibrotic kidneys, Piezo1 protein expression was markedly upregulated. The abundance of Piezo1 protein in kidneys of mice with UUO or with folic-acid treatment was significantly increased. Inhibition of Piezo1 with GsMTx4 markedly ameliorated UUO or folic acid-induced kidney fibrosis. Mechanical stretch, compression or stiffness induced Piezo1 activation and pro-fibrotic responses in human HK2 cells and primary cultured mouse proximal tubular cells (mPTCs), which were greatly prevented by inhibition or silence of Piezo1. TGFβ-1 induced increased Piezo1 expression and pro-fibrotic phenotypic alterations in HK2 cells and mPTCs, which was again markedly prevented by inhibition of Piezo1. Activation of Piezo1 by Yoda1, a Piezo1 agonist, caused calcium influx and profibrotic responses in HK2 cells and induced calpain2 activation, followed by talin1 cleavage and upregulation of integrinβ1. Also, Yoda1 promoted the link between ECM and integrinβ1. In conclusion, Piezo1 is involved in the progression of kidney fibrosis and pro-fibrotic alterations in renal proximal tubular cells, likely through activating calcium-calpain2-integrinβ1 pathway.
Xiaoduo Zhao, Yonglun Kong, Baien Liang, Jinhai Xu, Yu Lin, Nan Zhou, Jing Li, Bin Jiang, Jianding Cheng, Chunling Li, Weidong Wang
Cytomegalovirus (CMV) is a globally ubiquitous pathogen with a seroprevalence of approximately 50% in the UK. CMV infection induces expansion of immunosenescent T cell and NK cell populations with these cells demonstrating lower responsiveness to activation and reduced functionality upon infection and vaccination. In this study, we found that CMV+ participants had normal T cell responses after single dose or homologous vaccination with the viral vector ChAdOx1. In contrast, CMV seropositivity was associated with a loss of T cell IFN-γ secretion following heterologous ChAd-MVA viral vector vaccination. Analysis of participants receiving a single dose of ChAdOx1 demonstrates that T cells from CMV+ donors have a more terminally differentiated profile of CD57+PD1+ CD4+ T cells and CD8+ T cells expressing less IL-2Rα (CD25), and fewer polyfunctional CD4+ T cells 14 days post-vaccination. NK cells from CMV-seropositive individuals also have a reduced activation profile. Overall, our data suggest that although CMV infection enhances immunosenescence of T and NK populations, it does not affect antigen-specific T cell IFN-γ secretion or antibody IgG production after vaccination with the current ChAdOx1 nCoV-19 vaccination regimen in the UK.
Hannah R. Sharpe, Nicholas M. Provine, Georgina S. Bowyer, Pedro Moreira Folegatti, Sandra Belij-Rammerstorfer, Amy Flaxman, Rebecca Makinson, Adrian V.S. Hill, Katie J. Ewer, Andrew J. Pollard, Paul Klenerman, Sarah Gilbert, Teresa Lambe
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
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
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