SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis.

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.

Tissue factor binds to and inhibits interferon-α receptor 1 signaling.

Tissue factor (TF), which is a member of the cytokine receptor family, promotes coagulation and coagulation-dependent inflammation. TF also exerts protective effects through unknown mechanisms. Here, we showed that TF bound to interferon-α receptor 1 (IFNAR1) and antagonized its signaling, preventing spontaneous sterile inflammation and maintaining immune homeostasis. Structural modeling and direct binding studies revealed binding of the TF C-terminal fibronectin III domain to IFNAR1, which restricted the expression of interferon-stimulated genes (ISGs). Podocyte-specific loss of TF in mice (PodΔF3) resulted in sterile renal inflammation, characterized by JAK/STAT signaling, proinflammatory cytokine expression, disrupted immune homeostasis, and glomerulopathy. Inhibiting IFNAR1 signaling or loss of Ifnar1 expression in podocytes attenuated these effects in PodΔF3 mice. As a heteromer, TF and IFNAR1 were both inactive, while dissociation of the TF-IFNAR1 heteromer promoted TF activity and IFNAR1 signaling. These data suggest that the TF-IFNAR1 heteromer is a molecular switch that controls thrombo-inflammation.

Publisher Correction: Human γδ T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.

In the version of this article initially published, a source of funding (Deutsche José Carreras Leukämie-Stiftung e.V. (DJCLS R12/29 to C.K. and I.P.)) was not included in the Acknowledgments section. The correct statement is as follows: "Supported by Deutsche Forschungsgemeinschaft, (SFB900/B8 to C.K. and I.P.; and PR727/4-1 to I.P.), Deutsche José Carreras Leukämie-Stiftung e.V. (DJCLS R12/29 to C.K. and I.P.) and the German Federal Ministry of Education and Research (01EO1302 to C.S.-F., C.K. and I.P.)." The error has been corrected in the HTML and PDF versions of the article.

Human γδ T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.

To investigate how the human γδ T cell pool is shaped during ontogeny and how it is regenerated after transplantation of hematopoietic stem cells (HSCs), we applied an RNA-based next-generation sequencing approach to monitor the dynamics of the repertoires of γδ T cell antigen receptors (TCRs) before and after transplantation in a prospective cohort study. We found that repertoires of rearranged genes encoding γδ TCRs (TRG and TRD) in the peripheral blood of healthy adults were stable over time. Although a large fraction of human TRG repertoires consisted of public sequences, the TRD repertoires were private. In patients undergoing HSC transplantation, γδ T cells were quickly reconstituted; however, they had profoundly altered TCR repertoires. Notably, the clonal proliferation of individual virus-reactive γδ TCR sequences in patients with reactivation of cytomegalovirus revealed strong evidence for adaptive anti-viral γδ T cell immune responses.

CD4+ T cell-induced inflammatory cell death controls immune-evasive tumours.

Most clinically applied cancer immunotherapies rely on the ability of CD8+ cytolytic T cells to directly recognize and kill tumour cells1-3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment4-6. The ability of CD4+ effector cells to contribute to antitumour immunity independently of CD8+ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified7-10. Here, we describe a mechanism whereby a small number of CD4+ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8+ T cell targeting. The CD4+ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II+CD11c+ antigen-presenting cells. We show that T helper type 1 cell-directed CD4+ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4+ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4+ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8+ T cells and natural killer cells and advance cancer immunotherapies.

A Direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration.

The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.

Neuropilin-1 identifies a subset of highly activated CD8+ T cells during parasitic and viral infections.

Neuropilin-1 (Nrp-1) expression on CD8+ T cells has been identified in tumor-infiltrating lymphocytes and in persistent murine gamma-herpes virus infections, where it interferes with the development of long-lived memory T cell responses. In parasitic and acute viral infections, the role of Nrp-1 expression on CD8+ T cells remains unclear. Here, we demonstrate a strong induction of Nrp-1 expression on CD8+ T cells in Plasmodium berghei ANKA (PbA)-infected mice that correlated with neurological deficits of experimental cerebral malaria (ECM). Likewise, the frequency of Nrp-1+CD8+ T cells was significantly elevated and correlated with liver damage in the acute phase of lymphocytic choriomeningitis virus (LCMV) infection. Transcriptomic and flow cytometric analyses revealed a highly activated phenotype of Nrp-1+CD8+ T cells from infected mice. Correspondingly, in vitro experiments showed rapid induction of Nrp-1 expression on CD8+ T cells after stimulation in conjunction with increased expression of activation-associated molecules. Strikingly, T cell-specific Nrp-1 ablation resulted in reduced numbers of activated T cells in the brain of PbA-infected mice as well as in spleen and liver of LCMV-infected mice and alleviated the severity of ECM and LCMV-induced liver pathology. Mechanistically, we identified reduced blood-brain barrier leakage associated with reduced parasite sequestration in the brain of PbA-infected mice with T cell-specific Nrp-1 deficiency. In conclusion, Nrp-1 expression on CD8+ T cells represents a very early activation marker that exacerbates deleterious CD8+ T cell responses during both, parasitic PbA and acute LCMV infections.

A bacterium from a mountain lake harvests light using both proton-pumping xanthorhodopsins and bacteriochlorophyll-based photosystems.

Photoheterotrophic bacteria harvest light energy using either proton-pumping rhodopsins or bacteriochlorophyll (BChl)-based photosystems. The bacterium Sphingomonas glacialis AAP5 isolated from the alpine lake Gossenköllesee contains genes for both systems. Here, we show that BChl is expressed between 4°C and 22°C in the dark, whereas xanthorhodopsin is expressed only at temperatures below 16°C and in the presence of light. Thus, cells grown at low temperatures under a natural light-dark cycle contain both BChl-based photosystems and xanthorhodopsins with a nostoxanthin antenna. Flash photolysis measurements proved that both systems are photochemically active. The captured light energy is used for ATP synthesis and stimulates growth. Thus, S. glacialis AAP5 represents a chlorophototrophic and a retinalophototrophic organism. Our analyses suggest that simple xanthorhodopsin may be preferred by the cells under higher light and low temperatures, whereas larger BChl-based photosystems may perform better at lower light intensities. This indicates that the use of two systems for light harvesting may represent an evolutionary adaptation to the specific environmental conditions found in alpine lakes and other analogous ecosystems, allowing bacteria to alternate their light-harvesting machinery in response to large seasonal changes of irradiance and temperature.

Comparative sequencing study of mismatch repair and homology-directed repair genes in endometrial cancer and breast cancer patients from Kazakhstan.

Endometrial cancer has been associated with pathogenic variants in mismatch repair (MMR) genes, especially in the context of the hereditary Lynch Syndrome. More recently, pathogenic variants in genes of homology-directed repair (HDR) have also been suggested to contribute to a subset of endometrial cancers. In the present hospital-based study, we investigated the relative distribution of pathogenic MMR or HDR gene variants in a series of 342 endometrial cancer patients from the Oncology Clinic in Almaty, Kazakhstan. In comparison, we also sequenced 178 breast cancer patients from the same population with the same gene panel. Identified variants were classified according to ClinVar, ESM1b, and AlphaMissense prediction tools. We found 10 endometrial cancer patients (2.9%) carrying pathogenic or likely pathogenic variants in MMR genes (7 MSH6, 1 MSH2, 2 MUTYH), while 14 endometrial cancer patients (4.1%) carried pathogenic variants in HDR genes (4 BRCA2, 3 BRCA1, 3 FANCM, 2 SLX4, 1 BARD1, 1 BRIP1). In the breast cancer series, we found 8 carriers (4.5%) of pathogenic or likely pathogenic variants in MMR genes (2 MSH2, 2 MSH6, 4 MUTYH) while 12 patients (6.7%) harbored pathogenic or likely pathogenic HDR gene variants (5 BRCA1, 3 BRCA2, 1 BRIP1, 1 ERRC4, 1 FANCM, 1 SLX4). One patient who developed breast cancer first and endometrial cancer later carried a novel frameshift variant in MSH6. Our results indicate that MMR and HDR gene variants with predicted pathogenicity occur at substantial frequencies in both breast and endometrial cancer patients from the Kazakh population.

Germline variants of homology-directed repair or mismatch repair genes in cervical cancer.

While cervical cancer is associated with a persistent human papillomavirus (HPV) infection, the progression to cancer is influenced by genomic risk factors that have remained largely obscure. Pathogenic variants in genes of the homology-directed repair (HDR) or mismatch repair (MMR) are known to predispose to diverse tumour entities including breast and ovarian cancer (HDR) or colon and endometrial cancer (MMR). We here investigate the spectrum of HDR and MMR germline variants in cervical cancer, with particular focus on the HPV status and histological subgroups. We performed targeted next-generation sequencing for 5 MMR genes and 12 HDR genes on 728 German patients with cervical dysplasia or invasive cancer. In total, 4% of our patients carried a pathogenic germline variant, based on ClinVar classifications and additional ESM1b and AlphaMissense predictions. These included 15 patients with truncating variants in HDR genes (BARD1, BRCA1, BRCA2, BRIP1, FANCM, RAD51D and SLX4). MMR-related gene variants were less prevalent and mainly of the missense type. While MMR-related gene variants tended to associate with adenocarcinomas, HDR gene variants were commonly observed in squamous cancers. While one patient with HPV-negative cancer carried a pathogenic MMR gene variant (in MSH6), the HDR germline variants were found in patients with HPV-positive cancers and tended to associate with HPV18. Taken together, our study supports a potentially risk-modifying role of MMR and HDR germline variants in cervical cancer but no association with HPV-negative status. These variants may be exploitable in future therapeutic managements.

DNA-binding protein-A promotes kidney ischemia/reperfusion injury and participates in mitochondrial function.

DNA-binding protein-A (DbpA; gene: Ybx3) belongs to the cold shock protein family with known functions in cell cycling, transcription, translation, and tight junction communication. In chronic nephritis, DbpA is upregulated. However, its activities in acute injury models, such as kidney ischemia/reperfusion injury (IRI), are unclear. To study this, mice harboring Ybx3+/+, Ybx3+/- or the Ybx3-/- genotype were characterized over 24 months and following experimental kidney IRI. Mitochondrial function, number and integrity were analyzed by mitochondrial stress tests, MitoTracker staining and electron microscopy. Western Blot, immunohistochemistry and flow cytometry were performed to quantify tubular cell damage and immune cell infiltration. DbpA was found to be dispensable for kidney development and tissue homeostasis under healthy conditions. Furthermore, endogenous DbpA protein localizes within mitochondria in primary tubular epithelial cells. Genetic deletion of Ybx3 elevates the mitochondrial membrane potential, lipid uptake and metabolism, oxygen consumption rates and glycolytic activities of tubular epithelial cells. Ybx3-/- mice demonstrated protection from IRI with less immune cell infiltration, endoplasmic reticulum stress and tubular cell damage. A presumed protective mechanism was identified via upregulated antioxidant activities and reduced ferroptosis, when Ybx3 was deleted. Thus, our studies reveal DbpA acts as a mitochondrial protein with profound adverse effects on cell metabolism and highlights a protective effect against IRI when Ybx3 is genetically deleted. Hence, preemptive DbpA targeting in situations with expected IRI, such as kidney transplantation or cardiac surgery, may preserve post-procedure kidney function.

Zfp362 potentiates murine colonic inflammation by constraining Treg cell function rather than promoting Th17 cell differentiation.

Mucosal barrier integrity and pathogen clearance is a complex process influenced by both Th17 and Treg cells. Previously, we had described the DNA methylation profile of Th17 cells and identified Zinc finger protein (Zfp)362 to be uniquely demethylated. Here, we generated Zfp362-/- mice to unravel the role of Zfp362 for Th17 cell biology. Zfp362-/- mice appeared clinically normal, showed no phenotypic alterations in the T-cell compartment, and upon colonization with segmented filamentous bacteria, no effect of Zfp362 deficiency on Th17 cell differentiation was observed. By contrast, Zfp362 deletion resulted in increased frequencies of colonic Foxp3+ Treg cells and IL-10+ and RORγt+ Treg cell subsets in mesenteric lymph nodes. Adoptive transfer of naïve CD4+ T cells from Zfp362-/- mice into Rag2-/- mice resulted in a significantly lower weight loss when compared with controls receiving cells from Zfp362+/+ littermates. However, this attenuated weight loss did not correlate with alterations of Th17 cells but instead was associated with an increase of effector Treg cells in mesenteric lymph nodes. Together, these results suggest that Zfp362 plays an important role in promoting colonic inflammation; however, this function is derived from constraining the effector function of Treg cells rather than directly promoting Th17 cell differentiation.

Methylation, Gene Expression, and Risk Genotypes at the TERT-CLPTM1L Locus in Cervical Cancer.

The reverse transcriptase subunit of telomerase, TERT, is frequently activated in high-grade dysplasia and invasive cancers of the uterine cervix. Telomerase activation through hypomethylation of the TERT promoter holds promise as a biomarker for cervical cancer progression, however, specific CpG sites involved in cervical cancer risk remain to be fully defined. A recent genome-wide association study on cervical cancer identified genetic polymorphisms at 5p13.33 (close to TERT-CLPTM1L) but the underlying mechanisms are undetermined. We investigated 529 CpG sites within the TERT promoter region and 3 CpG islands nearby, and 21 CpG sites within CLPTM1L in 190 bisulfite-converted cervical tumor DNA samples from BioRAIDs (NCT02428842). We identified eight CpG sites within TERT intron 2 where methylation was significantly associated with the genotypes of cervical cancer risk variants rs27070 and rs459961 in cervical tumors after multiple testing correction (p < 9.4 × 10E-5). Hypermethylation at chr5:1289663 correlated with decreased TERT mRNA levels. In an independent series of 188 normal or dysplastic cervical tissues, rare alleles of rs27070 and rs459961 were associated with low basal CLPTM1L levels and with the absence of TERT mRNA in HPV-negative samples, consistent with their proposed role as protective variants for cervical cancer. HPV infection was associated with increased CLPTM1L and TERT levels. Collectively, our results provide a link between cervical cancer risk variants, methylation, and gene expression and implicate both TERT and CLPTM1L as genes modulated by genomic background and HPV infection during cervical cancer development.

Mineralocorticoid receptor promotes cardiac macrophage inflammaging.

Inflammaging, a pro-inflammatory status that characterizes aging and primarily involving macrophages, is a master driver of age-related diseases. Mineralocorticoid receptor (MR) activation in macrophages critically regulates inflammatory and fibrotic processes. However, macrophage-specific mechanisms and the role of the macrophage MR for the regulation of inflammation and fibrotic remodeling in the aging heart have not yet been elucidated. Transcriptome profiling of cardiac macrophages from male/female young (4 months-old), middle (12 months-old) and old (18 and 24 months-old) mice revealed that myeloid cell-restricted MR deficiency prevents macrophage differentiation toward a pro-inflammatory phenotype. Pathway enrichment analysis showed that several biological processes related to inflammation and cell metabolism were modulated by the MR in aged macrophages. Further, transcriptome analysis of aged cardiac fibroblasts revealed that macrophage MR deficiency reduced the activation of pathways related to inflammation and upregulation of ZBTB16, a transcription factor involved in fibrosis. Phenotypic characterization of macrophages showed a progressive replacement of the TIMD4+MHC-IIneg/low macrophage population by TIMD4+MHC-IIint/high and TIMD4-MHC-IIint/high macrophages in the aging heart. By integrating cell sorting and transwell experiments with TIMD4+/TIMD4-macrophages and fibroblasts from old MRflox/MRLysMCre hearts, we showed that the inflammatory crosstalk between TIMD4- macrophages and fibroblasts may imply the macrophage MR and the release of mitochondrial superoxide anions. Macrophage MR deficiency reduced the expansion of the TIMD4- macrophage population and the emergence of fibrotic niches in the aging heart, thereby protecting against cardiac inflammation, fibrosis, and dysfunction. This study highlights the MR as an important mediator of cardiac macrophage inflammaging and age-related fibrotic remodeling.

Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection.

Excessive inflammation is a major cause of morbidity and mortality in many viral infections including influenza. Therefore, there is a need for therapeutic interventions that dampen and redirect inflammatory responses and, ideally, exert antiviral effects. Itaconate is an immunomodulatory metabolite which also reprograms cell metabolism and inflammatory responses when applied exogenously. We evaluated effects of endogenous itaconate and exogenous application of itaconate and its variants dimethyl- and 4-octyl-itaconate (DI, 4OI) on host responses to influenza A virus (IAV). Infection induced expression of ACOD1, the enzyme catalyzing itaconate synthesis, in monocytes and macrophages, which correlated with viral replication and was abrogated by DI and 4OI treatment. In IAV-infected mice, pulmonary inflammation and weight loss were greater in Acod1-/- than in wild-type mice, and DI treatment reduced pulmonary inflammation and mortality. The compounds reversed infection-triggered interferon responses and modulated inflammation in human cells supporting non-productive and productive infection, in peripheral blood mononuclear cells, and in human lung tissue. All three itaconates reduced ROS levels and STAT1 phosphorylation, whereas AKT phosphorylation was reduced by 4OI and DI but increased by itaconate. Single-cell RNA sequencing identified monocytes as the main target of infection and the exclusive source of ACOD1 mRNA in peripheral blood. DI treatment silenced IFN-responses predominantly in monocytes, but also in lymphocytes and natural killer cells. Ectopic synthesis of itaconate in A549 cells, which do not physiologically express ACOD1, reduced infection-driven inflammation, and DI reduced IAV- and IFNγ-induced CXCL10 expression in murine macrophages independent of the presence of endogenous ACOD1. The compounds differed greatly in their effects on cellular gene homeostasis and released cytokines/chemokines, but all three markedly reduced release of the pro-inflammatory chemokines CXCL10 (IP-10) and CCL2 (MCP-1). Viral replication did not increase under treatment despite the dramatically repressed IFN responses. In fact, 4OI strongly inhibited viral transcription in peripheral blood mononuclear cells, and the compounds reduced viral titers (4OI>Ita>DI) in A549 cells whereas viral transcription was unaffected. Taken together, these results reveal itaconates as immunomodulatory and antiviral interventions for influenza virus infection.

Correction: Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection.

[This corrects the article DOI: 10.1371/journal.ppat.1010219.].

Differential transcriptomic host responses in the early phase of viral and bacterial infections in human lung tissue explants ex vivo.

BACKGROUND: The first 24 h of infection represent a critical time window in interactions between pathogens and host tissue. However, it is not possible to study such early events in human lung during natural infection due to lack of clinical access to tissue this early in infection. We, therefore, applied RNA sequencing to ex vivo cultured human lung tissue explants (HLTE) from patients with emphysema to study global changes in small noncoding RNA, mRNA, and long noncoding RNA (lncRNA, lincRNA) populations during the first 24 h of infection with influenza A virus (IAV), Mycobacterium bovis Bacille Calmette-Guerin (BCG), and Pseudomonas aeruginosa. RESULTS: Pseudomonas aeruginosa caused the strongest expression changes and was the only pathogen that notably affected expression of microRNA and PIWI-associated RNA. The major classes of long RNAs (> 100 nt) were represented similarly among the RNAs that were differentially expressed upon infection with the three pathogens (mRNA 77-82%; lncRNA 15-17%; pseudogenes 4-5%), but lnc-DDX60-1, RP11-202G18.1, and lnc-THOC3-2 were part of an RNA signature (additionally containing SNX10 and SLC8A1) specifically associated with IAV infection. IAV infection induced brisk interferon responses, CCL8 being the most strongly upregulated mRNA. Single-cell RNA sequencing identified airway epithelial cells and macrophages as the predominant IAV host cells, but inflammatory responses were also detected in cell types expressing few or no IAV transcripts. Combined analysis of bulk and single-cell RNAseq data identified a set of 6 mRNAs (IFI6, IFI44L, IRF7, ISG15, MX1, MX2) as the core transcriptomic response to IAV infection. The two bacterial pathogens induced qualitatively very similar changes in mRNA expression and predicted signaling pathways, but the magnitude of change was greater in P. aeruginosa infection. Upregulation of GJB2, VNN1, DUSP4, SerpinB7, and IL10, and downregulation of PKMYT1, S100A4, GGTA1P, and SLC22A31 were most strongly associated with bacterial infection. CONCLUSIONS: Human lung tissue mounted substantially different transcriptomic responses to infection by IAV than by BCG and P. aeruginosa, whereas responses to these two divergent bacterial pathogens were surprisingly similar. This HLTE model should prove useful for RNA-directed pathogenesis research and tissue biomarker discovery during the early phase of infections, both at the tissue and single-cell level.

Excessive sodium chloride ingestion promotes inflammation and kidney fibrosis in aging mice.

In aging kidneys, a decline of function resulting from extracellular matrix (ECM) deposition and organ fibrosis is regarded as "physiological." Whether a direct link between high salt intake and fibrosis in aging kidney exists autonomously from arterial hypertension is unclear. This study explores kidney intrinsic changes (inflammation, ECM derangement) induced by a high-salt diet (HSD) in a murine model lacking arterial hypertension. The contribution of cold shock Y-box binding protein (YB-1) as a key orchestrator of organ fibrosis to the observed differences is determined by comparison with a knockout strain (Ybx1ΔRosaERT+TX). Comparisons of tissue from mice fed with normal-salt diet (NSD, standard chow) or high-salt diet (HSD, 4% NaCl in chow; 1% NaCl in water) for up to 16 mo revealed that with HSD tubular cell numbers decrease and tubulointerstitial scarring [periodic acid-Schiff (PAS), Masson's trichrome, Sirius red staining] prevails. In Ybx1ΔRosaERT+TX animals tubular cell damage, a loss of cell contacts with profound tubulointerstitial alterations, and tubular cell senescence was seen. A distinct tubulointerstitial distribution of fibrinogen, collagen type VI, and tenascin-C was detected under HSD, transcriptome analyses determined patterns of matrisome regulation. Temporal increase of immune cell infiltration was seen under HSD of wild type, but not Ybx1ΔRosaERT+TX animals. In vitro Ybx1ΔRosaERT+TX bone marrow-derived macrophages exhibited a defect in polarization (IL-4/IL-13) and abrogated response to sodium chloride. Taken together, HSD promotes progressive kidney fibrosis with premature cell aging, ECM deposition, and immune cell recruitment that is exacerbated in Ybx1ΔRosaERT+TX animals.NEW & NOTEWORTHY Short-term experimental studies link excessive sodium ingestion with extracellular matrix accumulation and inflammatory cell recruitment, yet long-term data are scarce. Our findings with a high-salt diet over 16 mo in aging mice pinpoints to a decisive tipping point after 12 mo with tubular stress response, skewed matrisome transcriptome, and immune cell infiltration. Cell senescence was aggravated in knockout animals for cold shock Y-box binding protein (YB-1), suggesting a novel protective protein function.

Heterozygous variants in the DVL2 interaction region of DACT1 cause CAKUT and features of Townes-Brocks syndrome 2.

Most patients with congenital anomalies of the kidney and urinary tract (CAKUT) remain genetically unexplained. In search of novel genes associated with CAKUT in humans, we applied whole-exome sequencing in a patient with kidney, anorectal, spinal, and brain anomalies, and identified a rare heterozygous missense variant in the DACT1 (dishevelled binding antagonist of beta catenin 1) gene encoding a cytoplasmic WNT signaling mediator. Our patient's features overlapped Townes-Brocks syndrome 2 (TBS2) previously described in a family carrying a DACT1 nonsense variant as well as those of Dact1-deficient mice. Therefore, we assessed the role of DACT1 in CAKUT pathogenesis. Taken together, very rare (minor allele frequency ≤ 0.0005) non-silent DACT1 variants were detected in eight of 209 (3.8%) CAKUT families, significantly more frequently than in controls (1.7%). All seven different DACT1 missense variants, predominantly likely pathogenic and exclusively maternally inherited, were located in the interaction region with DVL2 (dishevelled segment polarity protein 2), and biochemical characterization revealed reduced binding of mutant DACT1 to DVL2. Patients carrying DACT1 variants presented with kidney agenesis, duplex or (multi)cystic (hypo)dysplastic kidneys with hydronephrosis and TBS2 features. During murine development, Dact1 was expressed in organs affected by anomalies in patients with DACT1 variants, including the kidney, anal canal, vertebrae, and brain. In a branching morphogenesis assay, tubule formation was impaired in CRISPR/Cas9-induced Dact1-/- murine inner medullary collecting duct cells. In summary, we provide evidence that heterozygous hypomorphic DACT1 variants cause CAKUT and other features of TBS2, including anomalies of the skeleton, brain, distal digestive and genital tract.

The Challenge of Long-Term Cultivation of Human Precision-Cut Lung Slices.

Human precision-cut lung slices (PCLS) have proven to be an invaluable tool for numerous toxicologic, pharmacologic, and immunologic studies. Although a cultivation period of <1 week is sufficient for most studies, modeling of complex disease mechanisms and investigating effects of long-term exposure to certain substances require cultivation periods that are much longer. So far, data regarding tissue integrity of long-term cultivated PCLS are incomplete. More than 1500 human PCLS from 16 different donors were cultivated under standardized, serum-free conditions for up to 28 days and the viability, tissue integrity, and the transcriptome was assessed in great detail. Even though viability of PCLS was well preserved during long-term cultivation, a continuous loss of cells was observed. Although the bronchial epithelium was well preserved throughout cultivation, the alveolar integrity was preserved for about 2 weeks, and the vasculatory system experienced significant loss of integrity within the first week. Furthermore, ciliary beat in the small airways gradually decreased after 1 week. Interestingly, keratinizing squamous metaplasia of the alveolar epithelium with significantly increasing manifestation were found over time. Transcriptome analysis revealed a significantly increased immune response and significantly decreased metabolic activity within the first 24 hours after PCLS generation. Overall, this study provides a comprehensive overview of histomorphologic and pathologic changes during long-term cultivation of PCLS.