MxA for differentiating viral and bacterial infections in adults: a prospective, exploratory study.

PURPOSE: Inappropriate antibiotic prescription in patients with viral infections contributes to the surge of antibiotic resistance. Viral infections induce the expression of the antiviral protein MxA in monocytes, which is a promising biomarker to differentiate between viral and bacterial diseases. In this prospective, exploratory study, we aimed to determine the diagnostic value of monocyte MxA expression in adults with viral, bacterial or co-infections. METHODS: We measured monocyte MxA expression using flow cytometry in a cohort of 61 adults with various viral, bacterial and co-infections including patients receiving immunosuppressive therapy. RESULTS: Monocyte MxA expression in virus-infected patients was significantly higher compared to bacterial infections (83.3 [66.8, 109.4] vs. 33.8 [29.3, 47.8] mean fluorescence intensity [MFI]; p < 0.0001) but not co-infections (53.1 [33.9, 88.9] MFI). At a threshold of 62.2 MFI, the area under the ROC curve (AUC) to differentiate between viral and bacterial infections was 0.9, with a sensitivity and specificity of 92.3% and 84.6%, respectively. Immunosuppressive therapy did not affect monocyte MxA expression in virus-infected patients. CONCLUSION: Our findings corroborate the diagnostic performance of MxA in differentiating viral and bacterial infections but also point to an important caveat of MxA in viral-bacterial co-infections. This study extends previous reports and indicates that MxA is also a useful biomarker in immunocompromised patients.

The energy sensor AMPK orchestrates metabolic and translational adaptation in expanding T helper cells.

The importance of cellular metabolic adaptation in inducing robust T cell responses is well established. However, the mechanism by which T cells link information regarding nutrient supply to clonal expansion and effector function is still enigmatic. Herein, we report that the metabolic sensor adenosine monophosphate-activated protein kinase (AMPK) is a critical link between cellular energy demand and translational activity and, thus, orchestrates optimal expansion of T cells in vivo. AMPK deficiency did not affect T cell fate decision, activation, or T effector cell generation; however, the magnitude of T cell responses in murine in vivo models of T cell activation was markedly reduced. This impairment was global, as all T helper cell subsets were similarly sensitive to loss of AMPK which resulted in reduced T cell accumulation in peripheral organs and reduced disease severity in pathophysiologically as diverse models as T cell transfer colitis and allergic airway inflammation. T cell receptor repertoire analysis confirmed similar clonotype frequencies in different lymphoid organs, thereby supporting the concept of a quantitative impairment in clonal expansion rather than a skewed qualitative immune response. In line with these findings, in-depth metabolic analysis revealed a decrease in T cell oxidative metabolism, and gene set enrichment analysis indicated a major reduction in ribosomal biogenesis and mRNA translation in AMPK-deficient T cells. We, thus, provide evidence that through its interference with these delicate processes, AMPK orchestrates the quantitative, but not the qualitative, manifestation of primary T cell responses in vivo.

Rhinovirus induces an anabolic reprogramming in host cell metabolism essential for viral replication.

Rhinoviruses (RVs) are responsible for the majority of upper airway infections; despite their high prevalence and the resulting economic burden, effective treatment is lacking. We report here that RV induces metabolic alterations in host cells, which offer an efficient target for antiviral intervention. We show that RV-infected cells rapidly up-regulate glucose uptake in a PI3K-dependent manner. In parallel, infected cells enhance the expression of the PI3K-regulated glucose transporter GLUT1. In-depth metabolomic analysis of RV-infected cells revealed a critical role of glucose mobilization from extracellular and intracellular pools via glycogenolysis for viral replication. Infection resulted in a highly anabolic state, including enhanced nucleotide synthesis and lipogenesis. Consistently, we observed that glucose deprivation from medium and via glycolysis inhibition by 2-deoxyglucose (2-DG) potently impairs viral replication. Metabolomic analysis showed that 2-DG specifically reverts the RV-induced anabolic reprogramming. In addition, treatment with 2-DG inhibited RV infection and inflammation in a murine model. Thus, we demonstrate that the specific metabolic fingerprint of RV infection can be used to identify new targets for therapeutic intervention.

Histone deacetylase 1 (HDAC1): A key player of T cell-mediated arthritis.

Rheumatoid Arthritis (RA) represents a chronic T cell-mediated inflammatory autoimmune disease. Studies have shown that epigenetic mechanisms contribute to the pathogenesis of RA. Histone deacetylases (HDACs) represent one important group of epigenetic regulators. However, the role of individual HDAC members for the pathogenesis of arthritis is still unknown. In this study we demonstrate that mice with a T cell-specific deletion of HDAC1 (HDAC1-cKO) are resistant to the development of Collagen-induced arthritis (CIA), whereas the antibody response to collagen type II was undisturbed, indicating an unaltered T cell-mediated B cell activation. The inflammatory cytokines IL-17 and IL-6 were significantly decreased in sera of HDAC1-cKO mice. IL-6 treated HDAC1-deficient CD4+ T cells showed an impaired upregulation of CCR6. Selective inhibition of class I HDACs with the HDAC inhibitor MS-275 under Th17-skewing conditions inhibited the upregulation of chemokine receptor 6 (CCR6) in mouse and human CD4+ T cells. Accordingly, analysis of human RNA-sequencing (RNA-seq) data and histological analysis of synovial tissue samples from human RA patients revealed the existence of CD4+CCR6+ cells with enhanced HDAC1 expression. Our data indicate a key role for HDAC1 for the pathogenesis of CIA and suggest that HDAC1 and other class I HDACs might be promising targets of selective HDAC inhibitors (HDACi) for the treatment of RA.

Novel insights into non-HLA alloimmunity in kidney transplantation.

Recognition of non-self structures on donor cells represents the main immunological barrier in solid organ transplantation. The human leukocyte antigens (HLA) are considered the most important non-self (allo)antigens in transplantation. Long-term graft attrition is mainly caused by the formation of alloreactive antibodies that are directed against non-self structures (i.e., epitopes) on cell surface proteins. Recently published data provided evidence for a similar importance of non-HLA mismatches between donors and recipients in acute rejection as well as long-term kidney allograft survival. These data suggest a broader concept of immunological non-self that goes beyond HLA incompatibility and expands the current concept of polymorphic non-self epitopes on cell surface molecules from HLA to non-HLA targets. Amino acid substitutions caused by single nucleotide variants in protein-coding genes or complete loss of gene expression represent the basis for polymorphic residues in both HLA and non-HLA molecules. To better understand these novel insights in non-HLA alloimmunity, we will first review basic principles of the alloimmune response with a focus on the HLA epitope concept in donor-specific antibody formation before discussing key publications on non-HLA antibodies.

Interleukin 1 Receptor Antagonist Anakinra, Intravenous Immunoglobulin, and Corticosteroids in the Management of Critically Ill Adult Patients With Hemophagocytic Lymphohistiocytosis.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) causes multiple organ dysfunction frequently leading to intensive care unit (ICU) referral and/or death. We report on a series of critically ill adult patients treated with a non-etoposide-based regimen including interleukin 1 antagonist anakinra, intravenous immunoglobulin (IVIG), and/or corticosteroids (CS) for HLH. METHODS: Eight adult (≥18 years) ICU patients having received treatment with anakinra ± IVIG ± CS for HLH between March 2014 and March 2016 at a large tertiary care university hospital (Medical University of Vienna, Vienna, Austria) were retrospectively analyzed. RESULTS: Eight patients (median age: 38 years; range: 20-58 years; 4 males and 4 females) received anakinra together with IVIG (n = 7) and/or high-dose CS (n = 5) for suspected reactive HLH (median H-score: 214; range: 171-288). Seven (88%) patients required vasopressors and invasive mechanical ventilation and 6 (75%) patients required renal replacement therapy (median Sequential Organ Failure Assessment [SOFA] score at HLH diagnosis: 9.5; range: 6-14). Six patients showed a significant decline in the SOFA score at 1 and 2 weeks following treatment initiation (P = .03), and the remainder 2 patients experienced early death. Five patients survived to ICU discharge, 4 of them could further be discharged from hospital (hospital survival rate: 50%). No overt treatment-related toxicity was noted. CONCLUSION: Anakinra in combination with IVIG and/or CS resulted in a hospital survival rate of 50% in 8 critically ill adult patients with HLH despite a vast degree of organ dysfunction and the need for aggressive ICU treatment. Further research on non-etoposide-based treatment strategies for HLH in critically ill adults is warranted.

Next generation sequencing based assessment of the alloreactive T cell receptor repertoire in kidney transplant patients during rejection: a prospective cohort study.

BACKGROUND: Kidney transplantation is the optimal treatment in end stage renal disease but the allograft survival is still hampered by immune reactions against the allograft. This process is driven by the recognition of allogenic antigens presented to T-cells and their unique T-cell receptor (TCR) via the major histocompatibility complex (MHC), which triggers a complex immune response potentially leading to graft injury. Although the immune system and kidney transplantation have been studied extensively, the subtlety of alloreactive immune responses has impeded sensitive detection at an early stage. Next generation sequencing of the TCR enables us to monitor alloreactive T-cell populations and might thus allow the detection of early rejection events. METHODS/DESIGN: This is a prospective cohort study designed to sequentially evaluate the alloreactive T cell repertoire after kidney transplantation. The TCR repertoire of patients who developed biopsy confirmed acute T cell mediated rejection (TCMR) will be compared to patients without rejection. To track the alloreactive subsets we will perform a mixed lymphocyte reaction between kidney donor and recipient before transplantation and define the alloreactive TCR repertoire by next generation sequencing of the complementary determining region 3 (CDR3) of the T cell receptor beta chain. After initial clonotype assembly from sequencing reads, TCR repertoire diversity and clonal expansion of T cells of kidney transplant recipients in periphery and kidney biopsy will be analyzed for changes after transplantation, during, prior or after a rejection. The goal of this study is to describe changes of overall T cell repertoire diversity, clonality in kidney transplant recipients, define and track alloreactive T cells in the posttransplant course and decipher patterns of expanded alloreactive T cells in acute cellular rejection to find an alternative monitoring to invasive and delayed diagnostic procedures. DISCUSSION: Changes of the T cell repertoire and tracking of alloreactive T cell clones after combined bone marrow and kidney transplant has proven to be of potential use to monitor the donor directed alloresponse. The dynamics of the donor specific T cells in regular kidney transplant recipients in rejection still rests elusive and can give further insights in human alloresponse. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03422224 , registered February 5th 2018.

Quantification of Torque Teno Virus Viremia as a Prospective Biomarker for Infectious Disease in Kidney Allograft Recipients.

Background: Drug-induced immunosuppression following kidney transplantation is crucial to prevent allograft rejection, but increases risk for infectious disease. Tailoring of drug dosing to prevent both rejection and infection is greatly desirable. The apathogenic and ubiquitous torque teno virus (TTV) reflects immunocompetence of the host and might be a potential candidate for immunologic monitoring. Methods: To assess TTV as an infection biomarker, virus load was prospectively quantified in peripheral blood of 169 consecutive renal allograft recipients at the Medical University Vienna. Results: Patients with infection showed higher TTV levels compared to patients without infection (4.2 × 108 copies/mL [interquartile range, IQR, 2.7 × 107-1.9 × 109] vs 2.9 × 107 [IQR 1.0 × 106-7.2 × 108]; P = .006). Differences in TTV load became evident almost 3 months before infection (median 77 days, IQR 19-98). Each log level of TTV copies/mL increased the odds ratio for infection by 23% (95% confidence interval 1.04-1.45; P = .014). TTV >3.1 × 109 copies/mL corresponded to 90% sensitivity to predict infections. Logistic regression demonstrated independent association between TTV levels and infection. Conclusions: TTV quantification predicts infection after kidney transplantation and might be a potential tool to tailor immunosuppressive drug therapy.

The AMP analog AICAR modulates the Treg/Th17 axis through enhancement of fatty acid oxidation.

T cells must tightly regulate their metabolic processes to cope with varying bioenergetic demands depending on their state of differentiation. The metabolic sensor AMPK is activated in states of low energy supply and modulates cellular metabolism toward a catabolic state. Although this enzyme is known to be particularly active in regulatory T (Treg) cells, its impact on T helper (Th)-cell differentiation is poorly understood. We investigated the impact of several AMPK activators on Treg-cell differentiation and found that the direct activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide), but not the indirect activators metformin and 2-deoxyglucose, strongly enhanced Treg-cell induction by specifically enhancing Treg-cell expansion. Conversely, Th17 generation was impaired by the agent. Further investigation of the metabolic background of our observations revealed that AICAR enhanced both cellular mitochondrogenesis and fatty acid uptake. Consistently, increased Treg induction was entirely reversible on inhibition of fatty acid oxidation, thus confirming the dependence of AICAR's effects on metabolic pathways alterations. Translating our findings to an in vivo model, we found that the substance enhanced Treg cell generation on IL-2 complex-induced immune stimulation. We provide a previously unrecognized insight into the delicate interplay between immune cell function and metabolism and delineate a potential novel strategy for metabolism-targeting immunotherapy.-Gualdoni, G. A., Mayer, K. A., Göschl, L., Boucheron, N., Ellmeier, W., Zlabinger, G. J. The AMP analog AICAR modulates the Treg/Th17 axis through enhancement of fatty acid oxidation.

Resveratrol enhances TNF-α production in human monocytes upon bacterial stimulation.

BACKGROUND: Resveratrol is a key component of red wine that has been reported to have anti-carcinogenic and anti-aging properties. Additional studies conducted in vitro and in animal models suggested anti-inflammatory properties. However, data from primary human immune cells and in vivo studies are limited. METHODS: A pilot study was performed including 10 healthy volunteers. Plasma cytokine levels were measured over 48h after oral application of 5g resveratrol. To verify the in vivo findings, cytokine release and gene expression in human peripheral blood mononuclear cells (PBMC) and/or monocytes was assessed after treatment with resveratrol or its metabolites and stimulation with several toll-like receptor (TLR)-agonists. Additionally, the impact on intracellular signaling pathways was analyzed using a reporter cell line and Western blotting. RESULTS: Resveratrol treated individuals showed a significant increase in tumor necrosis factor-α (TNF-α) levels 24h after treatment compared to baseline. Studies using human PBMC or isolated monocytes confirmed potentiation of TNF-α production with different TLR agonists, while interleukin (IL)-10 was inhibited. Moreover, we observed significantly enhanced nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) activation using a reporter cell line and found increased phosphorylation of p105, which is indicative of alternative NF-κB pathway activation. GENERAL SIGNIFICANCE: By administering resveratrol to healthy humans and utilizing primary immune cells we were able to detect TNF-α enhancing properties of the agent. In parallel, we found enhanced alternative NF-κB activation. We report on a novel pro-inflammatory property of resveratrol which has to be considered in concepts of its biologic activity.