MicroRNAs (miRNA) are considered promising biomarkers for inflammatory and neoplastic diseases in fecal samples, but despite reproducible miRNA detection, there is no consensus on appropriate normalization in fecal samples. In this study, we aimed to explore an endogenous normalizer for miRNA analysis in fecal samples. For this purpose, we performed a multistep study with 3 independent cohorts. In the first step, we investigated miRNA profiling using serial dilutions of blood and fecal samples from healthy subjects. In the second part, we tested the analysis using a cohort of IBD patients (n = 30) with active disease and in remission and 59 patients with different liver pathologies or healthy subjects. Finally, the data were validated using an independent cohort of patients with liver disease (n = 360). After extraction, miRNAs were analyzed using Affymetrix GeneChip microarray technology in the screening and confirmation cohorts, and TaqMan qPCR was performed for validation. Analysis of the blood stool mixture cohort revealed 5 miRNAs that were stable in all three subgroups. Among them, miR-638 was found to have a Log2FC of 0.0004 and was selected for further analysis as one of the best studied miRNAs from previous reports. The stability of miR-638 in microarray analysis was demonstrated in two independent cohorts of patients with IBD and liver disease. We observed no effect of age or other factors on miR-638 levels in fecal samples. Furthermore, its relatively high concentration in feces and its stability against potential blood contamination may offer great advantages over the previously used miR-16, suggesting miR-638 as a potential endogenous fecal normalizer.

The ongoing emergence of novel SARS-CoV-2 variants due to viral mutations poses a persistent challenge to the efficacy of existing vaccines. To address this challenge, we engineered and comprehensively tested three optimized mRNA vaccine candidates, evaluating the kinetics, quality, and magnitude of antibody responses as well as antigen-specific T cell immunity during a prime-boost vaccination regimen in mice. Among the tested candidates, TP2A encoding secreted receptor-binding domains (RBDs) derived from SARS-CoV-2 wild type (WT), Delta and Omicron variants demonstrated superior immunogenicity, inducing an early IgG2a-dominated antibody response against distinct SARS-CoV-2 spike (S) glycoprotein variants. In addition, TP2A elicited IFN-γ-producing T cells in both spleen and draining lymph nodes and antigen-specific cytotoxic T lymphocytes. Notably, beyond broad immunity induced by the vaccine, TP2A functions as a modular platform, thus enabling flexible antigen assembly and rapid vaccine adaptation to newly emerging variants or even other viral pathogens. These findings position TP2A as a promising next-generation mRNA vaccine candidate.

Background Pneumococcal pneumonia following influenza A virus (IAV) infection is a synergistic complication with high mortality in which IAV infection modulates host antibacterial responses and affects bacterial invasiveness of Streptococcus pneumoniae ( S. pn. ). IAV-mediated effects can last beyond viral clearance. In acute IAV pneumonia, alveolar type II epithelial cells (AECII) are primary targets for viral replication and contribute to the immune response. Our study addresses sustained effects of IAV infection on AECII and consequences for their response towards different serotypes of S. pn. Methods We analyzed bacterial loads, respiratory inflammation and AECII gene transcription profiling in mice infected with IAV and/or one of three S. pn. serotypes of varying invasiveness (4 > 7F > 19F). We inferred a scale-free-like ARACNE gene co-expression network on AECII transcriptional regulation under these conditions. We performed Western blotting for protein expression of interferon signaling components in AECII. We additionally performed ATAC-seq analysis of AECII isolated 14 days following IAV infection. Results Previous IAV infection rendered the lung susceptible to invasive S. pn. infection with serotype 4 and the mildly invasive 7F but not 19F. Particularly secondary infection with 7F induced exacerbated inflammatory responses as compared to bacterial infection alone, marked by increased protein expression of type I and II interferons. AECII gene co-expression network revealed interferon-response network modules. Network-mapping unfolded S. pn. serotype-specific transcriptional network responses/usage and secondary S. pn. infection was found to abrogate an IAV-induced AECII proliferative configuration. Enhanced expression of several ARACNE network genes were found to be associated with increased chromatin accessibility at their promoter regions. Conclusions Our study demonstrates AECII to retain a sustained IAV-associated configuration with epigenetic involvement, affecting their proliferation and serotype-specifically intensifying their transcriptional response, mainly to interferons, in secondary S. pn. infection. In a broader context, our results suggest the concepts of peripheral inflammatory imprinting and trained innate immunity to apply to cells of the respiratory epithelium in the context of subsequent viral/bacterial challenges.

Background: Patients with ulcerative colitis (UC) suffer from a chronic relapsing-remitting inflammatory bowel disease and show heterogeneous disease severity and therapy response. It was recently reported that rectal biopsies from patients with UC show a downregulation of various mitochondrial genes during active UC. Methods: We used dextran sulfate sodium (DSS)-induced colitis as a preclinical mouse model for UC to study metabolic characteristics of ex vivo colonic lamina propria CD4+ and CD8+ T cells, B cells, eosinophils, neutrophils and intestinal epithelial cells during experimental acute and chronic inflammatory bowel disease and remission state. Results: Our results indicate that CD4+ and CD8+ T cells in the colon produce significantly less mitochondrial reactive oxygen species and possess smaller mitochondria during chronic DSS-induced colitis, which is resolved during remission state. In addition, CD4+ and CD8+ T cells exhibit increased glucose uptake during acute but defective glucose consumption during chronic DSS colitis. Conclusions: Together, our data provide evidence for an atypical mitochondrial phenotype of colonic CD4+ and CD8+ T cells during chronic colitis that is resolved during the remission phase of the disease.

Astrocytes are central to the pathogenesis of multiple sclerosis; however, their regulation by intrinsic post-translational ubiquitination and deubiquitination is unresolved. This study shows that the deubiquitinating enzyme OTUD7B in astrocytes confers protection against murine experimental autoimmune encephalomyelitis, a model of MS, by limiting neuroinflammation. RNA-sequencing of isolated astrocytes and spatial transcriptomics showed that in EAE OTUD7B downregulates the expression of chemokines in astrocytes of inflammatory lesions, which is associated with reduced recruitment of encephalitogenic CD4 + T cells. Furthermore, OTUD7B was essential for GFAP protein expression of astrocytes bordering inflammatory lesions. Mechanistically, OTUD7B (i) restricted TNF-induced chemokine production of astrocytes by sequential K63- and K48-deubiquitination of RIPK1 limiting NF-κB and MAPK activation and (ii) enabled GFAP protein expression by supporting GFAP mRNA expression and preventing its proteasomal degradation through K48-deubiquitination of GFAP. This dual action on TNF signaling and GFAP identifies astrocyte-intrinsic OTUD7B as a central inhibitor of astrocyte-mediated inflammation.

Background: Fibrosis is characterized by excessive extracellular matrix formation in solid organs, disrupting tissue architecture and function. The Y-box binding protein-1 (YB-1) regulates fibrosis-related genes (e.g., Col1a1, Mmp2, and Tgfβ1) and contributes significantly to disease progression. This study aims to identify fibrogenic signatures and the underlying signaling pathways modulated by YB-1. Methods: Transcriptomic changes associated with matrix gene patterns in human chronic kidney diseases and murine acute injury models were analyzed with a focus on known YB-1 targets. Ybx1-knockout mouse strains (Ybx1ΔRosaERT+TX and Ybx1ΔLysM) were subjected to various kidney injury models. Fibrosis patterns were characterized by histopathological staining, transcriptome analysis, qRT-PCR, methylation analysis, zymography, and Western blotting. Results: Integrative transcriptomic analyses revealed that YB-1 is involved in several fibrogenic signatures related to the matrisome, the WNT, YAP/TAZ, and TGFß pathways, and regulates Klotho expression. Changes in the methylation status of the Klotho promoter by specific methyltransferases (DNMT) are linked to YB-1 expression, extending to other fibrogenic genes. Notably, kidney-resident cells play a significant role in YB-1-modulated fibrogenic signaling, whereas infiltrating myeloid immune cells have a minimal impact. Conclusions: YB-1 emerges as a master regulator of fibrogenesis, guiding DNMT1 to fibrosis-related genes. This highlights YB-1 as a potential target for epigenetic therapies interfering in this process.

IntroductionAllergic asthma has been mainly attributed to T helper type 2 (Th2) and proinflammatory responses but many cellular processes remain elusive. There is increasing evidence for distinct roles for macrophage and dendritic cell (DC) subsets in allergic airway inflammation (AAI). At the same time, there are various mouse models for allergic asthma that have been of utmost importance in identifying key inflammatory pathways in AAI but that differ in the allergen and/or route of sensitization. It is unclear whether and how the accumulation and activation of specialized macrophage and DC subsets depend on the experimental model chosen for analyses.MethodsIn our study, we employed high-parameter spectral flow cytometry to comprehensively assess the accumulation and phenotypic alterations of different macrophage- and DC-subsets in the lung in an OVA- and an HDM-mediated mouse model of AAI.ResultsWe observed subset-specific as well as model-specific characteristics with respect to cell numbers and functional marker expression. Generally, alveolar as opposed to interstitial macrophages showed increased MHCII surface expression in AAI. Between the models, we observed significantly increased numbers of alveolar macrophages, CD103+ DC and CD11b+ DC in HDM-mediated AAI, concurrent with significantly increased airway interleukin-4 but decreased total serum IgE levels. Further, increased expression of CD80 and CD86 on DC was exclusively detected in HDM-mediated AAI.DiscussionOur study demonstrates a model-specific involvement of macrophage and DC subsets in AAI. It further highlights spectral flow cytometry as a valuable tool for their comprehensive analysis under inflammatory conditions in the lung.

Pneumococcal infection following influenza A virus (IAV) pneumonia is a synergistic complication with high mortality. IAV modulates host antibacterial responses and invasiveness of pneumococcal serotypes and is an important pathogenic factor 2 . Yet, serotype-specifc immediate-early responses of the IAV-perturbed alveolar epithelium have not been adressed. We analyzed gene transcription in alveolar type II epithelial cells (AECII) from mice infected with IAV and/or one of three S. pneumoniae ( S.pn. ) serotypes of varying invasiveness (4 > 7F > 19F). IAV, 14 days post infection, rendered the lung susceptible to invasive S.pn. infection with serotype 4 and the mildly invasive 7F but not 19F. Only 7F secondary infection induced exacerbated cytokine/chemokine responses. IAV/7F infection induced superior protein expression of type I and II interferons, exceeding that in IAV/serotype 4 infection. Inference of a scale-free-like ARACNE gene co-expression network revealed interferon-response network modules. Network-mapping unfolded S.pn. serotype-specific transcriptional network responses/usage. Secondary S.pn. infection abrogated the IAV-induced pneumocyte proliferative configuration and IAV infection rendered the transcriptional response to 7F comparable to that of serotype 4. This related to network genes correlating with the expression of two master regulators of interferon responses: Irf7 and Stat1 . Epigenetic ATAC-seq analysis of AECII in resolved IAV infection identified enhanced expression of ARACNE network genes Hist1h2bf , Igtp , Mki67 , Rasl10b , H2-Q6 and H2-Q7 to be associated with increased chromatin accessability at promoter regions. We show that AECII retain a sustained IAV-associated transcriptional configuration with epigenetic involvement that serotype-specifically affects proliferation and intensifies the AECII transcriptional response, mainly to interferons, in S.pn. infection.

Mitochondrial functionality is crucial for the execution of physiologic functions of metabolically active cells in the respiratory tract including airway epithelial cells (AECs). Cigarette smoke is known to impair mitochondrial function in AECs. However, the potential contribution of mitochondrial dysfunction in AECs to airway infection and airway epithelial barrier dysfunction is unknown. In this study, we used an in vitro model based on AECs exposed to cigarette smoke extract (CSE) followed by an infection with Streptococcus pneumoniae (Sp). The levels of oxidative stress as an indicator of mitochondrial stress were quantified upon CSE and Sp treatment. In addition, expression of proteins associated with mitophagy, mitochondrial content, and biogenesis as well as mitochondrial fission and fusion was quantified. Transcriptional AEC profiling was performed to identify the potential changes in innate immune pathways and correlate them with indices of mitochondrial function. We observed that CSE exposure substantially altered mitochondrial function in AECs by suppressing mitochondrial complex protein levels, reducing mitochondrial membrane potential and increasing mitochondrial stress and mitophagy. Moreover, CSE-induced mitochondrial dysfunction correlated with reduced enrichment of genes involved in apical junctions and innate immune responses to Sp, particularly type I interferon responses. Together, our results demonstrated that CSE-induced mitochondrial dysfunction may contribute to impaired innate immune responses to Sp.

IκB proteins regulate the inhibition and activation of NF-κB transcription factor complexes. While classical IκB proteins keep NF-κB complexes inactive in the cytoplasm, atypical IκB proteins act on activated NF-κB complexes located in the nucleus. Most of the knowledge regarding the function of IκB proteins has been collected in vitro, while far less is known regarding their impact on activation and regulation of immune responses during in vivo infections. Combining in vivo Listeria monocytogenes (Lm) infection with comparative ex vivo transcriptional profiling of the hepatic response to the pathogen we observed that in contrast to wild type mice that mounted a robust inflammatory response, IκBNS-deficiency was generally associated with a transcriptional repression of innate immune responses. Whole tissue transcriptomics revealed a pronounced IκBNS-dependent reduction of myeloid cell-associated transcripts in the liver together with an exceptionally high Nfkbid promoter activity uncovered in Ly6Chigh inflammatory monocytes prompted us to further characterize the specific contribution of IκBNS in the inflammatory response of monocytes to the infectious agent. Indeed, Ly6Chigh monocytes primed during Lm infection in the absence of IκBNS displayed a blunted response compared to wild type-derived Ly6Chigh monocytes as evidenced by the reduced early expression of hallmark transcripts of monocyte-driven inflammation such as Il6, Nos2 and Il1β. Strikingly, altered monocyte activation in IκBNS-deficient mice was associated with an exceptional resistance against Lm infection and protection was associated with a strong reduction in immunopathology in Lm target organs. Of note, mice lacking IκBNS exclusively in myeloid cells failed to resist Lm infection, indicating that the observed effect was not monocyte intrinsic but monocyte extrinsic. While serum cytokine-profiling did not discover obvious differences between wild type and IκBNS-/- mice for most of the analyzed mediators, IL-10 was virtually undetectable in IκBNS-deficient mice, both in the steady state and following Lm infection. Together, we show here a crucial role for IκBNS during Lm infection with IκBNS-deficient mice showing an overall blunted pro-inflammatory immune response attributed to a reduced pro-inflammatory signature in Ly6Chigh monocytes. Reduced immunopathology and complete protection of mice against an otherwise fatal Lm infection identified IκBNS as molecular driver of inflammation in listeriosis.