Multiple Cell Types Research Articles

Immune and inflammatory insights in atherosclerosis: development of a risk prediction model through single-cell and bulk transcriptomic analyses.

Investigation into the immune heterogeneity linked with atherosclerosis remains understudied. This knowledge gap hinders the creation of a robust theoretical framework essential for devising personalized immunotherapies aimed at combating this disease. Single-cell RNA sequencing (scRNA-seq) analysis was employed to delineate the immune cell-type landscape within atherosclerotic plaques, followed by assessments of cell-cell interactions and phenotype characteristics using scRNA-seq datasets. Subsequently, pseudotime trajectory analysis was utilized to elucidate the heterogeneity in cell fate and differentiation among macrophages. Through integrated approaches, including single-cell sequencing, Weighted Gene Co-expression Network Analysis (WGCNA), and machine learning techniques, we identified hallmark genes. A risk score model and a corresponding nomogram were developed and validated using these genes, confirmed through Receiver Operating Characteristic (ROC) curve analysis. Additionally, enrichment and immune characteristic analyses were conducted based on the risk score model. The model's applicability was further corroborated by in vitro and in vivo validation of specific genes implicated in atherosclerosis. This comprehensive scRNA-seq analysis has shed new light on the intricate immune landscape and the role of macrophages in atherosclerotic plaques. The presence of diverse immune cell populations, with a particularly enriched macrophage population, was highlighted by the results. Macrophage heterogeneity was intricately characterized, revealing four distinct subtypes with varying functional attributes that underscore their complex roles in atherosclerotic pathology. Intercellular communication analysis revealed robust macrophage interactions with multiple cell types and detailed pathways differing between proximal adjacent and atherosclerotic core groups. Furthermore, pseudotime trajectories charted the developmental course of macrophage subpopulations, offering insights into their differentiation fates within the plaque microenvironment. The use of machine learning identified potential diagnostic markers, culminating in the identification of RNASE1 and CD14. The risk score model based on these biomarkers exhibited high accuracy in diagnosing atherosclerosis. Immune characteristic analysis validated the risk score model's efficacy in defining patient profiles, distinguishing high-risk individuals with pronounced immune cell activities. Finally, experimental validation affirmed RNASE1's involvement in atherosclerotic progression, suggesting its potential as a therapeutic target. Our findings have advanced our understanding of atherosclerosis immunopathology and paved the way for novel diagnostic and therapeutic strategies.

Transcriptional Patterns in Stages of Alzheimer's Disease Are Cell-Type-Specific and Partially Converge with the Effects of Alcohol Use Disorder in Humans.

Advances in single-cell technologies have led to the discovery and characterization of new brain cell types, which in turn lead to a better understanding of the pathogenesis of Alzheimer's disease (AD). Here, we present a detailed analysis of single-nucleus (sn)RNA-seq data for three stages of AD from middle temporal gyrus and compare it with snRNA-seq data from the prefrontal cortices from individuals with alcohol use disorder (AUD). We observed a significant decrease in both inhibitory and excitatory neurons, in general agreement with previous reports. We observed several cell-type-specific gene expressions and pathway dysregulations that delineate AD stages. Endothelial and vascular leptomeningeal cells showed the greatest degree of gene expression changes. Cell-type-specific evidence of neurodegeneration was seen in multiple neuronal cell types particularly in somatostatin and Layer 5 extratelencephalic neurons, among others. Evidence of inflammatory responses was seen in non-neuronal cells, particularly in intermediate and advanced AD. We observed common perturbations in AD and AUD, particularly in pathways, like transcription, translation, apoptosis, autophagy, calcium signaling, neuroinflammation, and phosphorylation, that imply shared transcriptional pathogenic mechanisms and support the role of excessive alcohol intake in AD progression. Major AUD gene markers form and perturb a network of genes significantly associated with intermediate and advanced AD. Master regulator analysis from AUD gene markers revealed significant correlation with advanced AD of transcription factors that have implications in intellectual disability, neuroinflammation, and other neurodegenerative conditions, further suggesting a shared nexus of transcriptional changes between AD and AUD.

Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

BackgroundThe IL-6 cytokine family, with its crucial and pleiotropic intracellular signaling pathway STAT3, is a promising target for treating vasoproliferative retinal diseases. Previous research has shown that IL-6 cis-signaling (via membrane-bound receptors) and trans-signaling (via soluble receptors) can have distinct effects on target cells, leading to their application in various disease treatments. While IL-6 has been extensively studied, less is known about the angiogenic effects of IL-11, another member of the IL-6 family, in the retina. Therefore, the aim of this study was to characterize the effects of IL-11 on retinal angiogenesis.Main textIn vitreous samples from proliferative diabetic retinopathy (PDR) patients, elevated levels of IL-11Rα, but not IL-11, were detected. In vitro studies using vascular endothelial cells revealed distinct effects of cis- and trans-signaling: cis-signaling (IL-11 alone) had antiangiogenic effects, while trans-signaling (IL-11 + sIL-11Rα) had proangiogenic and pro-migratory effects. These differences can be attributed to their individual signaling responses and associated transcriptomic changes. Notably, no differences in cis- and trans-signaling were detected in primary mouse Müller cell cultures. STAT3 and STAT1 siRNA knockdown experiments revealed opposing effects on IL-11 signaling, with STAT3 functioning as an antiproliferative and proapoptotic player while STAT1 acts in opposition to STAT3. In vivo, both IL-11 and IL-11 + sIL-11Rα led to a reduction in retinal neovascularization. Immunohistochemical staining revealed Müller cell activation in response to treatment, suggesting that IL-11 affects multiple retinal cell types in vivo beyond vascular endothelial cells.ConclusionsCis- and trans-signaling by IL-11 have contrasting angiomodulatory effects on endothelial cells in vitro. In vivo, cis- and trans-signaling also influence Müller cells, ultimately determining the overall angiomodulatory impact on the retina, highlighting the intricate interplay between vascular and glial cells in the retina.

Retention of ES cell-derived 129S genome drives NLRP1 hypersensitivity and transcriptional deregulation in Nlrp3tm1Flv mice

Immune response genes are highly polymorphic in humans and mice, with heterogeneity amongst loci driving strain-specific host defence responses. The inadvertent retention of polymorphic loci can introduce confounding phenotypes, leading to erroneous conclusions, and impeding scientific advancement. In this study, we employ a combination of RNAseq and variant calling analyses to identify a substantial region of 129S genome, including the highly polymorphic Nlrp1 locus, proximal to Nlrp3, in one of the most commonly used mouse models of NLRP3 deficiency (Nlrp3tm1Flv). We show that the presence of the Nlrp1129S locus leads to an increase in NLRP1B protein expression, and a sensitising of Nlrp3tm1Flv macrophages to NLRP1 inflammasome activation, independent of NLRP3 deficiency. Retention of 129S genome further leads to protein sequence differences and altered gene regulation across multiple cell types, including of the key tissue-resident macrophage marker, TIM4. Using alternative models of NLRP3 deficiency, including a previously undescribed conditional Nlrp3 allele enabling precise temporal and cell-type specific control over Nlrp3 deletion, we further show that NLRP3 contributes to Talabostat-driven IL-1β release. Our study also establishes a generic framework to identify functionally relevant SNPs and assess genomic contamination in transgenic mice using RNAseq data. This allows for unambiguous attribution of phenotypes to the target gene and advances the precision and reliability of research in the field of host defence responses.

Atomic vacancies of molybdenum disulfide nanoparticles stimulate mitochondrial biogenesis

Diminished mitochondrial function underlies many rare inborn errors of energy metabolism and contributes to more common age-associated metabolic and neurodegenerative disorders. Thus, boosting mitochondrial biogenesis has been proposed as a potential therapeutic approach for these diseases; however, currently we have a limited arsenal of compounds that can stimulate mitochondrial function. In this study, we designed molybdenum disulfide (MoS2) nanoflowers with predefined atomic vacancies that are fabricated by self-assembly of individual two-dimensional MoS2 nanosheets. Treatment of mammalian cells with MoS2 nanoflowers increased mitochondrial biogenesis by induction of PGC-1α and TFAM, which resulted in increased mitochondrial DNA copy number, enhanced expression of nuclear and mitochondrial-DNA encoded genes, and increased levels of mitochondrial respiratory chain proteins. Consistent with increased mitochondrial biogenesis, treatment with MoS2 nanoflowers enhanced mitochondrial respiratory capacity and adenosine triphosphate production in multiple mammalian cell types. Taken together, this study reveals that predefined atomic vacancies in MoS2 nanoflowers stimulate mitochondrial function by upregulating the expression of genes required for mitochondrial biogenesis.

Abstract B035: Nanoparticle delivery of innate immune agonists combines with senescence- inducing agents to mediate T cell control of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma has quickly risen to become the 3rd leading cause of cancer- related death. This is in part due to its fibrotic tumor microenvironment (TME) that contributes to poor vascularization and immune infiltration and subsequent chemo- and immunotherapy failure. Here we investigated an innovative immunotherapy approach combining delivery of STING and TLR4 innate immune agonists via lipid-based nanoparticles (NPs) co-encapsulation with senescence-inducing RAS-targeted therapies that can remodel the immune suppressive PDAC TME through the senescence-associated secretory phenotype. Treatment of transplanted and autochthonous PDAC mouse models with these regimens led to enhanced uptake of NPs by multiple cell types in the PDAC TME, induction of type I interferon and other pro-inflammatory signaling, increased antigen presentation by tumor cells and antigen presenting cells, and subsequent activation of both innate and adaptive immune responses. This two-pronged approach produced potent T cell-driven and Type I interferon-mediated tumor regressions and long-term survival in preclinical PDAC models dependent on both tumor and host STING activation. STING and TLR4-mediated Type I interferon signaling were also associated with enhanced NK and CD8+ T cell immunity in human PDAC. Thus, combining localized immune agonist delivery with systemic tumor-targeted therapy can synergize to orchestrate a coordinated Type I interferon-driven innate and adaptive immune assault to overcome immune suppression and activate durable anti-tumor T cell responses against PDAC. Citation Format: Kelly D DeMarco, Loretah Chibaya, Christina F Lusi, Griffin I Kane, Meghan L Brassil, Chaitanya N Parikh, Katherine C Murphy, Shreya R Chowdury, Junhui Li, Boyang Ma, Tiana E Taylor, Julia Cerrutti, Haruka Mori, Miranda Diaz-Infante, Jessica Peura, Jason R Pitarresi, Lihua Julie Zhu, Katherine A Fitzgerald, Prabhani U Atukorale, Marcus Ruscetti. Nanoparticle delivery of innate immune agonists combines with senescence- inducing agents to mediate T cell control of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B035.

Abstract A045: Disrupting local immunosuppression through combination myCAF/myeloid targeting in pancreatic cancer

Abstract Immunotherapy has revolutionized clinical care for many cancers, yet the approach has not been fully realized in immunologically cold tumors like Pancreatic Ductal Adenocarcinoma (PDAC). One striking hallmark of PDAC is local immunosuppression (LIS) which leads to cytotoxic T cell inactivation and exclusion. There is great need for an increased understanding of the cellular crosstalk within tumors and the identification of stromal and immune populations involved in shaping the tumor microenvironment (TME). Oncogenic KRAS activation in tumor cells promotes the invasion and proliferation of tumor-supporting stromal cells, while excluding cancer-targeted cytotoxic T cells. LIS is mediated by multiple subtypes of cancer-associated fibroblasts (CAFs) and myeloid cells resident within the tumor parenchyma. Multiple prior attempts to reverse LIS in PDAC by targeting individual stromal cell populations have been unsuccessful, alluding to the complexity of stromal crosstalk within the TME. The stromal diversity of PDAC complicates investigating paracrine cascades involving multiple cell types. To decipher diverse drug effects on altering the TME, we employ in vivo studies in mouse models recapitulating the human disease, as well as a novel tumor explant model that enables the short-term culture of intact human or murine PDAC. Importantly, PDAC explants maintain their histopathological architecture and cellular diversity over time. This medium-throughput platform allows for testing of multiple drugs and mechanistic hypotheses in the native PDAC TME. We show in preliminary data that Smoothened inhibition (SMOi) decreases proliferation and activity of myCAFs, but provokes the expansion of CD11b-positive myeloid cells in vivo. Thus, we hypothesize that LIS in PDAC is maintained by a delicate balance between myCAFs and myeloid cells, preventing effective T cell invasion. Single cell RNA-seq data comparing ctrl vs. SMOi-treated murine PDAC elucidates stromal subpopulations involved in the LIS phenotype and guides the identification of myeloid subtypes emerging after SMOi. Strikingly, we demonstrated that simultaneous SMOi and targeting myeloid cells via anti-Gr1 or CCR1 inhibition (CCR1i) significantly elevates cytotoxic T cell numbers up to 20x within the TME. We are currently investigating whether the activity of these T cells may be further potentiated through combination with immunomodulatory agents. By testing various treatment combination in the same TME, we will identify the best synergistic effects for future immunotherapy approaches in human PDAC. In summary, we are elucidating the complex mechanism behind LIS in PDAC by employing our novel explant culture system alongside in vivo studies. We aim to develop a translatable regimen to neutralize LIS, reactivating the cytotoxic T cells in the tumor periphery to invade, proliferate, and attack cancer cells. Citation Format: Marie C Hasselluhn, Lukas Vlahos, Dafydd Thomas, Quin T Waterbury, Alvaro Curiel Garcia, Amanda R Decker-Farrell, Tanner C Dalton, Stephen A Sastra, Carmine F Palermo, Timothy C Wang, Andrea Califano, Kenneth P Olive. Disrupting local immunosuppression through combination myCAF/myeloid targeting in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A045.

HFD feeding for seven months abolishes STING disruption-driven but not female sex-based protection against hepatic steatosis and inflammation in mice

Stimulator of interferon genes (STING) is positively correlated with the degrees of liver inflammation in human metabolic dysfunction-associated steatotic liver disease (MASLD). In addition, STING disruption alleviates MASLD in mice fed a high-fat diet (HFD) for 3 months (3-m-HFD). Here we investigated the role of the duration of dietary feeding in regulating MASLD in mice and explored the involvement of STING in sex differences in MASLD. Both male and female STING-disrupted (STINGgt) and wild-type C57BL/6J mice were fed an HFD for 3 or 7 months (7-m-HFD). Additionally, female STINGgt mice upon ovariectomy (OVX) and 3-m-HFD were analyzed for MASLD. Upon 3-m-HFD, STINGgt mice exhibited decreased severity of MASLD compared to control. However, upon 7-m-HFD, STINGgt mice were comparable with wild-type mice in body weight, fat mass, and MASLD. Regarding regulating the liver RNA transcriptome, 7-m-HFD increased the expression of genes indicating proinflammatory activation of various liver cells. Interestingly, the severity of MASLD in female mice was much lighter than in male mice, regardless of STING disruption. Upon OVX, female STINGgt mice showed significantly increased severity of MASLD relative to sham control but were comparable with male STINGgt mice. Upon treatment with 17-beta estradiol (E2), hepatocytes revealed decreased fat deposition while macrophages displayed decreases in lipopolysaccharide-induced phosphorylation of Nfkb p65 and Jnk p46 independent of STING. These results suggest that 7-m-HFD, without altering female sex-based protection, abolishes STING disruption-driven protection of MASLD, likely through causing proinflammatory activation of multiple types of liver cells to offset the effect of STING disruption.

166 Development of non-model organoids in animal research: A paradigm shift in in vitro models

Abstract For decades, two-dimensional (2D) cell cultures have been used as the gold standard in vitro models for basic research, drug discovery and preclinical screening applications. However, since 2009, the field has quickly shifted towards the use of three-dimensional (3D) stem cell-derived organoids. Organoids offer a variety of advantages compared with conventional 2D models, including being self-organized, retaining their cellular polarity, and being simplified versions of organs in vitro. Adult stem cells can self-renew, differentiate into multiple cell types, and are genomically stable over multiple passage lines, providing a variety of technical benefits and offering the closest recapitulation of the in vivo environment. Our laboratory has focused on expanding, characterizing, and utilizing animal-derived 3D organoid models, specifically focusing on canines; but also including other animals such as swine, turtles, snakes, and squirrels to study their unique adaptations. Practically, we can use 3D canine colonoids for the screening of anti-inflammatory candidates using our tumor necrosis factor (TNF)-α stimulation model. Additionally, we have grown and used swine lung organoids as a model for studying H1N1 viral uptake. Furthermore, we have grown and bio-banked tumor organoids/spheroids from a variety of canine cancers, including urothelial carcinoma (derived from urine and biopsies) and nasal carcinoma, among others, with future applications in precision veterinary medicine and comparative oncology. To accomplish this goal, we have carried out in vitro screening of a variety of chemotherapies and combination therapies at different concentrations across patients to predict in vivo responses and demonstrate the predictive ability of the organoid model. Finally, our laboratory has derived and characterized a variety of healthy organoid lines from canines including the pancreas, lung, liver, bladder, colon, and pituitary to act as healthy controls in mechanistic studies looking at disease pathogenesis and for uses in basic biology. In summary, in vitro organoid models from various animal species and tissues can be used in a variety of basic and applied research fields, including virology, toxicology, and oncology, with the potential to broaden our scientific understanding of disease and overall improve human and veterinary medicine.

Differential restriction of chikungunya virus in primary human cardiac endothelial cells occurs at multiple steps in the viral life cycle.

Arthropod-borne viruses (arboviruses) constitute a significant ongoing public health threat, as the mechanisms of pathogenesis remain incompletely understood. Cardiovascular symptomatology is emerging as an important manifestation of arboviral infection. We have recently studied the cardiac tropism and mechanisms implicated in cardiac damage in mice for the alphavirus chikungunya virus (CHIKV), and we therefore sought to evaluate the cardiac tropism of other emerging alphaviruses and arboviruses. Using human primary cardiac cells, we found that arboviruses from diverse viral families were able to replicate within these cells. Interestingly, we noted that while the closely related alphavirus Mayaro virus (MAYV) could replicate to high titers in primary human cardiac microvascular endothelial cells, pulmonary, and brain endothelial cells, the Indian Ocean Lineage of CHIKV (CHIKV-IOL) was completely restricted in all endothelial cells tested. Upon further investigation, we discovered that this restriction occurs at both entry and egress stages. Additionally, we observed that compared to CHIKV, MAYV may antagonize or evade the innate immune response more efficiently in human cardiac endothelial cells to increase infection. Overall, this study explores the tropism of arboviruses in human primary cardiac cells and characterizes the strain-specific restriction of CHIKV-IOL in human endothelial cells. Further work is needed to understand how the differential restriction of alphaviruses in human endothelial cells impacts pathogenesis in a living model, as well as the specific host factors responsible.

Organization of a functional glycolytic metabolon on mitochondria for metabolic efficiency.

Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, and is crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyses reversible O-GlcNAcylation, a post-translational modification influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of the regulatory domain of HK1, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances the mitochondrial association with HK1, orchestrating glycolytic and mitochondrial ATP production. Mutation in HK1's O-GlcNAcylation site reduces ATP generation in multiple cell types, specifically affecting metabolic efficiency in neurons. This study reveals a previously unappreciated pathway that links neuronal metabolism and mitochondrial function through OGT and the formation of the glycolytic metabolon, providing potential strategies for tackling metabolic and neurological disorders.

Stem‐Cell‐Based Small‐Diameter Blood Vessels with 3D Printing

Cardiovascular disease has emerged as the leading cause of death worldwide. Since coronary arteries, carotid arteries, and other blood vessels are prone to narrowing, small‐diameter artificial blood channels offer a crucial solution for restoring blood flow. Ideal grafts must emulate the structure of natural blood vessels, possess adequate mechanical strength, ensure long‐term patency, and incorporate functional cells with minimal immunogenicity. Enhanced cell sources and engineering methods are vital for the creation of functional small‐diameter blood vessels (SDBVs). Among potential cell sources, stem cells stand out due to their ability to differentiate into multiple cell types, self‐renew, and exhibit low immunogenicity. Additionally, three‐dimensionally (3D) printed vascular stents have attracted widespread attention for their precision and controllable bioink application. The need for tissue‐engineered blood vessels is currently rising, and innovative design concepts integrating stem cells and 3D printing present promising solutions. Herein, the construction requirements of vascular grafts are reviewed, current status of using stem cells as a cell source and 3D printing as an engineering strategy is described, and prospects and challenges for the development of SDBVs in the medical field are discussed.

An Enhanced Retroviral Vector for Efficient Genetic Manipulation and Selection in Mammalian Cells.

Introducing genetic material into hard-to-transfect mammalian cell lines and primary cells is often best achieved through retroviral infection. An ideal retroviral vector should offer a compact, selectable, and screenable marker while maximizing transgene delivery capacity. However, a previously published retroviral vector featuring an EGFP/Puromycin fusion protein failed to meet these criteria in our experiments. We encountered issues such as low infection efficiency, weak EGFP fluorescence, and selection against infected cells. To address these shortcomings, we developed a novel retroviral vector based on the Moloney murine leukemia virus. This vector includes a compact bifunctional EGFP and Puromycin resistance cassette connected by a 2A peptide. Our extensively tested vector demonstrated superior EGFP expression, efficient Puromycin selection, and no growth penalty in infected cells compared with the earlier design. These benefits were consistent across multiple mammalian cell types, underscoring the versatility of our vector. In summary, our enhanced retroviral vector offers a robust solution for efficient infection, reliable detection, and effective selection in mammalian cells. Its improved performance and compact design make it an ideal choice for a wide range of applications involving precise genetic manipulation and characterization in cell-based studies.

Dexamethasone Impairs ATP Production and Mitochondrial Performance in Human Trabecular Meshwork Cells.

Mitochondrial damage occurs in human trabecular meshwork (HTM) cells as a result of normal aging and in open angle glaucoma. Using an HTM cell model, we quantified mitochondrial function and ATP generation rates after dexamethasone (Dex) and TGF-β2 treatments, frequently used as in vitro models of glaucoma. Primary HTM cells were assayed for metabolic function using a Seahorse XFp Analyzer. We additionally assessed the mitochondrial copy number and the expression of transcripts associated with mitochondrial biogenesis and oxidative stress regulation. Cells treated with Dex, but not TGF-β2, exhibited a significant decrease in total ATP production and ATP from oxidative phosphorylation relative to that of the control. Dex treatment also resulted in significant decreases in maximal respiration, ATP-linked O2 consumption, and non-mitochondrial O2 consumption. We did not observe significant changes in the level of mitochondrial genomes or mRNA transcripts of genes involved in mitochondrial biogenesis and oxidative stress regulation. Decreased mitochondrial performance and ATP production are consistent with the results of prior studies identifying the effects of Dex on multiple cell types, including HTM cells. Our results are also consistent with in vivo evidence of mitochondrial damage in open-angle glaucoma. Overall, these results demonstrate a decrease in mitochondrial performance in Dex-induced glaucomatous models in vitro, meriting further investigation.

Phenotypic Variation in Staphylococcus aureus during Colonisation Involves Antibiotic-Tolerant Cell Types.

Staphylococcus aureus is a bacterial species that is commonly found colonising healthy individuals but that presents a paradoxical nature: simultaneously, it can migrate within the body and cause a range of diseases. Many of these become chronic by resisting immune responses, antimicrobial treatment, and medical intervention. In part, this ability to persist can be attributed to the adoption of multiple cell types within a single cellular population. These dynamics in the S. aureus cell population could be the result of its interplay with host cells or other co-colonising bacteria-often coagulase-negative Staphylococcal (CoNS) species. Further understanding of the unique traits of S. aureus alternative cell types, the drivers for their selection or formation during disease, as well as their presence even during non-pathological colonisation could advance the development of diagnostic tools and drugs tailored to target specific cells that are eventually responsible for chronic infections.

Single-nucleus transcriptomic profiling of human orbitofrontal cortex reveals convergent effects of aging and psychiatric disease

Aging is a complex biological process and represents the largest risk factor for neurodegenerative disorders. The risk for neurodegenerative disorders is also increased in individuals with psychiatric disorders. Here, we characterized age-related transcriptomic changes in the brain by profiling ~800,000 nuclei from the orbitofrontal cortex from 87 individuals with and without psychiatric diagnoses and replicated findings in an independent cohort with 32 individuals. Aging affects all cell types, with LAMP5+LHX6+ interneurons, a cell-type abundant in primates, by far the most affected. Disrupted synaptic transmission emerged as a convergently affected pathway in aged tissue. Age-related transcriptomic changes overlapped with changes observed in Alzheimer’s disease across multiple cell types. We find evidence for accelerated transcriptomic aging in individuals with psychiatric disorders and demonstrate a converging signature of aging and psychopathology across multiple cell types. Our findings shed light on cell-type-specific effects and biological pathways underlying age-related changes and their convergence with effects driven by psychiatric diagnosis.

Sparsentan improves glomerular hemodynamics, cell functions, and tissue repair in a mouse model of FSGS.

Dual endothelin-1 (ET-1) and angiotensin II (AngII) receptor antagonism with sparsentan has strong antiproteinuric actions via multiple potential mechanisms that are more pronounced, or additive, compared with current standard of care using angiotensin receptor blockers (ARBs). Considering the many actions of ET-1 and AngII on multiple cell types, this study aimed to determine glomeruloprotective mechanisms of sparsentan compared to the ARB losartan by direct visualization of its effects in the intact kidney in focal segmental glomerulosclerosis (FSGS) using intravital multiphoton microscopy. In both healthy and FSGS models, sparsentan treatment increased afferent/efferent arteriole diameters; increased or preserved blood flow and single-nephron glomerular filtration rate; attenuated acute ET-1 and AngII-induced increases in podocyte calcium; reduced proteinuria; preserved podocyte number; increased both endothelial and renin lineage cells and clones in vasculature, glomeruli, and tubules; restored glomerular endothelial glycocalyx; and attenuated mitochondrial stress and immune cell homing. These effects were either not observed or of smaller magnitude with losartan. The pleiotropic nephroprotective effects of sparsentan included improved hemodynamics, podocyte and endothelial cell functions, and tissue repair. Compared with losartan, sparsentan was more effective in the sustained preservation of kidney structure and function, which underscores the importance of the ET-1 component in FSGS pathogenesis and therapy.

Recruitment of CXCR4+ type 1 innate lymphoid cells distinguishes sarcoidosis from other skin granulomatous diseases.

Sarcoidosis is a multiorgan granulomatous disease that lacks diagnostic biomarkers and targeted treatments. Using blood and skin from patients with sarcoid and non-sarcoid skin granulomas, we discovered that skin granulomas from different diseases exhibit unique immune cell recruitment and molecular signatures. Sarcoid skin granulomas were specifically enriched for type 1 innate lymphoid cells (ILC1s) and B cells and exhibited molecular programs associated with formation of mature tertiary lymphoid structures (TLSs), including increased CXCL12/CXCR4 signaling. Lung sarcoidosis granulomas also displayed similar immune cell recruitment. Thus, granuloma formation was not a generic molecular response. In addition to tissue-specific effects, patients with sarcoidosis exhibited an 8-fold increase in circulating ILC1s, which correlated with treatment status. Multiple immune cell types induced CXCL12/CXCR4 signaling in sarcoidosis, including Th1 T cells, macrophages, and ILCs. Mechanistically, CXCR4 inhibition reduced sarcoidosis-activated immune cell migration, and targeting CXCR4 or total ILCs attenuated granuloma formation in a noninfectious mouse model. Taken together, our results show that ILC1s are a tissue and circulating biomarker that distinguishes sarcoidosis from other skin granulomatous diseases. Repurposing existing CXCR4 inhibitors may offer a new targeted treatment for this devastating disease.

RAPIDASH: Tag-free enrichment of ribosome-associated proteins reveals composition dynamics in embryonic tissue, cancer cells, and macrophages

Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translation. Nevertheless, a lack of technologies to enrich RAPs across sample types has prevented systematic analysis of RAP identities, dynamics, and functions. We have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including Dhx30 and Llph, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development linked to the translation of genes with long coding sequences. In addition, we showed that RAPIDASH can identify ribosome changes in cancer cells. Finally, we characterized ribosome composition remodeling during immune cell activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs in multiple cell types, tissues, and stimuli and is adaptable to characterize ribosome remodeling in several contexts.

Lack of factor VIII detection in humans and dogs with an intron 22 inversion challenges hypothesis regarding inhibitor risk

BackgroundAlmost half of severe hemophilia A (HA) cases are caused by an intron 22 inversion (Int22Inv) mutation, which truncates the 26-exon F8 messenger RNA (mRNA) after exon 22. Another F8 transcript, F8B, is initiated from within F8-intron-22. F8B mRNA consists of a short exon spliced to exons 23 to 26 and is expressed in multiple human cell types. It has been hypothesized that Int22Inv patients have self-tolerance to partial factor (F)VIII proteins expressed from these 2 transcripts. FVIII is expressed in endothelial cells, primarily in the liver and lungs. Several studies have reported FVIII expression in other cell types, although this has been controversial. ObjectivesTo determine if partial FVIII proteins are expressed from intron 22–inverted and/or F8B mRNA and if FVIII is expressed in nonendothelial cells. MethodsA panel of FVIII-specific antibodies was validated and employed to label FVIII in cells and tissues and for immunoprecipitation followed by western blots and mass spectrometry proteomics analysis. ResultsImmunofluorescent staining localized FVIII to endothelial cells in liver sections from non-HA but not HA-Int22Inv dogs. Neither FVIII nor FVIIIB was detected in human peripheral blood mononuclear cells, B cell or T cell lines, or cell lines expanded from peripheral blood mononuclear cells, whereas FVIII antigen and activity were readily detected in primary nonhemophilic liver sinusoidal endothelial cells. ConclusionIf FVIII is expressed in nonendothelial cells or if partial FVIII proteins are expressed in HA-Int22Inv, the concentrations are below the detection limits of these sensitive assays. Our results argue against promotion of immune tolerance through expression of partial FVIII proteins in Int-22Inv patients.