Targeting CNS myeloid infiltrates provides neuroprotection in a progressive multiple sclerosis model.

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.

Recycled melanoma-secreted melanosomes regulate tumor-associated macrophage diversification.

Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as "second-hand" EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.

Senescence of human pancreatic beta cells enhances functional maturation through chromatin reorganization and promotes interferon responsiveness.

Senescent cells can influence the function of tissues in which they reside, and their propensity for disease. A portion of adult human pancreatic beta cells express the senescence marker p16, yet it is unclear whether they are in a senescent state, and how this affects insulin secretion. We analyzed single-cell transcriptome datasets of adult human beta cells, and found that p16-positive cells express senescence gene signatures, as well as elevated levels of beta-cell maturation genes, consistent with enhanced functionality. Senescent human beta-like cells in culture undergo chromatin reorganization that leads to activation of enhancers regulating functional maturation genes and acquisition of glucose-stimulated insulin secretion capacity. Strikingly, Interferon-stimulated genes are elevated in senescent human beta cells, but genes encoding senescence-associated secretory phenotype (SASP) cytokines are not. Senescent beta cells in culture and in human tissue show elevated levels of cytoplasmic DNA, contributing to their increased interferon responsiveness. Human beta-cell senescence thus involves chromatin-driven upregulation of a functional-maturation program, and increased responsiveness of interferon-stimulated genes, changes that could increase both insulin secretion and immune reactivity.

AN1284 attenuates steatosis, lipogenesis, and fibrosis in mice with pre-existing non-alcoholic steatohepatitis and directly affects aryl hydrocarbon receptor in a hepatic cell line.

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.

Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Modulate Apoptosis, TNF Alpha and Interferon Gamma Response Gene mRNA Expression in T Lymphocytes.

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.

Amyloidogenic light chains impair plasma cell survival.

Systemic light chain amyloidosis (AL) is a clonal plasma cell 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 LC-producing plasma cell lines and use them to investigate the biology of the amyloidogenic clone. We used lentiviral vectors to generate cell lines expressing LC 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 LC-producing cells. According to the results of RNA sequencing the AL LC-producing lines showed higher mitochondrial oxidative stress, and decreased activity of the Myc and cholesterol pathways. The neoplastic behavior of plasma cells 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 the unique cellular pathways of AL, thus expediting the development of specific treatments for patients with this disorder.

Identification of global transcriptional variations in human peripheral blood mononuclear cells two months postheat injury helps categorization heat-tolerant or heat-intolerant phenotypes.

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.

Combined hepatocellular-cholangiocarcinoma derives from liver progenitor cells and depends on senescence and IL-6 trans-signaling.

BACKGROUND & AIMS: Primary liver cancers include hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCA) and combined HCC-CCA tumors (cHCC-CCA). It has been suggested, but not unequivocally proven, that hepatic progenitor cells (HPCs) can contribute to hepatocarcinogenesis. We aimed to determine whether HPCs contribute to HCC, cHCC-CCA or both types of tumors. METHODS: To trace progenitor cells during hepatocarcinogenesis, we generated Mdr2-KO mice that harbor a yellow fluorescent protein (YFP) reporter gene driven by the Foxl1 promoter which is expressed specifically in progenitor cells. These mice (Mdr2-KOFoxl1-CRE;RosaYFP) develop chronic inflammation and HCCs by the age of 14-16 months, followed by cHCC-CCA tumors at the age of 18 months. RESULTS: In this Mdr2-KOFoxl1-CRE;RosaYFP mouse model, liver progenitor cells are the source of cHCC-CCA tumors, but not the source of HCC. Ablating the progenitors, caused reduction of cHCC-CCA tumors but did not affect HCCs. RNA-sequencing revealed enrichment of the IL-6 signaling pathway in cHCC-CCA tumors compared to HCC tumors. Single-cell RNA-sequencing (scRNA-seq) analysis revealed that IL-6 is expressed by immune and parenchymal cells during senescence, and that IL-6 is part of the senescence-associated secretory phenotype. Administration of an anti-IL-6 antibody to Mdr2-KOFoxl1-CRE;RosaYFP mice inhibited the development of cHCC-CCA tumors. Blocking IL-6 trans-signaling led to a decrease in the number and size of cHCC-CCA tumors, indicating their dependence on this pathway. Furthermore, the administration of a senolytic agent inhibited IL-6 and the development of cHCC-CCA tumors. CONCLUSION: Our results demonstrate that cHCC-CCA, but not HCC tumors, originate from HPCs, and that IL-6, which derives in part from cells in senescence, plays an important role in this process via IL-6 trans-signaling. These findings could be applied to develop new therapeutic approaches for cHCC-CCA tumors. LAY SUMMARY: Combined hepatocellular carcinoma-cholangiocarcinoma is the third most prevalent type of primary liver cancer (i.e. a cancer that originates in the liver). Herein, we show that this type of cancer originates in stem cells in the liver and that it depends on inflammatory signaling. Specifically, we identify a cytokine called IL-6 that appears to be important in the development of these tumors. Our results could be used for the development of novel treatments for these aggressive tumors.

Single-cell transcriptomics reveals a senescence-associated IL-6/CCR6 axis driving radiodermatitis.

Irradiation-induced alopecia and dermatitis (IRIAD) are two of the most visually recognized complications of radiotherapy, of which the molecular and cellular basis remains largely unclear. By combining scRNA-seq analysis of whole skin-derived irradiated cells with genetic ablation and molecular inhibition studies, we show that senescence-associated IL-6 and IL-1 signaling, together with IL-17 upregulation and CCR6+ -mediated immune cell migration, are crucial drivers of IRIAD. Bioinformatics analysis colocalized irradiation-induced IL-6 signaling with senescence pathway upregulation largely within epidermal hair follicles, basal keratinocytes, and dermal fibroblasts. Loss of cytokine signaling by genetic ablation in IL-6-/- or IL-1R-/- mice, or by molecular blockade, strongly ameliorated IRIAD, as did deficiency of CCL20/CCR6-mediated immune cell migration in CCR6-/- mice. Moreover, IL-6 deficiency strongly reduced IL-17, IL-22, CCL20, and CCR6 upregulation, whereas CCR6 deficiency reciprocally diminished IL-6, IL-17, CCL3, and MHC upregulation, suggesting that proximity-dependent cellular cross talk promotes IRIAD. Therapeutically, topical application of Janus kinase blockers or inhibition of T-cell activation by cyclosporine effectively reduced IRIAD, suggesting the potential of targeted approaches for the treatment of dermal side effects in radiotherapy patients.

Amniotic fluid biomarkers predict the severity of congenital cytomegalovirus infection.

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.

Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions.

OBJECTIVE: Cellular senescence limits tumourigenesis by blocking the proliferation of premalignant cells. Additionally, however, senescent cells can exert paracrine effects influencing tumour growth. Senescent cells are present in premalignant pancreatic intraepithelial neoplasia (PanIN) lesions, yet their effects on the disease are poorly characterised. It is currently unknown whether senolytic drugs, aimed at eliminating senescent cells from lesions, could be beneficial in blocking tumour development. DESIGN: To uncover the functions of senescent cells and their potential contribution to early pancreatic tumourigenesis, we isolated and characterised senescent cells from PanINs formed in a Kras-driven mouse model, and tested the consequences of their targeted elimination through senolytic treatment. RESULTS: We found that senescent PanIN cells exert a tumour-promoting effect through expression of a proinflammatory signature that includes high Cox2 levels. Senolytic treatment with the Bcl2-family inhibitor ABT-737 eliminated Cox2-expressing senescent cells, and an intermittent short-duration treatment course dramatically reduced PanIN development and progression to pancreatic ductal adenocarcinoma. CONCLUSIONS: These findings reveal that senescent PanIN cells support tumour growth and progression, and provide a first indication that elimination of senescent cells may be effective as preventive therapy for the progression of precancerous lesions.

MTADV 5-MER peptide suppresses chronic inflammations as well as autoimmune pathologies and unveils a new potential target-Serum Amyloid A.

Despite the existence of potent anti-inflammatory biological drugs e.g., anti-TNF and anti IL-6 receptor antibodies, for treating chronic inflammatory and autoimmune diseases, these are costly and not specific. Cheaper oral available drugs remain an unmet need. Expression of the acute phase protein Serum Amyloid A (SAA) is dependent on release of pro-inflammatory cytokines IL-1, IL-6 and TNF-α during inflammation. Conversely, SAA induces pro-inflammatory cytokine secretion, including Th17, leading to a pathogenic vicious cycle and chronic inflammation. 5- MER peptide (5-MP) MTADV (methionine-threonine-alanine-aspartic acid-valine), also called Amilo-5MER, was originally derived from a sequence of a pro-inflammatory CD44 variant isolated from synovial fluid of a Rheumatoid Arthritis (RA) patient. This human peptide displays an efficient anti-inflammatory effects to ameliorate pathology and clinical symptoms in mouse models of RA, Inflammatory Bowel Disease (IBD) and Multiple Sclerosis (MS). Bioinformatics and qRT-PCR revealed that 5-MP, administrated to encephalomyelytic mice, up-regulates genes contributing to chronic inflammation resistance. Mass spectrometry of proteins that were pulled down from an RA synovial cell extract with biotinylated 5-MP, showed that it binds SAA. 5-MP disrupted SAA assembly, which is correlated with its pro-inflammatory activity. The peptide MTADV (but not scrambled TMVAD) significantly inhibited the release of pro-inflammatory cytokines IL-6 and IL-1ß from SAA-activated human fibroblasts, THP-1 monocytes and peripheral blood mononuclear cells. 5-MP suppresses the pro-inflammatory IL-6 release from SAA-activated cells, but not from non-activated cells. 5-MP could not display therapeutic activity in rats, which are SAA deficient, but does inhibit inflammations in animal models of IBD and MS, both are SAA-dependent, as shown by others in SAA knockout mice. In conclusion, 5-MP suppresses chronic inflammation in animal models of RA, IBD and MS, which are SAA-dependent, but not in animal models, which are SAA-independent.

Slow Transcription of the 99a/let-7c/125b-2 Cluster Results in Differential MiRNA Expression and Promotes Melanoma Phenotypic Plasticity.

Almost half of the human microRNAs (miRNAs) are encoded in clusters. Although transcribed as a single unit, the levels of individual mature miRNAs often differ. The mechanisms underlying differential biogenesis of clustered miRNAs and the resulting physiological implications are mostly unknown. In this study, we report that the melanoma master transcription regulator MITF regulates the differential expression of the 99a/let-7c/125b-2 cluster by altering the distribution of RNA polymerase II along the cluster. We discovered that MITF interacts with TRIM28, a known inhibitor of RNA polymerase II transcription elongation, at the mIR-let-7c region, resulting in the pausing of RNA polymerase II activity and causing an elevation in mIR-let-7c expression; low levels of RNA polymerase II occupation over miR-99a and miR-125b-2 regions decreases their biogenesis. Furthermore, we showed that this differential expression affects the phenotypic state of melanoma cells. RNA-sequencing analysis of proliferative melanoma cells that express miR-99a and miR-125b mimics revealed a transcriptomic shift toward an invasive phenotype. Conversely, expression of a mIR-let-7c mimic in invasive melanoma cells induced a shift to a more proliferative state. We confirmed direct target genes of these miRNAs, including FGFR3, BAP1, Bcl2, TGFBR1, and CDKN1A. Our study demonstrates an MITF-governed biogenesis mechanism that results in differential expression of clustered 99a/let-7c/125b-2 miRNAs that control melanoma progression.

Multiple Roles of IL6 in Hepatic Injury, Steatosis, and Senescence Aggregate to Suppress Tumorigenesis.

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.

Human Nasal and Lung Tissues Infected Ex Vivo with SARS-CoV-2 Provide Insights into Differential Tissue-Specific and Virus-Specific Innate Immune Responses in the Upper and Lower Respiratory Tract.

The nasal mucosa constitutes the primary entry site for respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the imbalanced innate immune response of end-stage coronavirus disease 2019 (COVID-19) has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here, we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo, coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with a rapid increase in tissue-associated viral subgenomic mRNA and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon-stimulated genes, cytokines, and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tracts that are specific to SARS-CoV-2. The studies shed light on the role of the nasal mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19. IMPORTANCE In order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here, we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues infected in parallel with SARS-CoV-2 and influenza virus, we found distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal mucosal infection model can be employed to assess the impact of viral evolutionary changes and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.

Single-cell transcriptomes of pancreatic preinvasive lesions and cancer reveal acinar metaplastic cells' heterogeneity.

Acinar metaplasia is an initial step in a series of events that can lead to pancreatic cancer. Here we perform single-cell RNA-sequencing of mouse pancreas during the progression from preinvasive stages to tumor formation. Using a reporter gene, we identify metaplastic cells that originated from acinar cells and express two transcription factors, Onecut2 and Foxq1. Further analyses of metaplastic acinar cell heterogeneity define six acinar metaplastic cell types and states, including stomach-specific cell types. Localization of metaplastic cell types and mixture of different metaplastic cell types in the same pre-malignant lesion is shown. Finally, single-cell transcriptome analyses of tumor-associated stromal, immune, endothelial and fibroblast cells identify signals that may support tumor development, as well as the recruitment and education of immune cells. Our findings are consistent with the early, premalignant formation of an immunosuppressive environment mediated by interactions between acinar metaplastic cells and other cells in the microenvironment.

Human Nasal Turbinate Tissues in Organ Culture as a Model for Human Cytomegalovirus Infection at the Mucosal Entry Site.

The initial events of viral infection at the primary mucosal entry site following horizontal person-to-person transmission have remained ill defined. Our limited understanding is further underscored by the absence of animal models in the case of human-restricted viruses, such as human cytomegalovirus (HCMV), a leading cause of congenital infection and a major pathogen in immunocompromised individuals. Here, we established a novel ex vivo model of HCMV infection in native human nasal turbinate tissues. Nasal turbinate tissue viability and physiological functionality were preserved for at least 7 days in culture. We found that nasal mucosal tissues were susceptible to HCMV infection, with predominant infection of ciliated respiratory epithelial cells. A limited viral spread was demonstrated, involving mainly stromal and vascular endothelial cells within the tissue. Importantly, functional antiviral and proleukocyte chemotactic signaling pathways were significantly upregulated in the nasal mucosa in response to infection. Conversely, HCMV downregulated the expression of nasal epithelial cell-related genes. We further revealed tissue-specific innate immune response patterns to HCMV, comparing infected human nasal mucosal and placental tissues, representing the viral entry and the maternal-to-fetal transmission sites, respectively. Taken together, our studies provide insights into the earliest stages of HCMV infection. Studies in this model could help evaluate new interventions against the horizontal transmission of HCMV.IMPORTANCE HCMV is a ubiquitous human pathogen causing neurodevelopmental disabilities in congenitally infected children and severe disease in immunocompromised patients. The earliest stages of HCMV infection in the human host have remained elusive in the absence of a model for the viral entry site. Here, we describe the establishment and use of a novel nasal turbinate organ culture to study the initial steps of viral infection and the consequent innate immune responses within the natural complexity and the full cellular repertoire of human nasal mucosal tissues. This model can be applied to examine new antiviral interventions against the horizontal transmission of HCMV and potentially that of other viruses.

Empty SV40 capsids increase survival of septic rats by eliciting numerous host signaling networks that participate in a number of systemic functions.

Sepsis is an excessive, dysregulated immune response to infection that activates inflammatory and coagulation cascades, which may lead to tissue injury, multiple organ dysfunction syndrome and death. Millions of individuals die annually of sepsis. To date, the only treatment available is antibiotics, drainage of the infection source when possible, and organ support in intensive care units. Numerous previous attempts to develop therapeutic treatments, directed at discreet targets of the sepsis cascade, could not cope with the complex pathophysiology of sepsis and failed. Here we describe a novel treatment, based on empty capsids of SV40 (nanocapsids - NCs). Studies in a severe rat sepsis model showed that pre-treatment by NCs led to a dramatic increase in survival, from zero to 75%. Transcript analyses (RNAseq) demonstrated that the NC treatment is a paradigm shift. The NCs affect multiple facets of biological functions. The affected genes are modified with time, adjusting to the recovery processes. The NCs effect on normal control rats was negligible. The study shows that the NCs are capable of coping with diseases with intricate pathophysiology. Further studies are needed to determine whether when applied after sepsis onset, the NCs still improve outcome.

Upregulation of Hallmark Muscle Genes Protects GneM743T/M743T Mutated Knock-In Mice From Kidney and Muscle Phenotype.

BACKGROUND: Mutations in GNE cause a recessive, adult onset myopathy characterized by slowly progressive distal and proximal muscle weakness. Knock-in mice carrying the most frequent mutation in GNE myopathy patients, GneM743T/M743T, usually die few days after birth from severe renal failure, with no muscle phenotype. However, a spontaneous sub-colony remains healthy throughout a normal lifespan without any kidney or muscle pathology. OBJECTIVE: We attempted to decipher the molecular mechanisms behind these phenotypic differences and to determine the mechanisms preventing the kidney and muscles from disease. METHODS: We analyzed the transcriptome and proteome of kidneys and muscles of sick and healthy GneM743T/M743T mice. RESULTS: The sick GneM743T/M743T kidney was characterized by up-regulation of extra-cellular matrix degradation related processes and by down-regulation of oxidative phosphorylation and respiratory electron chain pathway, that was also observed in the asymptomatic muscles. Surprisingly, the healthy kidneys of the GneM743T/M743T mice were characterized by up-regulation of hallmark muscle genes. In addition the asymptomatic muscles of the sick GneM743T/M743T mice showed upregulation of transcription and translation processes. CONCLUSIONS: Overexpression of muscle physiology genes in healthy GneM743T/M743T mice seems to define the protecting mechanism in these mice. Furthermore, the strong involvement of muscle related genes in kidney may bridge the apparent phenotypic gap between GNE myopathy and the knock-in GneM743T/M743T mouse model and provide new directions in the study of GNE function in health and disease.

Gut microbiota shape 'inflamm-ageing' cytokines and account for age-dependent decline in DNA damage repair.

OBJECTIVE: Failing to properly repair damaged DNA drives the ageing process. Furthermore, age-related inflammation contributes to the manifestation of ageing. Recently, we demonstrated that the efficiency of repair of diethylnitrosamine (DEN)-induced double-strand breaks (DSBs) rapidly declines with age. We therefore hypothesised that with age, the decline in DNA damage repair stems from age-related inflammation. DESIGN: We used DEN-induced DNA damage in mouse livers and compared the efficiency of their resolution in different ages and following various permutations aimed at manipulating the liver age-related inflammation. RESULTS: We found that age-related deregulation of innate immunity was linked to altered gut microbiota. Consequently, antibiotic treatment, MyD88 ablation or germ-free mice had reduced cytokine expression and improved DSBs rejoining in 6-month-old mice. In contrast, feeding young mice with a high-fat diet enhanced inflammation and facilitated the decline in DSBs repair. This latter effect was reversed by antibiotic treatment. Kupffer cell replenishment or their inactivation with gadolinium chloride reduced proinflammatory cytokine expression and reversed the decline in DSBs repair. The addition of proinflammatory cytokines ablated DSBs rejoining mediated by macrophage-derived heparin-binding epidermal growth factor-like growth factor. CONCLUSIONS: Taken together, our results reveal a previously unrecognised link between commensal bacteria-induced inflammation that results in age-dependent decline in DNA damage repair. Importantly, the present study support the notion of a cell non-autonomous mechanism for age-related decline in DNA damage repair that is based on the presence of 'inflamm-ageing' cytokines in the tissue microenvironment, rather than an intrinsic cellular deficiency in the DNA repair machinery.