Given the resurgence of pertussis, several countries have introduced maternal tetanus, diphtheria, and acellular pertussis (aP) vaccination during pregnancy to protect young infants against severe pertussis. Although protective against the disease, the effect of maternal aP vaccination on bacterial colonization of the offspring is unknown. Here, we used a mouse model to demonstrate that maternal aP immunization, either before or during pregnancy, protects pups from lung colonization by Bordetella pertussis. However, it substantially prolongs nasal carriage by inhibiting the recruitment of IL-17-producing resident memory T cells and ensuing neutrophil influx in the nasal tissue upon B. pertussis infection, especially of those with pro-inflammatory and cytotoxic properties. Prolonged nasal carriage after aP vaccination is due to IL-4 signaling, as it is abolished in IL-4Ra-/- mice. The effect of maternal aP vaccination can be transferred transplacentally to the offspring or by breastfeeding and is long-lasting, as it persists into adulthood. Maternal aP vaccination may thus potentially augment the B. pertussis reservoir.
Violaine Dubois, Jonathan Chatagnon, Manon Depessemier, Camille Locht
Glutaminolysis is a hallmark of the activation and metabolic reprogramming of T cells. Isotopic tracer analyses of antigen-activated effector CD8+ T cells revealed that glutamine is the principal carbon source for the biosynthesis of polyamines putrescine, spermidine and spermine. These metabolites play critical roles in activation-induced T-cell proliferation, as well as for the production of hypusine, which is derived from spermidine and is covalently linked to the translation elongation factor eIF5A. Here, we demonstrated that the glutamine-polyamine-hypusine axis controls the expression of CD69, an important regulator of tissue resident memory T cells (TRM). Inhibition of this circuit augmented the development of TRM cells ex vivo and in vivo in the bone marrow, a well-established niche for TRM cells. Furthermore, blocking the polyamine-hypusine axis augmented CD69 expression and IFN-γ and TNF-α production in human CD8+ T cells from peripheral blood and sarcoma tumor infiltrating lymphocytes, as well as in human CD8+ CAR-T cells. Collectively, these findings support the notion that the polyamine-hypusine circuit can be exploited to modulate TRM cells for therapeutic benefit.
Aya G. Elmarsafawi, Rebecca S. Hesterberg, Mario R. Fernandez, Chunying Yang, Lancia N.F. Darville, Min Liu, John M. Koomen, Otto Phanstiel IV, Reginald Atkins, John E. Mullinax, Shari A. Pilon-Thomas, Frederick L. Locke, Pearlie K. Epling-Burnette, John L. Cleveland
DNASE1L3, an enzyme highly expressed in dendritic cells, is functionally important for regulating autoimmune responses to self-DNA and chromatin. Deficiency of DNASE1L3 leads to development of autoimmune diseases in both humans and mice. However, despite the well-established causal relationship between DNASE1L3 and immunity, little is known about the involvement of DNASE1L3 in regulation of anti-tumor immunity, the foundation of modern anti-tumor immunotherapy. In this study, we identify DNASE1L3 as a new regulator of anti-tumor immunity and a tumor suppressor in colon cancer. In humans, DNASE1L3 is downregulated in tumor-infiltrating dendritic cells, and this downregulation is associated with poor patient prognosis and reduced tumor immune cell infiltration in many cancer types. In mice, Dnase1l3 deficiency in the tumor microenvironment enhances tumor formation and growth in several colon cancer models. Notably, the increased tumor formation and growth in Dnase1l3-deficient mice are associated with impaired anti-tumor immunity, including a substantial reduction of cytotoxic T cells and a unique subset of dendritic cells. Consistently, Dnase1l3-deficient dendritic cells directly modulate cytotoxic T cells in vitro. Collectively, our study unveils a previously unknown link between DNASE1L3 and anti-tumor immunity, and further suggests that restoration of DNASE1L3 activity may represent a potential therapeutic approach for anti-cancer therapy.
Wenling Li, Hideki Nakano, Wei Fan, Yuanyuan Li, Payel Sil, Keiko Nakano, Fei Zhao, Peer W. Karmaus, Sara A. Grimm, Min Shi, Xin Xu, Ryushin Mizuta, Daisuke Kitamura, Yisong Wan, Michael B. Fessler, Donald N. Cook, Igor Shats, Xiaoling Li, Leping Li
Denosumab is an anti-RANKL antibody that potently suppresses bone resorption, increases bone mass, and reduces fracture risk. Discontinuation of denosumab causes rapid rebound bone resorption and bone loss but the molecular mechanisms are unclear. We generated humanized RANKL mice and treated them with denosumab to examine the cellular and molecular conditions associated with rebound resorption. Denosumab potently suppressed both osteoclast and osteoblast number in cancellous bone in humanized RANKL mice. The decrease in osteoclast number was not associated with changes in osteoclast progenitors in bone marrow. Long-term but not short-term denosumab administration reduced OPG mRNA in bone. Localization of OPG expression revealed that OPG mRNA is produced by a subpopulation of osteocytes. Long-term denosumab administration reduced osteocyte OPG mRNA suggesting that OPG expression declines as osteocytes age. Consistent with this, osteocyte expression of OPG was more prevalent near the surface of cortical bone in humans and mice. These results suggest that new osteocytes are an important source of OPG in remodeling bone and that suppression of remodeling reduces OPG abundance by reducing new osteocyte formation. The lack of new osteocytes and the OPG they produce may contribute to rebound resorption after denosumab discontinuation.
Qiang Fu, Nancy C. Bustamante-Gomez, Humberto Reyes-Pardo, Igor Gubrij, Diana Escalona-Vargas, Jeff D. Thostenson, Michela Palmieri, Joseph J. Goellner, Intawat Nookaew, C. Lowry Barnes, Jeffrey B. Stambough, Elena Ambrogini, Charles A. O'Brien
Inadequate adaption to mechanical forces, including blood pressure, contributes to development of arterial aneurysms. Recent studies have pointed to a mechano-protective role of YAP and TAZ in vascular smooth muscle cells (SMCs). Here, we identified reduced expression of YAP1 in human aortic aneurysms. Vascular SMC-specific knockouts (KOs) of YAP/TAZ were thus generated using the novel integrin α8 (Itga8)-Cre mouse model (i8-YT-KO). i8-YT-KO mice spontaneously developed aneurysms in the abdominal aorta within two weeks of knockout induction and in smaller arteries at later times. The vascular specificity of the Itga8-Cre circumvented gastrointestinal effects. Aortic aneurysms were characterized by elastin disarray, SMC apoptosis, and accumulation of proteoglycans and immune cell populations. RNA-sequencing, proteomics, and myography demonstrated decreased contractile differentiation of SMCs and impaired vascular contractility. This associated with partial loss of myocardin expression, reduced blood pressure, and edema. Mediators in the inflammatory cGAS-STING pathway, were increased. A sizeable increase of SOX9, along with several direct target genes, including aggrecan (Acan), contributed to proteoglycan accumulation. This was the earliest detectable change, occurring three days after knockout induction and before the pro-inflammatory transition. In conclusion, Itga8-Cre deletion of YAP and TAZ represents a rapid and spontaneous aneurysm model that recapitulates features of human abdominal aortic aneurysms.
Marycarmen Arévalo Martínez, Olivia Ritsvall, Joakim A. Bastrup, Selvi Celik, Gabriel Jakobsson, Fatima Daoud, Christopher Winqvist, Anders Aspberg, Catarina Rippe, Lars Maegdefessel, Alexandru Schiopu, Thomas A. Jepps, Johan Holmberg, Karl Swärd, Sebastian Albinsson
Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, hampering accurate diagnosis and individualized therapy development. Here, bi-allelic variants in the highly conserved flightless-1 (FLII) gene were identified in three families with idiopathic early-onset dilated CM. We demonstrate that patient-specific FLII variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, result in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provide insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identify Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM, and report bi-allelic variants as a novel genetic cause of pediatric CM.
Claudine W.B. Ruijmbeek, Filomena Housley, Hafiza Idrees, Michael P. Housley, Jenny Pestel, Leonie Keller, Jason Kuan Han Lai, Herma C. van der Linde, Rob Willemsen, Janett Piesker, Zuhair N. Al-Hassnan, Abdulrahman Almesned, Michiel Dalinghaus, Lisa M. van den Bersselaar, Marjon A. van Slegtenhorst, Federico Tessadori, Jeroen Bakkers, Tjakko J. van Ham, Didier Y.R. Stainier, Judith M.A. Verhagen, Sven Reischauer
Mutation of the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) was linked to Darier disease more than two decades ago; however, there remain no targeted therapies for this disorder causing recurrent skin blistering and infections. Since Atp2a2 knockout mice do not phenocopy its pathology, we established a human tissue model of Darier disease to elucidate its pathogenesis and identify potential therapies. Leveraging CRISPR/Cas9, we generated human keratinocytes lacking SERCA2, which replicated features of Darier disease, including weakened intercellular adhesion and defective differentiation in organotypic epidermis. To identify pathogenic drivers downstream of SERCA2 depletion, we performed RNA sequencing and proteomic analysis. SERCA2-deficient keratinocytes lacked desmosomal and cytoskeletal proteins required for epidermal integrity and exhibited excess MAP kinase signaling, which modulates keratinocyte adhesion and differentiation. Immunostaining patient biopsies substantiated these findings with lesions showing keratin deficiency, cadherin mis-localization, and ERK hyper-phosphorylation. Dampening ERK activity with MEK inhibitors rescued adhesive protein expression and restored keratinocyte sheet integrity despite SERCA2 depletion or chemical inhibition. In sum, coupling multi-omic analysis with human organotypic epidermis as a pre-clinical model, we found that SERCA2 haploinsufficiency disrupts critical adhesive components in keratinocytes via ERK signaling and identified MEK inhibition as a treatment strategy for Darier disease.
Shivam A. Zaver, Mrinal K. Sarkar, Shaun Egolf, Jonathan Zou, Afua Tiwaa, Brian C. Capell, Johann E. Gudjonsson, Cory L. Simpson
Acyl-CoA thioesterase 1 (ACOT1) catalyzes the hydrolysis of long-chain acyl-CoAs to free fatty acids and coenzyme A and is typically upregulated in obesity. Whether targeting ACOT1 in the setting of high-fat diet induced obesity would be metabolically beneficial is not known. Here we report that male and female ACOT1KO mice are partially protected from high-fat diet induced obesity, an effect associated with increased energy expenditure without alterations in physical activity or food intake. In males, ACOT1 deficiency increased mitochondrial uncoupling protein-2 (UCP2) protein abundance, while reducing 4-hydroxynonenal (4-HNE), a marker of oxidative stress, in white adipose tissue and liver of high-fat fed mice. Moreover, concurrent knockdown of UCP2 with ACOT1 in hepatocytes prevented increases in oxygen consumption observed with ACOT1 knockdown during high lipid loading, suggesting that UCP2-induced uncoupling may increase energy expenditure to attenuate weight gain. Together, these data indicate that targeting ACOT1 may be effective for obesity prevention during caloric excess by increasing energy expenditure.
Timothy D. Heden, Mallory P. Franklin, Christina Dailey, Mara T. Mashek, Chen Chen, Douglas G. Mashek
Metabolic mechanisms underlying the heterogeneity of major adverse cardiovascular events (MACE) risk in individuals with type 2 diabetes mellitus (T2D) remain unclear. We hypothesized that circulating metabolites reflecting mitochondrial dysfunction predict incident MACE in T2D. Targeted mass-spectrometry profiling of 60 metabolites was performed on baseline plasma from TECOS (discovery) and EXSCEL (validation) trial biomarker substudy cohorts. A principal components analysis metabolite factor comprised of medium-chain acylcarnitines was associated with MACE in TECOS and validated in EXSCEL, with higher levels associated with higher MACE risk. Meta-analysis showed that long-chain acylcarnitines and dicarboxylacylcarnitines were also associated with MACE. Metabolites remained associated with MACE in multivariate models and favorably changed with exenatide therapy. A third cohort (CATHGEN) with T2D assessed whether these metabolites improved discriminative capability multivariate for MACE; nine metabolites (medium- and long-chain acylcarnitines and one dicarboxylacylcarnitine) were associated with time-to-MACE in CATHGEN. Addition of these metabolites to clinical models minimally improved the discriminative capability for MACE but did significantly down reclassify risk. Thus, metabolites reporting on dysregulated mitochondrial fatty acid oxidation are higher in individuals with T2D who experience subsequent MACE. These biomarkers may improve CV risk prediction models, be therapy responsive, and highlight emerging risk mechanisms.
Jessica A. Regan, Robert J. Mentz, Maggie Nguyen, Jennifer B. Green, Lauren K. Truby, Olga Ilkayeva, Christopher Newgard, John B. Buse, Harald Sourij, C. David Sjöström, Naveed Sattar, Robert W. McGarrah, Yinggan Zheng, Darren K. McGuire, Eberhard Standl, Paul Armstrong, Eric Peterson, Adrian Hernandez, Rury R. Holman, Svati H. Shah
The growth of skeletal muscle relies on a delicate equilibrium between protein synthesis and degradation; however, how proteostasis is managed in the endoplasmic reticulum is largely unknown. Here, we report that the SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD) complex, the primary molecular machinery that degrades misfolded proteins in the ER is vital to maintain postnatal muscle growth and systemic energy balance. Myocyte-specific SEL1L deletion blunts the hypertrophic phase of muscle growth, resulting in a net zero gain of muscle mass during this developmental period and a 30% reduction in overall body growth. In addition, myocyte-specific SEL1L deletion triggered a systemic reprogramming of metabolism characterized by improved glucose sensitivity, enhanced beiging of adipocytes, and resistance to diet induced obesity. These effects were partially mediated by the upregulation of the myokine FGF21. These findings highlight the pivotal role of SEL1L-HRD1 ERAD activity in skeletal myocytes for postnatal muscle growth, and its physiological integration in maintaining whole-body energy balance.
Benedict Abdon, Yusheng Liang, Débora da Luz Scheffer, Mauricio Torres, Neha Shrestha, Rachel B. Reinert, You Lu, Brent Pederson, Amara Bugarin-Lapuz, Sander Kersten, Ling Qi
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