Mucosal infections pose a significant global health burden. Antigen-specific tissue resident T cells are critical to maintaining barrier immunity. Previous studies in the context of systemic infection suggest that memory CD8 T cells may also provide innate-like protection against antigenically unrelated pathogens independent of TCR engagement. Whether "bystander T cell activation" is also an important defense mechanism in the mucosa is poorly understood. Here, we investigated if innate-like memory CD8 T cells could protect against a model mucosal virus infection, herpes simplex virus 2 (HSV-2). We found that immunization with an irrelevant antigen delayed disease progression from lethal HSV-2 challenge, suggesting that memory CD8 T cells may mediate protection despite the lack of antigen-specificity. Upon HSV-2 infection, we observed an early infiltration, rather than substantial local proliferation, of antigen-non-specific CD8 T cells, which became bystander-activated only within the infected mucosal tissue. Critically, we show that bystander-activated CD8 T cells are sufficient to reduce early viral burden after HSV-2 infection. Finally, local cytokine cues within the tissue microenvironment after infection were sufficient for bystander activation of mucosal tissue memory CD8 T cells from mice and humans. Altogether, our findings suggest that local bystander-activation of CD8 memory T cells contribute a fast and effective innate-like response to infection in mucosal tissue.
Tanvi Arkatkar, Veronica A. Davé, Irene Cruz Talavera, Jessica B. Graham, Jessica L. Swarts, Sean M. Hughes, Timothy A. Bell, Pablo Hock, Joe Farrington, Ginger D. Shaw, Anna C. Kirby, Michael Fialkow, Meei-Li Huang, Keith R. Jerome, Martin T. Ferris, Florian Hladik, Joshua T. Schiffer, Martin Prlic, Jennifer M. Lund
Sphingolipids function as membrane constituents and signaling molecules, with crucial roles in human diseases, from neurodevelopmental to cancer, best exemplified in the inborn errors of sphingolipid metabolism in lysosomes. The dihydroceramide desaturase DEGS1 acts in the last step of a sector of the sphingolipid pathway, de novo ceramide biosynthesis. Defects in DEGS1 cause the recently described hypomyelinating leukodystrophy-18 (HLD18, OMIM #618404). Here, we reveal that DEGS1 is a mitochondria-associated endoplasmic reticulum membrane (MAM)-resident enzyme, refining previous reports locating DEGS1 at the endoplasmic reticulum only. Using patient fibroblasts, multi-omics and enzymatic assays, we show that DEGS1 deficiency disrupts the main core functions of the MAM: i) mitochondrial dynamics, with a hyperfused mitochondrial network associated with decreased activation of dynamin-related protein 1; ii) cholesterol metabolism, with impaired sterol O-acyltransferase activity and decreased cholesteryl esters; iii) phospholipid metabolism, with increased phosphatidic acid and phosphatidylserine and decreased phosphatidylethanolamine; iv) biogenesis of lipid droplets, with increased size and numbers. Moreover, we detected increased mitochondrial superoxide species production in fibroblasts and mitochondrial respiration impairment in patient muscle biopsy tissues. Our findings shed light on the pathophysiology of HLD18 and broaden our understanding of the role of sphingolipid metabolism in MAMs function.
Laura Planas-Serra, Nathalie Launay, Leire Goicoechea, Bénédicte Heron, Cristina Jou, Natalia Juliá-Palacios, Montserrat Ruiz, Stéphane Fourcade, Carlos Casasnovas, Carolina De La Torre, Antoinette Gelot, Maria Marsal, Pablo Loza-Alvarez, Àngels García-Cazorla, Ali Fatemi, Isidre Ferrer, Manuel Portero-Otin, Estela Area-Gómez, Aurora Pujol
BACKGROUND. Maintaining durable immunity to vaccination represents a major challenge, but whether booster mRNA vaccination improves durability is unknown. METHODS. We measured antibody responses in 55 healthy adults who received a booster dose of Pfizer-BioNTech or Moderna vaccine against SARS-CoV-2 and calculated the half-life of antibody titers. We also measured memory B and T cell responses in a subset of 28 participants. In 13 volunteers who received a second booster, we measured serum antibody titers, and memory B and T cell responses. RESULTS. The booster (3rd immunization) dose at 6 - 10 months increased the half-life of serum neutralizing antibody (nAb) titers to 76 days from 56 - 66 days after the primary two-dose vaccination. A second booster dose (4th immunization) a year after the primary vaccination increased the half-life further to 88 days. However, despite this modestly improved durability in nAb responses against the ancestral (WA.1) strain, there was a loss in neutralization capacity against Omicron subvariants BA.2.75.2, BQ.1.1, and XBB.1.5 (48, 71, and 66-fold drop in titers respectively, relative to the WA.1 strain). While only 45 – 65% of participants demonstrated a detectable nAb titer against the newer variants after the booster (3rd dose), the response declined to below the detection limit in almost all individuals by 6 months. In contrast, booster vaccination induced antigen-specific memory B and T cells that persisted for at least 6 months. CONCLUSION. The durability of serum antibody responses improves only marginally following booster immunizations with the Pfizer-BioNTech or Moderna mRNA vaccines.
Prabhu S Arunachalam, Lilin Lai, Hady Samaha, Yupeng Feng, Mengyun Hu, Harold Sai-yin Hui, Bushra Wali, Madison L. Ellis, Meredith E. Davis-Gardner, Christopher M. Huerta, Kareem Bechnak, Sarah Bechnak, Matthew Lee, Matthew B. Litvack, Cecilia Losada, Alba Grifoni, Alessandro Sette, Veronika I. Zarnitsyna, Nadine Rouphael, Mehul S. Suthar, Bali Pulendran
Cutaneous Skeletal Hypophosphatemia Syndrome (CSHS) is a mosaic RASopathy characterized by the association of dysplastic skeletal lesions, congenital skin nevi of epidermal and/or melanocytic origin, and fibroblast growth factor-23 (FGF23)-mediated hypophosphatemia. The primary physiological source of circulating FGF23 is bone cells. However, several reports have suggested skin lesions as the source of excess FGF23 in CSHS. Consequently, without convincing evidence of efficacy, many patients with CSHS have undergone painful removal of cutaneous lesions in an effort to normalize blood phosphate levels.This study aims to elucidate whether the source of FGF23 excess in CSHS is RAS mutation-bearing bone or skin lesions. Towards this end, we analyzed the expression and activity of Fgf23 in two mouse models expressing similar HRAS/Hras activating mutations in a mosaic-like fashion in either bone or epidermal tissue. We found that HRAS hyperactivity in bone, not skin, caused excess of bioactive intact FGF23, hypophosphatemia and osteomalacia. Our findings support RAS-mutated dysplastic bone as the primary source of physiologically active FGF23 excess in patients with CSHS. This evidence informs the care of patients with CSHS, arguing against the practice of nevi removal to decrease circulating, physiologically active FGF23.
Diana Ovejero, Zachary Michel, Christophe Cataisson, Amanda Saikali, Rebeca Galisteo, Stuart H. Yuspa, Michael T. Collins, Luis F. de Castro
Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 and how its production is regulated is largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model. IL-6, measured in various cell types in post MI hearts by qPCR, RNAscope and at protein level, was preferentially formed by fibroblasts (CFs). scRNAseq in infarcted mouse and human hearts confirmed this finding. Adenosine stimulated fibroblast IL-6 formation via A2bR in a Gq-dependent manner. CFs highly expressed Adora2b, rapidly degraded extracellular ATP to AMP but lacked CD73. In mice and humans Adora2B was also mainly expressed by fibroblasts (scRNAseq). Global IL-6 formation was assessed in isolated hearts in mice lacking CD73 on T-cells (CD4CD73-/-) a condition known to be associated with adverse cardiac remodeling. The ischemia-induced release of IL-6 was strongly attenuated in CD4CD73-/- mice, suggesting adenosine-mediated modulation. Together this demonstrates that post-MI IL-6 is mainly derived from activated CFs and is controlled by T-cell derived adenosine. Purinergic metabolic cooperation between CFs and T-cells is a novel mechanism with therapeutic potential which modulates IL6 formation by the heart.
Christina Alter, Anne Sophie Henseler, Christoph Owenier, Julia Hesse, Zhaoping Ding, Tobias Lautwein, Jasmin Bahr, Sikander Hayat, Rafael Kramann, Eva Kostenis, Jürgen Scheller, Jürgen Schrader
KRASG12C inhibitors have revolutionized the clinical management of patients with KRASG12C-mutant lung adenocarcinoma. However, patient exposure to these inhibitors leads to the rapid onset of resistance. In this study, we have used genetically engineered mice to compare the therapeutic efficacy and the emergence of tumor resistance between genetic ablation of mutant Kras expression and pharmacological inhibition of oncogenic KRAS activity. Whereas Kras ablation induces massive tumor regression and prevents the appearance of resistant cells in vivo, treatment of KrasG12C/Trp53-driven lung adenocarcinomas with sotorasib, a selective KRASG12C inhibitor, caused a limited anti-tumor response similar to that observed in the clinic, including the rapid onset of resistance. Unlike in human tumors, we did not observe mutations in components of the RAS signaling pathways. Instead, sotorasib resistant tumors displayed amplification of the mutant Kras allele and activation of xenobiotic metabolism pathways, suggesting that reduction of the on-target activity of KRASG12C inhibitors is the main mechanism responsible for the onset of resistance. In sum, our results suggest that resistance to KRAS inhibitors could be prevented by achieving a more robust inhibition of KRAS signaling mimicking the results obtained upon Kras ablation.
Marina Salmón, Ruth Álvarez-Díaz, Coral Fustero-Torre, Oksana Brehey, Carmen G. Lechuga, Manuel Sanclemente, Fernando Fernández-García, Alejandra López-García, María Carmen Martín-Guijarro, Sandra Rodríguez-Perales, Emily Bousquet-Mur, Lucía Morales-Cacho, Francisca Mulero, Fátima Al-Shahrour, Lola Martínez, Orlando Domínguez, Eduardo Caleiras, Sagrario Ortega, Carmen Guerra, Monica Musteanu, Matthias Drosten, Mariano Barbacid
CRISPR-Cas9 has been proposed as a treatment for genetically inherited skin disorders. Here we report that CRISPR transfection activates STING-dependent antiviral responses in keratinocytes, resulting in heightened endogenous interferon (IFN) responses through induction of IFN-κ leading to decreased plasmid stability secondary to induction of the cytidine deaminase APOBEC3G. Notably, CRISPR-generated KO keratinocytes had permanent suppression of IFN-κ and IFN-stimulated gene (ISG) expression, secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. JAK inhibition via baricitinib prior to CRISPR transfection increased transfection efficiency, prevented IFNK promoter hypermethylation, and restored normal IFN-κ activity and ISG responses. This work shows that CRISPR-mediated gene correction alters antiviral responses in keratinocytes, has implications for future gene therapies of inherited skin diseases using CRISPR technology, and suggests pharmacologic JAK inhibition as a tool for facilitating and attenuating inadvertent selection effects in CRISPR-Cas9 therapeutic approaches.
Mrinal K. Sarkar, Ranjitha Uppala, Chang Zeng, Allison C. Billi, Lam C. Tsoi, Austin Kidder, Xianying Xing, Bethany E. Perez White, Shuai Shao, Olesya Plazyo, Sirisha Sirobhushanam, Enze Xing, Yanyun Jiang, Katherine A. Gallagher, John J. Voorhees, J. Michelle Kahlenberg, Johann E. Gudjonsson
During development of heart failure, capacity for cardiomyocyte fatty acid oxidation (FAO) and ATP production is progressively diminished contributing to pathologic cardiac hypertrophy and contractile dysfunction. Receptor interacting protein 140 (RIP140; Nrip1) has been shown to function as a transcriptional co-repressor of oxidative metabolism. We found that mice with striated muscle deficiency of RIP140 (strNrip1-/-) exhibit increased expression of a broad array of genes involved in mitochondrial energy metabolism and contractile function in heart and skeletal muscle. strNrip1-/- mice were resistant to the development of pressure overload-induced cardiac hypertrophy, and cardiomyocyte-specific RIP140 deficient (csNrip1-/-) mice were defended against development of heart failure caused by pressure overload combined with myocardial infarction. Genomic enhancers activated by RIP140 deficiency in cardiomyocytes were enriched in binding motifs for transcriptional regulators of mitochondrial function (estrogen-related receptor) and cardiac contractile proteins (myocyte enhancer factor 2). Consistent with a role in the control of cardiac fatty acid oxidation, loss of RIP140 in heart resulted in augmented triacylglyceride turnover and FA utilization. We conclude that RIP140 functions as a suppressor of a transcriptional regulatory network that controls cardiac fuel metabolism and contractile function, representing a potential therapeutic target for heart failure.
Tsunehisa Yamamoto, Santosh K. Maurya, Elizabeth Pruzinsky, Kirill Batmanov, Yang Xiao, Sarah M. Sulon, Tomoya Sakamoto, Yang Wang, Ling Lai, Kendra S. McDaid, Swapnil V. Shewale, Teresa C. Leone, Timothy R. Koves, Deborah M. Muoio, Pieterjan Dierickx, Mitchell A. Lazar, E. Douglas Lewandowski, Daniel P. Kelly
BACKGROUND. Hepatic de novo lipogenesis (DNL) and β-oxidation are tightly coordinated, and their dysregulation is thought to contribute to the pathogenesis of non-alcoholic fatty liver (NAFL). Fasting normally relaxes DNL-mediated inhibition of hepatic β-oxidation, dramatically increasing ketogenesis and decreasing reliance on the TCA cycle. Thus, we tested whether aberrant oxidative metabolism in fasting NAFL subjects is related to the inability to halt fasting DNL. METHODS. Forty consecutive non-diabetic individuals with and without a history of NAFL were recruited for this observational study. After phenotyping, subjects fasted for 24-hr, and hepatic metabolism was interrogated using a combination of 2H2O and 13C tracers, magnetic resonance spectroscopy, and high-resolution mass spectrometry. RESULTS. Within a subset of subjects, DNL was detectable after a 24-hr fast and was more prominent in those with NAFL, though it was poorly correlated with steatosis. However, fasting DNL negatively correlated with hepatic β-oxidation and ketogenesis and positively correlated with citrate synthesis. Subjects with NAFL but undetectable fasting DNL (25th percentile) were comparatively normal. However, those with the highest fasting DNL (75th percentile) were intransigent to the effects of fasting on the concentration of insulin, NEFA, and ketones. Additionally, they sustained glycogenolysis and spared the loss of oxaloacetate to gluconeogenesis in favor of citrate synthesis, which correlated with DNL and diminished ketogenesis. CONCLUSION. Metabolic flux analysis in fasted subjects indicates that shared metabolic mechanisms link the dysregulations of hepatic DNL, ketogenesis, and the TCA cycle in NAFL. TRIAL REGISTRATION. Data obtained during the enrollment/non-intervention phase of Effect of Vitamin E on Non-Alcoholic Fatty Liver Disease; ClinicalTrials.gov NCT02690792.
Xiaorong Fu, Justin A. Fletcher, Stanisław Deja, Melissa Inigo-Vollmer, Shawn C. Burgess, Jeffrey D. Browning
Anti-tumor activity of CD8+ T cells is potentially restrained by a variety of negative regulatory pathways that are triggered in tumor microenvironment, yet exact mechanisms remain incompletely defined. Here we report that intrinsic RIG-I in CD8+ T cells represents such a factor, as evidenced by observations that tumor-restricting effect of endogenous or adoptively transferred CD8+ T cells was enhanced by intrinsic Rig-I deficiency or inhibition, with the increased accumulation, survival, and cytotoxicity of tumor-infiltrating CD8+ T cells. Mechanistically, T cell activation-induced RIG-I upregulation restrained STAT5 activation via competitively sequestering HSP90. In accordance, the frequency of RIG-I+ tumor-infiltrating CD8+ T cells in human colon cancer positively correlated with attenuated survival and effector signatures of CD8+ T cells as well as poor prognosis. Collectively, these results implicate RIG-I as a potentially druggable factor for improving CD8+ T cells-based tumor immunotherapy.
Xinyi Jiang, Jian Lin, Chengfang Shangguan, Xiaoyao Wang, Bin Xiang, Juan Chen, Hezhou Guo, Wu Zhang, Jun Zhang, Yan Shi, Jiang Zhu, Hui Yang
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