Demyelination and axonal injury in chronic-progressive Multiple Sclerosis (MS) are presumed to be driven by a neurotoxic bystander effect of meningeal-based myeloid infiltrates. There is an unmet clinical need to attenuate disease progression in such forms of CNS-compartmentalized MS. The failure of systemic immune suppressive treatments has highlighted the need for neuroprotective and repair-inducing strategies. Here, we examined whether direct targeting of CNS myeloid cells and modulating their toxicity may prevent irreversible tissue injury in chronic immune-mediated demyelinating disease. To that end, we utilized the experimental autoimmune encephalomyelitis (EAE) model in Biozzi mice, a clinically relevant MS model. We continuously delivered intracerebroventricularly (ICV) a retinoic acid receptor alpha agonist (RARα), as a potent regulator of myeloid cells, in the chronic phase of EAE. We assessed disease severity and performed pathological evaluations, functional analyses of immune cells, and single-cell RNA sequencing on isolated spinal CD11b+ cells. Although initiating treatment in the chronic phase of the disease, the RARα agonist successfully improved clinical outcomes and prevented axonal loss. ICV RARα agonist treatment inhibited pro-inflammatory pathways and shifted CNS myeloid cells toward neuroprotective phenotypes without affecting peripheral infiltrating myeloid cell phenotypes, or peripheral immunity. The treatment regulated cell-death pathways across multiple myeloid cell populations and suppressed apoptosis, resulting in paradoxically marked increased neuroinflammatory infiltrates, consisting mainly of microglia and CNS / border-associated macrophages. This work establishes the notion of bystander neurotoxicity by CNS immune infiltrates in chronic demyelinating disease. Furthermore, it shows that targeting compartmentalized neuroinflammation by selective regulation of CNS myeloid cell toxicity and survival reduces irreversible tissue injury, and may serve as a novel disease-modifying approach.

This study sheds light on the central role of adenine nucleotide translocase 2 (ANT2) in the pathogenesis of obesity-induced CKD. Our data demonstrate that ANT2 depletion in renal proximal tubule cells (RPTCs) leads to a shift in their primary metabolic program from fatty acid oxidation to aerobic glycolysis, resulting in mitochondrial protection, cellular survival, and preservation of renal function. These findings provide new insights into the underlying mechanisms of obesity-induced CKD and have the potential to be translated toward the development of targeted therapeutic strategies for this debilitating condition.

The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.

The MAPK p38α was proposed to be a prominent promoter of skeletal muscle aging. The skeletal muscle tissue is composed of various muscle types, and it is not known if p38α is associated with aging in all of them. It is also not known if p38α is associated with aging of other tissues. JNK and ERK were also proposed to be associated with aging of several tissues. Nevertheless, the pattern of p38α, JNK, and ERK activity during aging was not documented. Here, we documented the levels of phosphorylated/active p38α, Erk1/2, and JNKs in several organs as well as the soleus, tibialis anterior, quadriceps, gastrocnemius, and EDL muscles of 1-, 3-, 6-, 13-, 18-, and 24-month-old mice. We report that in most tissues and skeletal muscles, the MAPKs' activity does not change in the course of aging. In most tissues and muscles, p38α is in fact active at younger ages. The quadriceps and the lungs are exceptions, where p38α is significantly active only in mice 13 months old or older. Curiously, levels of active JNK and ERKs are also elevated in aged lungs and quadriceps. RNA-seq analysis of the quadriceps during aging revealed downregulation of proteins related to the extra-cellular matrix (ECM) and ERK signaling. A panel of mRNAs encoding cell cycle inhibitors and senescence-associated proteins, considered to be aging markers, was not found to be elevated. It seems that the pattern of MAPKs' activation in aging, as well as expression of known 'aging' components, are tissue- and muscle type-specific, supporting a notion that the process of aging is tissue- and even cell-specific.

The postnatal interaction between microbiota and the immune system establishes lifelong homeostasis at mucosal epithelial barriers, however, the barrier-specific physiological activities that drive the equilibrium are hardly known. During weaning, the oral epithelium, which is monitored by Langerhans cells (LC), is challenged by the development of a microbial plaque and the initiation of masticatory forces capable of damaging the epithelium. Here we show that microbial colonization following birth facilitates the differentiation of oral LCs, setting the stage for the weaning period, in which adaptive immunity develops. Despite the presence of the challenging microbial plaque, LCs mainly respond to masticatory mechanical forces, inducing adaptive immunity, to maintain epithelial integrity that is also associated with naturally occurring alveolar bone loss. Mechanistically, masticatory forces induce the migration of LCs to the lymph nodes, and in return, LCs support the development of immunity to maintain epithelial integrity in a microbiota-independent manner. Unlike in adult life, this bone loss is IL-17-independent, suggesting that the establishment of oral mucosal homeostasis after birth and its maintenance in adult life involve distinct mechanisms.

Recent studies have highlighted the therapeutic potential of small extracellular bodies derived from mesenchymal stem cells (MSC-sEVs) for various diseases, notably through their ability to alter T-cell differentiation and function. The current study aimed to explore immunomodulatory pathway alterations within T cells through mRNA sequencing of activated T cells cocultured with bone marrow-derived MSC-sEVs. mRNA profiling of activated human T cells cocultured with MSC-sEVs or vehicle control was performed using the QIAGEN Illumina sequencing platform. Pathway networks and biological functions of the differentially expressed genes were analyzed using Ingenuity pathway analysis (IPA) software, KEGG pathway, GSEA and STRING database. A total of 364 differentially expressed genes were identified in sEV-treated T cells. Canonical pathway analysis highlighted the RhoA signaling pathway. Cellular development, movement, growth and proliferation, cell-to-cell interaction and inflammatory response-related gene expression were altered. KEGG enrichment pathway analysis underscored the apoptosis pathway. GSEA identified enrichment in downregulated genes associated with TNF alpha and interferon gamma response, and upregulated genes related to apoptosis and migration of lymphocytes and T-cell differentiation gene sets. Our findings provide valuable insights into the mechanisms by which MSC-sEVs implement immunomodulatory effects on activated T cells. These findings may contribute to the development of MSC-sEV-based therapies.

Non-alcoholic steatohepatitis (NASH) is an aggressive form of fatty liver disease with hepatic inflammation and fibrosis for which there is currently no drug treatment. This study determined whether an indoline derivative, AN1284, which significantly reduced damage in a model of acute liver disease, can reverse steatosis and fibrosis in mice with pre-existing NASH and explore its mechanism of action. The mouse model of dietary-induced NASH reproduces most of the liver pathology seen in human subjects. This was confirmed by RNA-sequencing analysis. The Western diet, given for 4 months, caused steatosis, inflammation, and liver fibrosis. AN1284 (1 mg or 5 mg/kg/day) was administered for the last 2 months of the diet by micro-osmotic-pumps (mps). Both doses significantly decreased hepatic damage, liver weight, hepatic fat content, triglyceride, serum alanine transaminase, and fibrosis. AN1284 (1 mg/kg/day) given by mps or in the drinking fluid significantly reduced fibrosis produced by carbon tetrachloride injections. In human HUH7 hepatoma cells incubated with palmitic acid, AN1284 (2.1 and 6.3 ng/ml), concentrations compatible with those in the liver of mice treated with AN1284, decreased lipid formation by causing nuclear translocation of the aryl hydrocarbon receptor (AhR). AN1284 downregulated fatty acid synthase (FASN) and sterol regulatory element-binding protein 1c (SREBP-1c) and upregulated Acyl-CoA Oxidase 1 and Cytochrome P450-a1, genes involved in lipid metabolism. In conclusion, chronic treatment with AN1284 (1mg/kg/day) reduced pre-existing steatosis and fibrosis through AhR, which affects several contributors to the development of fatty liver disease. Additional pathways are also influenced by AN1284 treatment.

Thermal intolerance may limit activity in hostile environments. After heat illness, two physiologically distinct phenotypes evolve: heat tolerant (HT) and heat intolerant (HI). The recognition that heat illness alters gene expression justified revisiting the established physiological concept of HI. We used a DNA microarray to examine the global transcriptional response in peripheral blood mononuclear cells (PMBCs) from HI and HT phenotypes, categorized 2-mo postheat injury using a functional physiological heat-tolerance test (HTT, 40°C)-Recovery (R, 24°C) protocol. The impact of recurrent heat stress was studied in vitro using peripheral blood mononuclear cells (PBMCs) from controls (participants with no history of heat injury), HI, and HT (categorized by functional HTT) with a customized NanoString array. There were significant differences under basal conditions between the HI and HT. HI were more immunological alerted. Almost no shared genes were found between end-HTT and recovery phases, suggesting vast cellular plasticity. In HI, mitochondrial function was dysregulated, canonical pathways associated with exercise endurance-NRF2 and insulin were downregulated, whereas AMPK and peroxisome proliferator-activated receptor (PPAR) were upregulated. HT exhibited reciprocal responses, suggesting that energy dysregulation found in HI interfered with performance in the heat. The endoplasmic-reticulum stress response was also suppressed in HI. In vitro HTT (43°C) abolished differences between HI and HT PBMCs including the HSPs genes, whereas controls showed profound HSPs upregulation.

Systemic light chain amyloidosis (AL) is a clonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin light chains (LC) as insoluble fibrils in organs. The lack of suitable models has hindered the investigation of the disease mechanisms. Our aim was to establish AL producing PC lines and to use them to investigate the biology of the amyloidogenic clone. We used lentiviral vectors to generate cell lines expressing LCs from patients suffering from AL amyloidosis. The AL LC producing cell lines showed a significant decrease in proliferation, cell cycle arrest, and an increase in apoptosis and autophagy as compared with the multiple myeloma (MM) LC producing cells. Using RNAsequencing the AL LC producing lines showed higher mitochondrial oxidative stress, decreased activity of the myc and cholesterol pathways. The neoplastic behavior of PCs is altered by the constitutive expression of amyloidogenic LC causing intracellular toxicity. This observation may explain the disparity in the malignant behavior of the amyloid clone compared to the myeloma clone. These findings should enable future in vitro studies and help delineate AL's unique cellular pathways, thus expediting the development of specific treatments for AL patients.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been employed in the past decade as therapeutic agents in various diseases, including central nervous system (CNS) disorders. We currently aimed to use MSC-EVs as potential treatment for cerebral small vessel disease (CSVD), a complex disorder with a variety of manifestations. MSC-EVs were intranasally administrated to salt-sensitive hypertension prone SBH/y rats that were DOCA-salt loaded (SBH/y-DS), which we have previously shown is a model of CSVD. MSC-EVs accumulated within brain lesion sites of SBH/y-DS. An in vitro model of an inflammatory environment in the brain demonstrated anti-inflammatory properties of MSC-EVs. Following in vivo MSC-EV treatment, gene set enrichment analysis (GSEA) of SBH/y-DS cortices revealed downregulation of immune system response-related gene sets. In addition, MSC-EVs downregulated gene sets related to apoptosis, wound healing and coagulation, and upregulated gene sets associated with synaptic signaling and cognition. While no specific gene was markedly altered upon treatment, the synergistic effect of all gene alternations was sufficient to increase animal survival and improve the neurological state of affected SBH/y-DS rats. Our data suggest MSC-EVs act as microenvironment modulators, through various molecular pathways. We conclude that MSC-EVs may serve as beneficial therapeutic measure for multifactorial disorders, such as CSVD.

To combat the various DNA lesions and their harmful effects, cells have evolved different strategies, collectively referred as DNA damage response (DDR). The DDR largely relies on intranuclear protein networks, which sense DNA lesions, recruit DNA repair enzymes, and coordinates several aspects of the cellular response, including a temporary cell cycle arrest. In addition, external cues mediated by the surface EGF receptor (EGFR) through downstream signaling pathways contribute to the cellular DNA repair capacity. However, cell cycle progression driven by EGFR activation should be reconciled with cell cycle arrest necessary for effective DNA repair. Here, we show that in damaged cells, the expression of Mig-6 (mitogen-inducible gene 6), a known regulator of EGFR signaling, is reduced resulting in heightened EGFR phosphorylation and downstream signaling. These changes in Mig-6 expression and EGFR signaling do not occur in cells deficient of Mre-11, a component of the MRN complex, playing a central role in double-strand break (DSB) repair or when cells are treated with the MRN inhibitor, mirin. RNAseq and functional analysis reveal that DNA damage induces a shift in cell response to EGFR triggering that potentiates DDR-induced p53 pathway and cell cycle arrest. These data demonstrate that the cellular response to EGFR triggering is skewed by components of the DDR, thus providing a plausible explanation for the paradox of the known role played by a growth factor such as EGFR in the DNA damage repair.

Oogenesis produces functional eggs and is essential for fertility, embryonic development, and reproduction. The zebrafish ovary is an excellent model to study oogenesis in vertebrates, and recent studies have identified multiple regulators in oocyte development through forward genetic screens, as well as reverse genetics by CRISPR mutagenesis. However, many developmental steps in oogenesis, in zebrafish and other species, remain poorly understood, and their underlying mechanisms are unknown. Here, we take a genomic approach to systematically uncover biological activities throughout oogenesis. We performed transcriptomic analysis on five stages of oogenesis, from the onset of oocyte differentiation through Stage III, which precedes oocyte maturation. These transcriptomes revealed thousands of differentially expressed genes across stages of oogenesis. We analyzed trends of gene expression dynamics along oogenesis, as well as their expression in pair-wise comparisons between stages. We determined their functionally enriched terms, identifying uniquely characteristic biological activities in each stage. These data identified two prominent developmental phases in oocyte differentiation and traced the accumulation of maternally deposited embryonic regulator transcripts in the developing oocyte. Our analysis provides the first molecular description for oogenesis in zebrafish, which we deposit online as a resource for the community. Further, the presence of multiple gene paralogs in zebrafish, and the exclusive curation by many bioinformatic tools of the single paralogs present in humans, challenge zebrafish genomic analyses. We offer an approach for converting zebrafish gene name nomenclature to the human nomenclature for supporting genomic analyses generally in zebrafish. Altogether, our work provides a valuable resource as a first step to uncover oogenesis mechanisms and candidate regulators and track accumulating transcripts of maternal regulators of embryonic development.

BACKGROUNDCytomegalovirus (CMV) is the most common intrauterine infection, leading to infant brain damage. Prognostic assessment of CMV-infected fetuses has remained an ongoing challenge in prenatal care, in the absence of established prenatal biomarkers of congenital CMV (cCMV) infection severity. We aimed to identify prognostic biomarkers of cCMV-related fetal brain injury.METHODSWe performed global proteome analysis of mid-gestation amniotic fluid samples, comparing amniotic fluid of fetuses with severe cCMV with that of asymptomatic CMV-infected fetuses. The levels of selected differentially excreted proteins were further determined by specific immunoassays.RESULTSUsing unbiased proteome analysis in a discovery cohort, we identified amniotic fluid proteins related to inflammation and neurological disease pathways, which demonstrated distinct abundance in fetuses with severe cCMV. Amniotic fluid levels of 2 of these proteins - the immunomodulatory proteins retinoic acid receptor responder 2 (chemerin) and galectin-3-binding protein (Gal-3BP) - were highly predictive of the severity of cCMV in an independent validation cohort, differentiating between fetuses with severe (n = 17) and asymptomatic (n = 26) cCMV, with 100%-93.8% positive predictive value, and 92.9%-92.6% negative predictive value (for chemerin and Gal-3BP, respectively). CONCLUSIONAnalysis of chemerin and Gal-3BP levels in mid-gestation amniotic fluids could be used in the clinical setting to profoundly improve the prognostic assessment of CMV-infected fetuses.FUNDINGIsrael Science Foundation (530/18 and IPMP 3432/19); Research Fund - Hadassah Medical Organization.

BACKGROUND: The dorsal domain of the neural tube is an excellent model to investigate the generation of complexity during embryonic development. It is a highly dynamic and multifaceted region being first transiently populated by prospective neural crest (NC) cells that sequentially emigrate to generate most of the peripheral nervous system. Subsequently, it becomes the definitive roof plate (RP) of the central nervous system. The RP, in turn, constitutes a patterning center for dorsal interneuron development. The factors underlying establishment of the definitive RP and its segregation from NC and dorsal interneurons are currently unknown. RESULTS: We performed a transcriptome analysis at trunk levels of quail embryos comparing the dorsal neural tube at premigratory NC and RP stages. This unraveled molecular heterogeneity between NC and RP stages, and within the RP itself. By implementing these genes, we asked whether Notch signaling is involved in RP development. First, we observed that Notch is active at the RP-interneuron interface. Furthermore, gain and loss of Notch function in quail and mouse embryos, respectively, revealed no effect on early NC behavior. Constitutive Notch activation caused a local downregulation of RP markers with a concomitant development of dI1 interneurons, as well as an ectopic upregulation of RP markers in the interneuron domain. Reciprocally, in mice lacking Notch activity, both the RP and dI1 interneurons failed to form and this was associated with expansion of the dI2 population. CONCLUSIONS: Collectively, our results offer a new resource for defining specific cell types, and provide evidence that Notch is required to establish the definitive RP, and to determine the choice between RP and interneuron fates, but not the segregation of RP from NC.

Hepatocellular carcinoma (HCC) typically develops on a background of chronic hepatitis for which the proinflammatory cytokine IL6 is conventionally considered a crucial driving factor. Paradoxically, IL6 also acts as a hepatoprotective factor in chronic liver injury. Here we used the multidrug-resistant gene 2 knockout (Mdr2(-/-)) mouse model to elucidate potential roles of IL6 in chronic hepatitis-associated liver cancer. Long-term analysis of three separate IL6/Stat3 signaling-deficient Mdr2(-/-) strains revealed aggravated liver injury with increased dysplastic nodule formation and significantly accelerated tumorigenesis in all strains. Tumorigenesis in the IL6/Stat3-perturbed models was strongly associated with enhanced macrophage accumulation and hepatosteatosis, phenotypes of nonalcoholic steatohepatitis (NASH), as well as with significant reductions in senescence and the senescence-associated secretory phenotype (SASP) accompanied by increased hepatocyte proliferation. These findings reveal a crucial suppressive role for IL6/Stat3 signaling in chronic hepatitis-associated hepatocarcinogenesis by impeding protumorigenic NASH-associated phenotypes and by reinforcing the antitumorigenic effects of the SASP. SIGNIFICANCE: These findings describe a context-dependent role of IL6 signaling in hepatocarcinogenesis and predict that increased IL6-neutralizing sgp130 levels in some patients with NASH may herald early HCC development.See related commentary by Huynh and Ernst, p. 4671.