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Chronic lung allograft dysfunction (CLAD) is a severe complication of lung transplantation limiting long-term survival. We studied correlations between CLAD after clinical lung transplantation and leukocyte-mediated development of transplant arteriosclerosis (TA) in a humanized mouse model. The pericardiophrenic artery was procured from surplus tissue of donor lungs (n = 22) transplanted in our clinical program and was implanted into the abdominal aorta of immune-deficient mice. Allogeneic human peripheral blood mononuclear cells (PBMCs) had been procured 1 day after lung transplantation from the respective recipients with or without enriching for CD4+CD25high T cells were used. TA was assessed in mice 28 days later by histology. The respective clinical lung recipients were later divided into 2 groups. Eight patients (36.3%) had developed CLAD 23 ± 5 months after lung transplantation, whereas the remaining 14 (63.6%) did not develop CLAD within 25 ± 5 months. In the PBMC CLAD+ group of mouse experiments, TA was significantly more severe than in the PBMC CLAD– group (39.9% ± 13% vs 14.9% ± 4% intimal thickening; P =.0081). Then, intimal thickening was significantly inhibited in the PBMC+ regulatory T cells CLAD+ group compared with the PBMC CLAD+ group (0.4% ± 4% vs 39.9% ± 13%; P =.003). In the experiments using PBMCs from lung recipients without CLAD, enriching regulatory T cells also suppressed the development of TA (0.9% ± 3% PBMC CLAD– vs 14.9% ± 4% PBMC+ regulatory T cells CLAD–; P =.001). Lung transplant recipients who later develop CLAD have peripheral leukocytes already at the time of transplant that transfer proinflammatory properties leading to TA in a humanized mouse model. TA remains sensitive to inhibition by autologous regulatory T cells, suggesting a cell therapy-based approach for the prevention of CLAD after lung transplantation. [ABSTRACT FROM AUTHOR]

Rationale and Objectives: To evaluate the feasibility of multiparametric magnetic resonance imaging (MRI) of the lungs to detect impaired organ function in a porcine model of ischemic injury within an ex-vivo lung perfusion system (EVLP) prior to transplantation.Materials and Methods: Twelve pigs were anesthetized, and left lungs were clamped to induce warm ischemia for 3 hours. Right lungs remained perfused as controls. Lungs were removed and installed in an EVLP for 12 hours. Lungs in the EVLP were imaged repeatedly using computed tomography, proton MRI (1H-MRI) and fluorine MRI (19F-MRI). Dynamic contrast-enhanced derived parenchymal blood volume, oxygen washout times, and 19F washout times were calculated. PaO2 was measured for ischemic and normal lungs, wet/dry ratio was determined, histologic samples were assessed, and cytokines in the lung tissue were analyzed. Statistical analysis was performed using nonparametric testing.Results: Eleven pigs were included in the final analysis. Ischemic lungs showed significantly higher wet/dry ratios (p = 0.024), as well as IL-8 tissue levels (p = 0.0098). Histologic assessment as well as morphologic scoring of computed tomography and 1H-MRI did not reveal significant differences between ischemic and control lungs. 19F washout (p = 0.966) and parenchymal blood flow (p = 0.32) were not significantly different. Oxygen washout was significantly prolonged in ischemic lungs compared to normal control lungs at the beginning (p = 0.018) and further prolonged at the end of the EVLP run (p = 0.005).Conclusion: Multiparametric pulmonary MRI is feasible in lung allografts within an EVLP system. Oxygen-enhanced imaging seems to be a promising marker for ischemic injury, enabling detection of affected lung segments prior to transplantation. [ABSTRACT FROM AUTHOR]

Summary Quiescent long-term hematopoietic stem cells (LT-HSCs) are efficiently activated by type I interferon (IFN-I). However, this effect remains poorly investigated in the context of IFN-I-inducing virus infections. Here we report that both vesicular stomatitis virus (VSV) and murine cytomegalovirus (MCMV) infection induce LT-HSC activation that substantially differs from the effects triggered upon injection of synthetic IFN-I-inducing agents. In both infections, inflammatory responses had to exceed local thresholds within the bone marrow to confer LT-HSC cell cycle entry, and IFN-I receptor triggering was not critical for this activation. After resolution of acute MCMV infection, LT-HSCs returned to phenotypic quiescence. However, non-acute MCMV infection induced a sustained inflammatory milieu within the bone marrow that was associated with long-lasting impairment of LT-HSC function. In conclusion, our results show that systemic virus infections fundamentally affect LT-HSCs and that also non-acute inflammatory stimuli in bone marrow donors can affect the reconstitution potential of bone marrow transplants. [ABSTRACT FROM AUTHOR]

BACKGROUND & AIMS: Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via noncytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes. METHODS: We performed a cytokine enzymelinked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBVinfected HepaRG cells, and primary human hepatocytes were incubated with interferon-γ (IFNg) or tumor necrosis factorα (TNFα), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA). RESULTS: Levels of IFNg and TNFα were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFNg and TNFα each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFNg and TNFα, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFNg and TNFa after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APOBEC3B. CONCLUSIONS: IFNg and TNFα, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay. [ABSTRACT FROM AUTHOR]

Background & Aims Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via noncytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes. Methods We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-γ (IFNγ) or tumor necrosis factor-α (TNF-α), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA). Results Levels of IFNγ and TNF-α were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFNγ and TNF-α each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFNγ and TNF-α, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFNγ and TNF-α after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APOBEC3B. Conclusions IFNγ and TNF-α, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay. [ABSTRACT FROM AUTHOR]

Abstract: There is accumulating evidence indicating that endothelial factors are involved in the pathogenesis of GVHD. We have recently shown that steroid-refractory, but not sensitive, GVHD is characterized by higher pretransplantation serum levels of angiopoetin-2 (ANG2), a hormone mediating endothelial vulnerability. To evaluate whether endothelial vulnerability is a risk factor for GVHD per se or becomes important only when noticeable GVHD is established, we measured ANG2 along with additional serum markers of endothelial stress, including soluble thrombomodulin (sTM), IL-8 (CXCL8), and hepatocyte growth factor (HGF), in patients with no, low-grade, or severe GVHD. Patients with refractory GVHD exhibited elevated serum levels of ANG2, sTM, HGF, and IL-8 posttransplantation compared with patients with sensitive GVHD and patients without GVHD. Pretransplantation ANG2 was the only growth factor correlated with the risk of refractoriness and mortality, and then only within the subset of patients who developed grade III-IV GVHD. In contrast, ANG2 was not predictive of GVHD or nonrelapse mortality (NRM) in patients with no GVHD or low-grade GVHD. These findings provide evidence that endothelial function plays an important role in the pathogenesis of steroid refractoriness in ongoing GVHD; however, endothelial vulnerability does not predict incidence of GVHD. [Copyright &y& Elsevier]

Abstract Schizophrenia and major depression are associated with alterations in peripheral inflammatory markers, and anti-inflammatory therapy has been proposed as a promising add-on approach in the pharmacologic treatment of both disorders. Second-generation atypical antipsychotics are currently first-line drugs in the treatment of schizophrenia and are also used as augmentation strategies in treatment-resistant major depression. Furthermore, these drugs have been reported to exhibit distinct metabolic side effects and to influence inflammatory processes. In this study, we used ex vivo stimulation of primary human peripheral blood mononuclear cells (PBMC) from healthy blood donors with atypical antipsychotics olanzapine or aripiprazole to examine effects on cytokine production independent from metabolic side effects and disease status. Both olanzapine and aripiprazole stimulation decreased mRNA levels of IL-1β, IL-6, and TNF-α and resulted in diminished protein concentrations of IL-6 and TNF-α in conditioned medium of stimulated PBMC. A multiplex approach revealed additional downregulation of IL-2; MIP-1β and IP-10 secretion. Similarly, olanzapine and aripiprazole stimulation of the human monocytic cell line THP-1 resulted in a significant decrease in expression and secretion of IL-1β and TNF-α. Our results suggest that atypical antipsychotics directly influence immune cell function and thereby highlight the importance to factor in potential side effects of drugs routinely used in treatment of schizophrenia and major depression on inflammatory processes when considering anti-inflammatory drug therapy as an additional treatment option. [ABSTRACT FROM AUTHOR]

Inflammation plays an important role in atherosclerosis, a notion supported by the beneficial effects of the IL-1β inhibitor canakinumab in the CANTOS trial. Sialic acids (Sias), components of the surface glycocalyx, regulate intercellular and intermolecular interactions. We investigated the expression of the Sia cleaving enzyme neuraminidase-1 (NEU1) in atherosclerotic plaques and its potential role in inflammatory processes. In isolated mononuclear blood cells from patients with myocardial infarction, NEU1 expression was increased compared to healthy controls. High expression of NEU1 in macrophages located on the intima layer, in calcified regions and the adventitia of the plaque was observed in human carotid arteries' atherectomies. IL-1β and LPS induced NEU1 expression in THP-1 monocytic cells. Lentiviral NEU1-overexpression in THP-1-cells enhanced expression of CD80, TNF-α, IL-1β, number of multinuclear cells, phagocytosis and chemotaxis indicative for M1 monocyte/macrophage polarization. CRISPR/Cas9-mediated knock-out of NEU1 in THP-1-cells did not affect differentiation of monocytes to macrophages but attenuated LPS- and IL-1β -induced TNF-α and IL-1β expression. SiRNA-mediated knock-down of NEU1 in M1-macrophages differentiated from primary human CD14 + monocytes reduced the expression of TNF-α and IL-1β. Thus, in monocytes/macrophages, LPS, NEU1 and IL-1β act in a positive feedback loop as enhancers of inflammation and may therefore promote atherosclerosis and plaque instability. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cell receptors (NKRs) play a crucial role in the homeostasis of antigen-experienced T cells. Indeed, prolonged antigen stimulation may induce changes in the receptor repertoire of T cells to a profile that features NKRs. Chronic antigen exposure, at the same time, has been shown to trigger the loss of costimulatory CD28 molecules with recently reported intensified antigen thresholds of antigen-experienced CD8 + T cells. In transplantation, NKRs have been shown to assist allograft rejection in a CD28-independent fashion. We discuss here a role for CD28-negative T cells that have acquired the competency of the NKR machinery, potentially promoting allorecognition either through T cell receptor (TCR) crossreactivity or independently from TCR recognition. Collectively, NKRs can bring about innate-like T cells by providing alternative costimulatory pathways that gain relevance in chronic inflammation, potentially leading to resistance to CD28-targeting immunosuppressants. [ABSTRACT FROM AUTHOR]

Inhibition of Janus-activated kinase-1 (JAK1) is a promising clinical concept for post-transplant immunosuppression and autoimmunity. However, it also raises concerns regarding possible immunosuppressive side effects. Our study investigates JAK1 signalling in the context of CD40L and bacterially activated human MoDC using siRNA and biological inhibitors. We demonstrate that strong stimuli (e.g. intact Escherichia coli or LPS in addition to IL-1β) induce IL-12p70 via a ROS/RELA/CDK9 pathway that is inhibited by simultaneous JAK1/STAT3 signalling. Transcription is effective if RELA recruits the positive transcription elongation factor b (P-TEFb) component CDK9 to a combined RELA/STAT3 binding site −50 to −20 bp upstream of the start site of the IL-12p35 promoter. STAT3 simultaneously attaches to this site and inhibits CDK9 binding. In the presence of IFNγ, JAK1/2 inhibitors block STAT1/IRF1/IRF8-dependent activation and simultaneously enhance CDK9-dependent activation signals. This inverse regulation of IFNγ- vs. E. coli -induced cytokine production by JAK inhibitors including Ruxolitinib was similarly observed for IL-6 and TNF-α production, but not for IL-10 production. Thus, JAK1 inhibition enhances IL-12p70 production in this context by increased DNA binding of CDK9. In contrast, weak RELA-activation signals (CD40L, LPS) depended on IFN-γ induced STAT1/IRF1/IRF8 co-signalling, which was completely blocked by JAK inhibitors as reported before. Our results suggest a novel molecular mechanism of how cytokine responses to invading pathogens are separable from IFNγ-dependent autoimmunity by targeting JAK1/STAT3 activation. [ABSTRACT FROM AUTHOR]

Suppressor of cytokine signaling 3 (SOCS-3) is an important intracellular negative regulator of several signaling pathways. We found that SOCS-3 is highly expressed in renal proximal tubules during acute kidney injury. To test the impact of this, conditional proximal tubular knockout mice (SOCS-3sglt2Δ/sglt2Δ) were created. These mice had better kidney function than their wild-type counterparts in aristolochic acid nephropathy and after ischemia/reperfusion injury. Kidneys of these knockout mice showed significantly more proximal tubular cell proliferation during the repair phase. A direct effect of SOCS-3 on tubular cell cycling was demonstrated by in vitro experiments showing a JAK/STAT pathway-dependent antimitotic effect of SOCS-3. Furthermore, acute damaged kidneys of the knockout mice contained increased numbers of F4/80+ cells. Phenotypic analysis of these F4/80+ cells indicated a polarization from classically activated to alternatively activated macrophages. In vitro, SOCS-3-overexpressing renal epithelial cells directly induced classical activation in cocultured macrophages, supporting the observed in vivo phenomenon. Thus, upregulation of SOCS-3 in stressed proximal tubules plays an important role during acute kidney injury by inhibition of reparative proliferation and by modulation of the macrophage phenotype. Antagonizing SOCS-3 could have therapeutic potential for acute kidney injury. [ABSTRACT FROM AUTHOR]

Low-dose cyclophosphamide (CP) therapy induces immunogenic tumor cell death and decreases regulatory T cell (Treg) numbers in mice with transplantable tumors. Using the ret transgenic murine melanoma model that resembles human melanoma, we detected no beneficial antitumor effects with such treatment, despite a decrease in Tregs. On the contrary, low-dose CP enhanced the production of chronic inflammatory mediators in melanoma lesions associated with increased accumulation of Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs), which exhibit elevated suppressive activity and nitric oxide (NO) production as well as inhibition of T-cell proliferation. Moreover, the frequencies of CD8+ T cells in the tumors and their ability to produce perforin were decreased. To study whether the observed CP-induced MDSC expansion and activation also occurs under chronic inflammatory tumor-free conditions, mice exhibiting chronic inflammation were treated with CP. Similar to tumor-bearing mice, CP-treated inflamed mice displayed elevated levels of MDSCs with enhanced production of NO, reactive oxygen species, and a suppressed in vivo natural killer (NK) cell cytotoxic activity indicating CP effects on the host immune system independent of the tumor. We suggest that melanoma therapy with low-dose CP could be efficient only when combined with the neutralization of MDSC immunosuppressive function and chronic inflammatory microenvironment. [ABSTRACT FROM AUTHOR]

Abstract: 3,4-dimethoxycinnamonyl-anthranilic acid (tranilast) is an orally available anti-allergic drug with structural and functional homologies to immunosuppressive catabolites of the essential amino acid tryptophan and broad anti-inflammatory properties. It has recently been shown to be effective in animal models of multiple sclerosis and rheumatoid arthritis, two autoimmune diseases that are mediated by auto-aggressive Th1-polarized CD4+ T lymphocytes. Here we demonstrate potent suppressive effects of tranilast on the function of naïve human CD4+ T cells. Tranilast inhibited inhibits activation and proliferation of purified CD4+ T cells stimulated through the T cell receptor with an EC50 of less than 10μM, a concentration that is well below plasma levels achieved after oral administration of approved doses of 200–600mg in humans. The antiproliferative effects were less potent on naïve CD8+ T cells. Suppression of CD4+ and CD8+ T cell proliferation was associated with an inhibition of T cell activation. Cytokine analyses of naïve CD4+ T cells revealed that tranilast interferes with the production of cyto- and chemokines driven by signal transducer and activator of transcription 1 (STAT1), notably chemokine (C-X-C motif) ligands (CXCL) 9 and 10. Tranilast limited STAT1 phosphorylation in activated T cells and supplementation of CXCL9 or CXCL10 reversed the anti-proliferative effects of tranilast. These data imply CXCL9 and CXCL10 as novel therapeutic targets of tranilast in Th1-mediated autoimmune diseases and identify phospho-STAT1 and its target chemokines CXCL9 and CXCL10 as potential markers for monitoring the bioactivity of tranilast in humans. [Copyright &y& Elsevier]

Abstract: CD40Ligand (CD40L) represents a strong endogenous danger signal associated with chronic inflammatory disease. CD40L induces activation of antigen-presenting cells (APCs) such as DCs, monocytes, B-cells and endothelial cells. However, CD40 activation alone, whilst inducing IL-10 production, is insufficient to induce interleukin (IL)-12p70 release in human APCs suggesting that additional cytokine signals (e.g. GM-CSF, IL-4 or IFN-γ) are required for the induction of a pro-inflammatory cytokine profile. We demonstrate that IFN-γ-induced Janus kinase 1 (JAK1) enhances CD40-induced IL-12p70 release whilst simultaneously inhibiting IL-10 synthesis, resulting in a pro-inflammatory phenotype of CD40L-activated dendritic cells (DCs). JAK2 mediated enhancing effects on IL-12p70 but did not inhibit IL-10 release, whereas Tyk2 mediated inhibitory effects on IL-12p70 release in this system. The mechanism by which complementary IFN-γ/JAK activities affect IL-12p70 production involves STAT1 activation and de novo induction of interferon-responsive factors (IRF)-1 and IRF-8. Simultaneously, JAK1 was unique in inhibiting IL-10 synthesis via STAT1 and IRF-8 with both transcription factors binding to the IL-10 promoter. We demonstrate that CD40- and JAK/STAT/IRF-signalling pathways are strictly complementary for the induction of a pro-inflammatory cytokine profile in human APCs. This suggests that a number of CD40 effects in chronic inflammatory diseases might be weakened by targeting JAK/STAT. [ABSTRACT FROM AUTHOR]

The preferential expression of the non-polymorphic human leukocyte antigen G (HLA-G) on invading extravillous cytotrophoblast cells that are, with the exception of HLA-C and -E, HLA class I negative led to the hypothesis that HLA-G plays a major role in controlling the effector functions of the large granular leukocytes (LGL), a specialized natural killer (NK) cell population present in large numbers in the decidua. Transcription of the HLA-G gene is characterized by extensive alternative splicing producing at least seven potentially membrane bound or secreted isoforms. Except for HLA-G1 and its soluble variant (HLA-G1s), there is still dispute as to whether any of the other isoforms displays a major immunological function. Here we describe that the membrane-bound isoforms HLA-G2, -G3, and G4 as well as the soluble variant of HLA-G2 (HLA-G2s) do not egress the endoplasmic reticulum as determined by Endo H sensitivity assays. Moreover these isoforms seem not to have a major immunological function with respect to NK cell inhibition by providing a ligand for HLA-E, which would allow the interaction of this molecule with the inhibitory CD94/NKG2A NK cell receptor. [Copyright &y& Elsevier]