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.

Systemic dysregulation and molecular insights into poor influenza vaccine response in the aging population.

Vaccination-induced protection against influenza is greatly diminished and increasingly heterogeneous with age. We investigated longitudinally (up to five time points) a cohort of 234 vaccinated >65-year-old vaccinees with adjuvanted vaccine FluAd across two independent seasons. System-level analyses of multiomics datasets measuring six modalities and serological data revealed that poor responders lacked time-dependent changes in response to vaccination as observed in responders, suggestive of systemic dysregulation in poor responders. Multiomics integration revealed key molecules and their likely role in vaccination response. High prevaccination plasma interleukin-15 (IL-15) concentrations negatively associated with antibody production, further supported by experimental validation in mice revealing an IL-15-driven natural killer cell axis explaining the suppressive role in vaccine-induced antibody production as observed in poor responders. We propose a subset of long-chain fatty acids as modulators of persistent inflammation in poor responders. Our findings provide a potential link between low-grade chronic inflammation and poor vaccination response and open avenues for possible pharmacological interventions to enhance vaccine responses.

Sex-specific epigenetic signatures of circulating urate and its increase after BCG vaccination.

Background: Urate concentration and the physiological regulation of urate homeostasis exhibit clear sex differences. DNA methylation has been shown to explain a substantial proportion of serum urate variance, mediate the genetic effect on urate concentration, and co-regulate with cardiometabolic traits. However, whether urate concentration is associated with DNA methylation in a sex-dependent manner is unknown. Additionally, it is worth investigating if urate changes after perturbations, such as vaccination, are associated with DNA methylation in a sex-specific manner. Methods: We investigated the association between DNA methylation and serum urate concentrations in a Dutch cohort of 325 healthy individuals. Urate concentration and DNA methylation were measured before and after Bacillus Calmette-Guérin (BCG) vaccination, used as a perturbation associated with increased gout flares. The association analysis included united, interaction, and sex-stratified analysis. Validation of the identified CpG sites was conducted using three independent cohorts. Results: 215 CpG sites were associated with serum urate in males, while 5 CpG sites were associated with serum urate in females, indicating sex-specific associations. Circulating urate concentrations significantly increased after BCG vaccination, and baseline DNA methylation was associated with differences in urate concentration before and after vaccination in a sex-specific manner. The CpG sites associated with urate concentration in males were enriched in neuro-protection pathways, whereas in females, the urate change-associated CpG sites were related to lipid and glucose metabolism. Conclusion: Our study enhances the understanding of how epigenetic factors contribute to regulating serum urate levels in a sex-specific manner. These insights have significant implications for the diagnosis, prevention, and treatment of various urate-related diseases and highlight the importance of personalized and sex-specific approaches in medicine.

Targeted metabolomics identifies accurate CSF metabolite biomarkers for the differentiation between COVID-19 with neurological involvement and CNS infections with neurotropic viral pathogens.

BACKGROUND: COVID-19 is primarily considered a respiratory tract infection, but it can also affect the central nervous system (CNS), which can result in long-term sequelae. In contrast to CNS infections by classic neurotropic viruses, SARS-CoV-2 is usually not detected in cerebrospinal fluid (CSF) from patients with COVID-19 with neurological involvement (neuro-COVID), suggesting fundamental differences in pathogenesis. METHODS: To assess differences in CNS metabolism in neuro-COVID compared to CNS infections with classic neurotropic viruses, we applied a targeted metabolomic analysis of 630 metabolites to CSF from patients with (i) COVID-19 with neurological involvement [n = 16, comprising acute (n = 13) and post-COVID-19 (n = 3)], (ii) viral meningitis, encephalitis, or myelitis (n = 10) due to herpes simplex virus (n = 2), varicella zoster virus (n = 6), enterovirus (n = 1) and tick-borne encephalitis virus (n = 1), and (iii) aseptic neuroinflammation (meningitis, encephalitis, or myelitis) of unknown etiology (n = 21) as additional disease controls. RESULTS: Standard CSF parameters indicated absent or low neuroinflammation in neuro-COVID. Indeed, CSF cell count was low in neuro-COVID (median 1 cell/µL, range 0-12) and discriminated it accurately from viral CNS infections (AUC = 0.99) and aseptic neuroinflammation (AUC = 0.98). 32 CSF metabolites passed quality assessment and were included in the analysis. Concentrations of differentially abundant (fold change ≥|1.5|, FDR ≤ 0.05) metabolites were both higher (9 and 5 metabolites) and lower (2 metabolites) in neuro-COVID than in the other two groups. Concentrations of citrulline, ceramide (d18:1/18:0), and methionine were most significantly elevated in neuro-COVID. Remarkably, triglyceride TG(20:1_32:3) was much lower (mean fold change = 0.09 and 0.11) in neuro-COVID than in all viral CNS infections and most aseptic neuroinflammation samples, identifying it as highly accurate biomarker with AUC = 1 and 0.93, respectively. Across all samples, TG(20:1_32:3) concentration correlated only moderately with CSF cell count (ρ = 0.65), protein concentration (ρ = 0.64), and Q-albumin (ρ = 0.48), suggesting that its low levels in neuro-COVID CSF are only partially explained by less pronounced neuroinflammation. CONCLUSIONS: The results suggest that CNS metabolite responses in neuro-COVID differ fundamentally from viral CNS infections and aseptic neuroinflammation and may be used to discover accurate diagnostic biomarkers in CSF and to gain insights into differences in pathophysiology between neuro-COVID, viral CNS infections and aseptic neuroinflammation.

Elevated phospholipids and acylcarnitines C4 and C5 in cerebrospinal fluid distinguish viral CNS infections from autoimmune neuroinflammation.

BACKGROUND: Viral and autoimmune encephalitis may present with similar symptoms, but require different treatments. Thus, there is a need for biomarkers to improve diagnosis and understanding of pathogenesis. We hypothesized that virus-host cell interactions lead to different changes in central nervous system (CNS) metabolism than autoimmune processes and searched for metabolite biomarkers in cerebrospinal fluid (CSF) to distinguish between the two conditions. METHODS: We applied a targeted metabolomic/lipidomic analysis to CSF samples from patients with viral CNS infections (n = 34; due to herpes simplex virus [n = 9], varicella zoster virus [n = 15], enteroviruses [n = 10]), autoimmune neuroinflammation (n = 25; autoimmune anti-NMDA-receptor encephalitis [n = 8], multiple sclerosis [n = 17), and non-inflamed controls (n = 31; Gilles de la Tourette syndrome [n = 20], Bell's palsy with normal CSF cell count [n = 11]). 85 metabolites passed quality screening and were evaluated as biomarkers. Standard diagnostic CSF parameters were assessed for comparison. RESULTS: Of the standard CSF parameters, the best biomarkers were: CSF cell count for viral infections vs. controls (area under the ROC curve, AUC = 0.93), Q-albumin for viral infections vs. autoimmune neuroinflammation (AUC = 0.86), and IgG index for autoimmune neuroinflammation vs. controls (AUC = 0.90). Concentrations of 2 metabolites differed significantly (p < 0.05) between autoimmune neuroinflammation and controls, with proline being the best biomarker (AUC = 0.77). In contrast, concentrations of 67 metabolites were significantly higher in viral infections than controls, with SM.C16.0 being the best biomarker (AUC = 0.94). Concentrations of 68 metabolites were significantly higher in viral infections than in autoimmune neuroinflammation, and the 10 most accurate metabolite biomarkers (AUC = 0.89-0.93) were substantially better than Q-albumin (AUC = 0.86). These biomarkers comprised six phosphatidylcholines (AUC = 0.89-0.92), two sphingomyelins (AUC = 0.89, 0.91), and acylcarnitines isobutyrylcarnitine (C4, AUC = 0.92) and isovalerylcarnitine (C5, AUC = 0.93). Elevated C4 and C5 concentrations suggested dysfunctional mitochondrial ß-oxidation and correlated only moderately with CSF cell count (Spearman ρ = 0.41 and 0.44), indicating that their increase is not primarily driven by inflammation. CONCLUSIONS: Changes in CNS metabolism differ substantially between viral CNS infections and autoimmune neuroinflammation and reveal CSF metabolites as pathophysiologically relevant diagnostic biomarkers for the differentiation between the two conditions. In viral CNS infections, the observed higher concentrations of free phospholipids are consistent with disruption of host cell membranes, whereas the elevated short-chain acylcarnitines likely reflect compromised mitochondrial homeostasis and energy generation.

The Changing Epidemiology of Viral Hepatitis in a Post-Soviet Country-The Case of Kyrgyzstan.

Historically, viral hepatitis has been a considerable public health problem in Central Asian countries, which may have worsened after the dissolution of the Soviet Union. However, up-to-date seroepidemiological studies are lacking. The aim of the present study was, therefore, to provide current estimates of the seroprevalence of viral hepatitis in Kyrgyzstan, one of the economically least developed countries in the region. We conducted a population-based cross-sectional study in 2018 in the capital of Kyrgyzstan, Bishkek (n = 1075). Participants, children and adults, were recruited from an outpatient clinic. The data were collected during face-to-face interviews. A blood sample (6 mL) was collected from each participant and tested with ELISA for the presence of serological markers for five viral hepatitides (A, B, C, D, and E). Post-stratification weighing was performed to obtain nationally representative findings. The overwhelming majority of the study participants were positive for anti-HAV (estimated seroprevalence, 75.3%; 95% confidence interval, 72.5-77.9%). The weighted seroprevalence estimates of HBsAg, anti-HCV, and anti-HDV were 2.2% (1.5-3.3%), 3.8% (2.8-5.1%), and 0.40% (0.15-1.01%), respectively. Anti-HEV seropositivity was 3.3% (2.4-4.5%). Of the 33 HBsAg-positive participants, five (15%) were anti-HDV-positive. Our study confirms that Kyrgyzstan remains a highly endemic country for hepatitis virus A and C infections. However, seroprevalences of HBV and HDV were lower than previously reported, and based on these data, the country could potentially be reclassified from high to (lower) intermediate endemicity. The observed anti-HEV seroprevalence resembles the low endemicity pattern characteristic of high-income countries.

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner.

In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.

Amino acid positions near the active site determine the reduced activity of human ACOD1 compared to murine ACOD1.

cis-Aconitate decarboxylase (ACOD1, IRG1) converts cis-aconitate to the immunomodulatory and antibacterial metabolite itaconate. Although the active site residues of human and mouse ACOD1 are identical, the mouse enzyme is about fivefold more active. Aiming to identify the cause of this difference, we mutated positions near the active site in human ACOD1 to the corresponding residues of mouse ACOD1 and measured resulting activities in vitro and in transfected cells. Interestingly, Homo sapiens is the only species with methionine instead of isoleucine at residue 154 and introduction of isoleucine at this position increased the activity of human ACOD1 1.5-fold in transfected cells and 3.5-fold in vitro. Enzyme activity of gorilla ACOD1, which is almost identical to the human enzyme but has isoleucine at residue 154, was similar to the mouse enzyme in vitro. Met154 in human ACOD1 forms a sulfur-π bond to Phe381, which is positioned to impede access of the substrate to the active site. It appears that the ACOD1 sequence has changed at position 154 during human evolution, resulting in a pronounced decrease in activity. This change might have offered a selective advantage in diseases such as cancer.

Generation of two human NRF2 knockout iPSC clones using CRISPR/Cas9 editing.

The nuclear factor erythroid 2-related factor 2 (NFE2L2, known as NRF2) regulates the expression of antioxidative and anti-inflammatory proteins. In order to investigate its impact during viral infections and testing of antiviral compounds, we applied CRISPR/Cas9 editing to eliminate NRF2 in the human iPS cell line MHHi001-A and generated two NRF2 knockout iPSC clones MHHi001-A-6 and MHHi001-A-7. After differentiation into epithelia or endothelial cells, these cells are useful tools to examine the antiviral effects of activators of the NRF2 signaling pathway.

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

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

Distinct immunological and molecular signatures underpinning influenza vaccine responsiveness in the elderly.

Seasonal influenza outbreaks, especially in high-risk groups such as the elderly, represent an important public health problem. Prevailing inadequate efficacy of seasonal vaccines is a crucial bottleneck. Understanding the immunological and molecular mechanisms underpinning differential influenza vaccine responsiveness is essential to improve vaccination strategies. Here we show comprehensive characterization of the immune response of randomly selected elderly participants (≥ 65 years), immunized with the adjuvanted influenza vaccine Fluad. In-depth analyses by serology, multi-parametric flow cytometry, multiplex and transcriptome analysis, coupled to bioinformatics and mathematical modelling, reveal distinguishing immunological and molecular features between responders and non-responders defined by vaccine-induced seroconversion. Non-responders are specifically characterized by multiple suppressive immune mechanisms. The generated comprehensive high dimensional dataset enables the identification of putative mechanisms and nodes responsible for vaccine non-responsiveness independently of confounding age-related effects, with the potential to facilitate development of tailored vaccination strategies for the elderly.

Itaconate Isomers in Bread.

The naturally occurring isomers itaconate, mesaconate and citraconate possess immunomodulatory, antioxidative and antimicrobial properties. However, it is not known whether they occur in commonly consumed human foods. Considering that they can arise as a result of heat conversion, we tested whether they occur in bread, representing a commonly consumed baked good. Using high-performance liquid chromatography−tandem mass spectrometry, we measured concentrations of the three isomers and their potential precursors, citrate and cis-aconitate, in unbaked sourdough and dough, and in crumb and crust of baked bread. All three isomers were detected at low concentrations (<20 pmol/mg dry weight) in sourdough, dough, crumb and crust. Concentrations of itaconate and citraconate were substantially higher in crust than in crumb of wheat and rye bread, and a modest increase in mesaconate was observed in crust of rye bread. In contrast, cis-aconitate concentrations were considerably lower in crust, which was consistent with the conversion of cis-aconitate to itaconate isomers due to higher temperature of the dough surface during baking. Based on data on the average consumption of bread and related baked goods in Germany, the daily intake of itaconate isomers was estimated to be roughly 7−20 µg. Thus, baked goods constitute a regular dietary source of low amounts of itaconate isomers. In order to enable studies on the impact of dietary intake of itaconate isomers on human health, their concentrations should be assessed in other foods that are subjected to high heating.

ISG15 deficiency features a complex cellular phenotype that responds to treatment with itaconate and derivatives.

BACKGROUND: Congenital ISG15 deficiency is a rare autoinflammatory disorder that is driven by chronically elevated systemic interferon levels and predominantly affects central nervous system and skin. METHODS AND RESULTS: We have developed induced pluripotent stem cell-derived macrophages and endothelial cells as a model to study the cellular phenotype of ISG15 deficiency and identify novel treatments. ISG15-/- macrophages exhibited the expected hyperinflammatory responses, but normal phagocytic function. In addition, they displayed a multifaceted pathological phenotype featuring increased apoptosis/pyroptosis, oxidative stress, glycolysis, and acylcarnitine levels, but decreased glutamine uptake, BCAT1 expression, branched chain amino acid catabolism, oxidative phosphorylation, ß-oxidation, and NAD(P)H-dependent oxidoreductase activity. Furthermore, expression of genes involved in mitochondrial biogenesis and respiratory chain complexes II-V was diminished in ISG15-/- cells. Defective mitochondrial respiration was restored by transduction with wild-type ISG15, but only partially by a conjugation-deficient variant, suggesting that some ISG15 functions in mitochondrial respiration require ISGylation to cellular targets. Treatment with itaconate, dimethyl-itaconate, 4-octyl-itaconate, and the JAK1/2 inhibitor ruxolitinib ameliorated increased inflammation, propensity for cell death, and oxidative stress. Furthermore, the treatments greatly improved mitochondria-related gene expression, BCAT1 levels, redox balance, and intracellular and extracellular ATP levels. However, efficacy differed among the compounds according to read-out and cell type, suggesting that their effects on cellular targets are not identical. Indeed, only itaconates increased expression of anti-oxidant genes NFE2L2, HMOX1, and GPX7, and dimethyl-itaconate improved redox balance the most. Even though itaconate treatments normalized the elevated expression of interferon-stimulated genes, ISG15-/- macrophages maintained their reduced susceptibility to influenza virus infection. CONCLUSIONS: These findings expand the cellular phenotype of human ISG15 deficiency and reveal the importance of ISG15 for regulating oxidative stress, branched chain amino acid metabolism, and mitochondrial function in humans. The results validate ruxolitinib as treatment for ISG15 deficiency and suggest itaconate-based medications as additional therapeutics for this rare disorder.

Reprogramming of Amino Acid Metabolism Differs between Community-Acquired Pneumonia and Infection-Associated Exacerbation of Chronic Obstructive Pulmonary Disease.

Amino acids and their metabolites are key regulators of immune responses, and plasma levels may change profoundly during acute disease states. Using targeted metabolomics, we evaluated concentration changes in plasma amino acids and related metabolites in community-acquired pneumonia (CAP, n = 29; compared against healthy controls, n = 33) from presentation to hospital through convalescence. We further aimed to identify biomarkers for acute CAP vs. the clinically potentially similar infection-triggered COPD exacerbation (n = 13). Amino acid metabolism was globally dysregulated in both CAP and COPD. Levels of most amino acids were markedly depressed in acute CAP, and total amino acid concentrations on admission were an accurate biomarker for the differentiation from COPD (AUC = 0.93), as were reduced asparagine and threonine levels (both AUC = 0.92). Reduced tryptophan and histidine levels constituted the most accurate biomarkers for acute CAP vs. controls (AUC = 0.96, 0.94). Only kynurenine, symmetric dimethyl arginine, and phenylalanine levels were increased in acute CAP, and the kynurenine/tryptophan ratio correlated best with clinical recovery and resolution of inflammation. Several amino acids did not reach normal levels by the 6-week follow-up. Glutamate levels were reduced on admission but rose during convalescence to 1.7-fold above levels measured in healthy control. Our data suggest that dysregulated amino acid metabolism in CAP partially persists through clinical recovery and that amino acid metabolism constitutes a source of promising biomarkers for CAP. In particular, total amino acids, asparagine, and threonine may constitute plasma biomarker candidates for the differentiation between CAP and infection-triggered COPD exacerbation and, perhaps, the detection of pneumonia in COPD.

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.

Congenital deficiency reveals critical role of ISG15 in skin homeostasis.

Ulcerating skin lesions are manifestations of human ISG15 deficiency, a type I interferonopathy. However, chronic inflammation may not be their exclusive cause. We describe two siblings with recurrent skin ulcers that healed with scar formation upon corticosteroid treatment. Both had a homozygous nonsense mutation in the ISG15 gene, leading to unstable ISG15 protein lacking the functional domain. We characterized ISG15-/- dermal fibroblasts, HaCaT keratinocytes, and human induced pluripotent stem cell-derived vascular endothelial cells. ISG15-deficient cells exhibited the expected hyperinflammatory phenotype, but also dysregulated expression of molecules critical for connective tissue and epidermis integrity, including reduced collagens and adhesion molecules, but increased matrix metalloproteinases. ISG15-/- fibroblasts exhibited elevated ROS levels and reduced ROS scavenger expression. As opposed to hyperinflammation, defective collagen and integrin synthesis was not rescued by conjugation-deficient ISG15. Cell migration was retarded in ISG15-/- fibroblasts and HaCaT keratinocytes, but normalized under ruxolitinib treatment. Desmosome density was reduced in an ISG15-/- 3D epidermis model. Additionally, there were loose architecture and reduced collagen and desmoglein expression, which could be reversed by treatment with ruxolitinib/doxycycline/TGF-ß1. These results reveal critical roles of ISG15 in maintaining cell migration and epidermis and connective tissue homeostasis, whereby the latter likely requires its conjugation to yet unidentified targets.

Differentiation of viral and autoimmune central nervous system inflammation by kynurenine pathway.

OBJECTIVE: To determine whether the metabolites of Kynurenine pathway (KP) could serve as biomarkers for distinguishing between viral CNS infections and autoimmune neuroinflammatory diseases, especially anti-N-methyl-D-aspartate receptor encephalitis (NMDARE) and herpes virus encephalitis (HSE). METHODS: This study enrolled CSF samples from 76 patients with viral CNS infections, autoimmune neuroinflammatory, and non-inflammatory neurological diseases. We measured cerebrospinal fluid (CSF) concentrations of tryptophan (Trp) and kynurenine (Kyn) by ELISA. RESULTS: Kyn concentrations and Kyn/Trp ratios were highly increased (p < 0.001, viral vs. autoimmune) in viral CNS infections, whereas patients with autoimmune neuroinflammatory and non-inflammatory diseases exhibited low concentrations. Furthermore, Kyn concentrations and Kyn/Trp ratio turned out to be excellent biomarkers to distinguish between herpes simplex encephalitis (HSE) and NMDARE (AUC 0.920 and AUC 0.906), whereas Trp concentrations were similar in all three groups. INTERPRETATION: The results suggest that elevated CSF Kyn concentrations and Kyn/Trp ratio may serve as biomarkers for distinguishing viral CNS infections from autoimmune neuroinflammatory diseases. In particular, the distinction between HSE and NMDARE is of great clinical relevance. Further studies are warranted to investigate the potential of CSF Kyn levels and Kyn/Trp ratio as routine parameters in patients with CNS diseases.

Transcriptomic Biomarkers for Tuberculosis: Validation of NPC2 as a Single mRNA Biomarker to Diagnose TB, Predict Disease Progression, and Monitor Treatment Response.

External validation in different cohorts is a key step in the translational development of new biomarkers. We previously described three host mRNA whose expression in peripheral blood is significantly higher (NPC2) or lower (DOCK9 and EPHA4) in individuals with TB compared to latent TB infection (LTBI) and controls. We have now conducted an independent validation of these genes by re-analyzing publicly available transcriptomic datasets from Brazil, China, Haiti, India, South Africa, and the United Kingdom. Comparisons between TB and control/LTBI showed significant differential expression of all three genes (NPC2high p < 0.01, DOCK9low p < 0.01, and EPHA4low p < 0.05). NPC2high had the highest mean area under the ROC curve (AUROC) for the differentiation of TB vs. controls (0.95) and LTBI (0.94). In addition, NPC2 accurately distinguished TB from the clinically similar conditions pneumonia (AUROC, 0.88), non-active sarcoidosis (0.87), and lung cancer (0.86), but not from active sarcoidosis (0.66). Interestingly, individuals progressing from LTBI to TB showed a constant increase in NPC2 expression with time when compared to non-progressors (p < 0.05), with a significant change closer to manifestation of active disease (≤3 months, p = 0.003). Moreover, NPC2 expression normalized with completion of anti-TB treatment. Taken together, these results validate NPC2 mRNA as a diagnostic host biomarker for active TB independent of host genetic background. Moreover, they reveal its potential to predict progression from latent to active infection and to indicate a response to anti-TB treatment.

Complete Genome Sequencing of Leptospira interrogans Isolates from Malaysia Reveals Massive Genome Rearrangement but High Conservation of Virulence-Associated Genes.

The ability of Leptospirae to persist in environments and animal hosts but to cause clinically highly variable disease in humans has made leptospirosis the most common zoonotic disease. Considering the paucity of data on variation in complete genomes of human pathogenic Leptospirae, we have used a combination of Single Molecule Real-Time (SMRT) and Illumina sequencing to obtain complete genome sequences of six human clinical L. interrogans isolates from Malaysia. All six contained the larger (4.28-4.56 Mb) and smaller (0.34-0.395 Mb) chromosome typical of human pathogenic Leptospirae and 0-7 plasmids. Only 24% of the plasmid sequences could be matched to databases. We identified a chromosomal core genome of 3318 coding sequences and strain-specific accessory genomes of 49-179 coding sequences. These sequences enabled detailed genomic strain typing (Genome BLAST Distance Phylogeny, DNA-DNA hybridization, and multi locus sequence typing) and phylogenetic classification (whole-genome SNP genotyping). Even though there was some shared synteny and collinearity across the six genomes, there was evidence of major genome rearrangement, likely driven by horizontal gene transfer and homologous recombination. Mobile genetic elements were identified in all strains in highly varying numbers, including in the rfb locus, which defines serogroups and contributes to immune escape and pathogenesis. On the other hand, there was high conservation of virulence-associated genes including those relating to sialic acid, alginate, and lipid A biosynthesis. These findings suggest (i) that the antigenic variation, adaption to various host environments, and broad spectrum of virulence of L. interrogans are in part due to a high degree of genomic plasticity and (ii) that human pathogenic strains maintain a core set of genes required for virulence.