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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.

BackgroundDuplications at the Xp21.2 locus have previously been linked to 46,XY gonadal dysgenesis (GD), which is thought to result from gene dosage effects of NR0B1 (DAX1), but the exact disease mechanism remains unknown.MethodsPatients with 46,XY GD were analysed by whole genome sequencing. Identified structural variants were confirmed by array CGH and analysed by high-throughput chromosome conformation capture (Hi-C).ResultsWe identified two unrelated patients: one showing a complex rearrangement upstream of NR0B1 and a second harbouring a 1.2 Mb triplication, including NR0B1. Whole genome sequencing and Hi-C analysis revealed the rewiring of a topological-associated domain (TAD) boundary close to NR0B1 associated with neo-TAD formation and may cause enhancer hijacking and ectopic NR0B1 expression. Modelling of previous Xp21.2 structural variations associated with isolated GD support our hypothesis and predict similar neo-TAD formation as well as TAD fusion.ConclusionHere we present a general mechanism how deletions, duplications or inversions at the NR0B1 locus can lead to partial or complete GD by disrupting the cognate TAD in the vicinity of NR0B1. This model not only allows better diagnosis of GD with copy number variations (CNVs) at Xp21.2, but also gives deeper insight on how spatiotemporal activation of developmental genes can be disrupted by reorganised TADs causing impairment of gonadal development.

Influenza A virus (IAV) causes respiratory tract disease and is responsible for seasonal and reoccurring epidemics affecting all age groups. Next to typical disease symptoms, such as fever and fatigue, IAV infection has been associated with behavioral alterations presumably contributing to the development of major depression. Influenza A virus (IAV) causes respiratory tract disease and is responsible for seasonal and reoccurring epidemics affecting all age groups. Next to typical disease symptoms, such as fever and fatigue, IAV infection has been associated with behavioral alterations presumably contributing to the development of major depression. Previous experiments using IAV/H1N1 infection models have shown impaired hippocampal neuronal morphology and cognitive abilities, but the underlying pathways have not been fully described. In this study, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes ample peripheral immune response followed by a temporary blood-brain barrier disturbance. Although histological examination did not reveal obvious pathological processes in the brains of IAV-infected mice, detailed multidimensional flow cytometric characterization of immune cells uncovered subtle alterations in the activation status of microglial cells. More specifically, we detected an altered expression pattern of major histocompatibility complex classes I and II, CD80, and F4/80 accompanied by elevated mRNA levels of CD36, CD68, C1QA, and C3, suggesting evolved synaptic pruning. To closer evaluate how these profound changes affect synaptic balance, we established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry. The introduction of this novel technique enabled us to simultaneously quantify the abundance of pre- and postsynapses from distinct brain regions. Our data reveal a significant reduction of VGLUT1 in excitatory presynaptic terminals in the cortex and hippocampus, identifying a subtle dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations. IMPORTANCE Influenza A virus (IAV) causes mainly respiratory tract disease with fever and fatigue but is also associated with behavioral alterations in humans. Here, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes peripheral immune response followed by a temporary blood-brain barrier disturbance. Characterization of immune cells uncovered subtle alterations in the activation status of microglia cells that might reshape neuronal synapses. We established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry to more closely study the synapses. Thus, we detected a specific dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations.

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. Plain english summary After an influenza A virus (IAV) infection, the lungs become more vulnerable to bacterial infections caused by Streptococcus pneumoniae ( S. pn. ). This effect can last even after the virus is cleared. Alveolar type II epithelial cells (AECII) in the lungs are primary targets for IAV and play a crucial role in the immune response. Our study examines how IAV infection affects AECII and their response to different S. pn. serotypes. We studied mice infected with IAV and three S. pn. serotypes to analyze bacterial loads, inflammation, and gene activity in AECII. We discovered that previous IAV infection made lungs more susceptible to certain S. pn. serotypes. Specifically, secondary infection with serotype 7F caused a stronger inflammatory response, marked by increased type I and II interferons. Our research revealed that AECII maintain changes caused by IAV, affecting their growth and response to bacteria. These changes are linked to alterations in gene accessibility within AECII. In summary, IAV leaves lasting effects on lung cells, making them more responsive to specific bacterial infections through enhanced interferon responses. This supports the idea that lung cells “remember” past infections, which affects their response to new ones, reflecting the concept of “trained immunity”.

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

Background: Clostridioides difficile infection (CDI) poses a health burden to patients and a financial burden to hospital systems. Timely identification of CDI patients can reduce the impacts by allowing for prompt treatment and ensuring that proper isolation precautions are in place to prevent spread. It also ensures correct CDI event categorization according to the NHSN. Community-onset (CO) CDI cases are tested on or prior to hospital day 3, and hospital-onset (HO) CDI are tested on or after hospital day 4. The objective of this study was to determine the effectiveness of utilizing an electronic health record (EHR) report to reduce CDI standard infection ratios (SIRs) by identifying potential CDI cases prior to hospital day 4. Methods: From August of 2021 to September 2022, an EHR report was implemented in a 5-hospital healthcare system in the Midwest to identify patients with 3 or more type 6 or 7 stools in a 24-hour period based on Bristol stool chart classification. All inpatients with 3 or more type 6 or 7 stools in 24 hours without an active order for a Clostridioides difficile test were listed. Patients with a laxative in the previous 48 hours, tube feedings without fever or leukocytosis, or a known cause of diarrhea were excluded. The attending provider of the patients meeting criteria were notified with a recommendation to test for C. difficile or provide alternative reason for symptoms. Results: In total, 26 patients were tested for C. difficile using polymerase chain reaction testing. Of those tested, 5 (19.2%) tested positive for C. difficile . There were 13 HO-CDI cases for the healthcare system during this period, for an SIR of 0.351. If the early identified cases were not identified until after hospital day 3, the SIR had the potential to have been 35.6% greater at 0.476. Conclusions: We were able to identify 5 CDI cases prior to hospital day 4 using an early identification report during this 13-month period. Although these cases may have been identified without the use of the EHR report, we were able to obtain a timely CDI diagnosis, potentially limiting the spread of C. difficile and preventing an increase in the CDI SIR by 35.6%. An EHR report to identify patients meeting C. difficile testing criteria may be an effective way to identify CO-CDI prior to HD 4 and thus reduce CDI SIR Disclosures: None

The adhesion molecule and co-receptor of receptor tyrosine kinases, CD44, is expressed in all cells of the immune system, but also in numerous non-immune cells. CD44 plays roles in the cellular response to different pathogens. The molecular actions of CD44 during these processes are by and large still unknown. The CD44 molecule undergoes a sequential proteolytic cleavage which leads to the release of a soluble intracellular domain (CD44-ICD). Previous reports had shown that the CD44-ICD is taken up into the nucleus where it enhances transcription of specific target genes. By RNA profiling we identified a CD44-dependent transcriptional increase of interferon-responsive genes, among them IFI16. IFI16 is important in the innate immune response. It senses and binds pathogenic DNA and, together with cGAS, activates the cGAS-cGAMP-STING pathway and induces the expression of genes relevant for the response, e.g. IFN-β. Our results show that the enhancement of IFI16 expression depended on CD44 cleavage. A CD44-negative tumor cell line, embryonic fibroblasts and bone marrow-derived macrophages from cd44-/- mice were reduced in their response to IFN-γ, to viral DNA fragments and to Listeria monocytogenes infection. We could rescue the deficiency of CD44 negative RPM-MC cells and cd44-/- MEFs by expressing only the soluble CD44-ICD in the absence of any other CD44 domain. Expression of the CD44-ICD carrying a mutation that prevented the uptake into the nucleus, could not rescue the absence of CD44. This molecular aspect of regulation by CD44 may explain part of the immune phenotypes of mice with cd44 gene disruption.

During the course of respiratory infections such as influenza, we do have a very limited view of immunological indicators to objectively and quantitatively evaluate the outcome of a host. Methods for monitoring immunological markers in a host’s lungs are invasive and expensive, and some of them are not feasible to perform. The tracking of pathogen burden and host responses with minimally invasive methods during respiratory infections is central for monitoring disease development and guiding treatment decisions. Utilizing a standardized murine model of respiratory influenza A virus (IAV) infection, we developed and tested different supervised machine learning models to predict viral burden and immune response markers, i.e., cytokines and leukocytes in the lung, from hematological data. We performed independently in vivo infection experiments to acquire extensive data for training and testing of the models. We show here that lung viral load, neutrophil counts, cytokines (such as gamma interferon [IFN-γ] and interleukin 6 [IL-6]), and other lung infection markers can be predicted from hematological data. Furthermore, feature analysis of the models showed that blood granulocytes and platelets play a crucial role in prediction and are highly involved in the immune response against IAV. The proposed in silico tools pave the path toward improved tracking and monitoring of influenza virus infections and possibly other respiratory infections based on minimally invasively obtained hematological parameters. IMPORTANCE During the course of respiratory infections such as influenza, we do have a very limited view of immunological indicators to objectively and quantitatively evaluate the outcome of a host. Methods for monitoring immunological markers in a host’s lungs are invasive and expensive, and some of them are not feasible to perform. Using machine learning algorithms, we show for the first time that minimally invasively acquired hematological parameters can be used to infer lung viral burden, leukocytes, and cytokines following influenza virus infection in mice. The potential of the framework proposed here consists of a new qualitative vision of the disease processes in the lung compartment as a noninvasive tool.

Kappa-opioid receptors (KORs) are important for motivation and other medial prefrontal cortex (mPFC)-dependent behaviors. Although KORs are present in the mPFC, their role in regulating transmission in this brain region and their contribution to KOR-mediated aversion are not known. Using in vivo microdialysis in rats and mice, we demonstrate that intra-mPFC administration of the selective KOR agonist U69,593 decreased local dopamine (DA) overflow, while reverse dialysis of the KOR antagonist nor-Binaltorphimine (nor-BNI) enhanced mPFC DA overflow. Extracellular amino-acid levels were also affected by KORs, as U69,593 reduced glutamate and GABA levels driven by the glutamate reuptake blocker, l-trans-pyrrolidine-2,4-dicarboxylate. Whole-cell recordings from mPFC layer V pyramidal neurons revealed that U69,593 decreased the frequency, but not amplitude, of glutamatergic mini EPSPs. To determine whether KOR regulation of mPFC DA overflow was mediated by KOR on DA terminals, we utilized a Cre recombinase-driven mouse line lacking KOR in DA neurons. In these mice, basal DA release or uptake was unaltered relative to controls, but attenuation of mPFC DA overflow by local U69,593 was not observed, indicating KOR acts directly on mPFC DA terminals to locally inhibit DA levels. Conditioning procedures were then used to determine whether mPFC KOR signaling was necessary for KOR-mediated aversion. U69,593-mediated conditioned place aversion was blocked by intra-mPFC nor-BNI microinjection. These findings demonstrate that mPFC KORs negatively regulate DA and amino-acid neurotransmission, and are necessary for KOR-mediated aversion.

During influenza A virus (IAV) infections, CD4+ T cell responses within infected lungs mainly involve T helper 1 (Th1) and regulatory T cells (Tregs). Th1-mediated responses favor the co-expression of T-box transcription factor 21 (T-bet) in Foxp3+ Tregs, enabling the efficient Treg control of Th1 responses in infected tissues. So far, the exact accumulation kinetics of T cell subsets in the lungs and lung-draining lymph nodes (dLN) of IAV-infected mice is incompletely understood, and the epigenetic signature of Tregs accumulating in infected lungs has not been investigated. Here, we report that the total T cell and the two-step Treg accumulation in IAV-infected lungs is transient, whereas the change in the ratio of CD4+ to CD8+ T cells is more durable. Within lungs, the frequency of Tregs co-expressing T-bet is steadily, yet transiently, increasing with a peak at Day 7 post-infection. Interestingly, T-bet+ Tregs accumulating in IAV-infected lungs displayed a strongly demethylated Tbx21 locus, similarly as in T-bet+ conventional T cells, and a fully demethylated Treg-specific demethylated region (TSDR) within the Foxp3 locus. In summary, our data suggest that T-bet+ but not T-bet− Tregs are epigenetically stabilized during IAV-induced infection in the lung.