BK polyomavirus-specific antibody and T-cell responses in kidney transplantation: update.

PURPOSE OF REVIEW: BK polyomavirus (BKPyV) has emerged as a significant cause of premature graft failure after kidney transplantation. Without effective antiviral drugs, treatment is based on reducing immunosuppression to regain immune control over BKPyV replication. The paradigm of high-level viruria/decoy cells, BKPyV-DNAemia, and proven nephropathy permits early interventions. Here, we review recent findings about BKPyV-specific antibody and T-cell responses and their potential role in risk stratification, immune monitoring, and therapy. RECENT FINDING: Kidney transplant recipients having low or undetectable BKPyV-specific IgG immunoglobulin G (IgG) are higher risk for developing BKPyV-DNAemia if the donor has high BKPyV-specific IgG. This observation has been extended to neutralizing antibodies. Immunosuppression, impaired activation, proliferation, and exhaustion of BKPyV-specific T cells may increase the risk of developing BKPyV-DNAemia and nephropathy. Clearance of BKPyV-DNAemia was correlated with high CD8 T cell responses to human leukocyte antigen (HLA)-types presenting BKPyV-encoded immunodominant 9mers. For clinical translation, these data need to be assessed in appropriately designed clinical studies, as outlined in recent guidelines on BKPyV in kidney transplantation. SUMMARY: Evaluation of BKPyV-specific immune responses in recipient and donor may help to stratify the risk of BKPyV-DNAemia, nephropathy, and graft loss. Future efforts need to evaluate clinical translation, vaccines, and immunotherapy to control BKPyV replication.

Adiponectin protects against Toll-like receptor 4-mediated cardiac inflammation and injury.

AIMS: Adiponectin (APN) is an immunomodulatory and cardioprotective adipocytokine. Toll-like receptor (TLR) 4 mediates autoimmune reactions that cause myocarditis resulting in inflammation-induced cardiac injury. Here, we investigated whether APN inhibits inflammation and injury in autoimmune myocarditis by interfering with TLR4 signalling. METHODS AND RESULTS: APN overexpression in murine experimental autoimmune myocarditis (EAM) down-regulated cardiac expression of TLR4 and its downstream targets tumour necrosis factor (TNF)α, interleukin (IL)-6, IL-12, CC chemokine ligand (CCL)2, and intercellular adhesion molecule (ICAM)-1 resulting in reduced infiltration with cluster of differentiation (CD)3+, CD14+, and CD45+ immune cells as well as diminished myocardial apoptosis. Expression of TLR4 signalling pathway components was unchanged in hearts and spleens of APN-knockout (APN-KO) mice. In vitro APN had no effect on TLR4 expression in cardiac and immune cells but induced dissociation of APN receptors from the activated TLR4/CD14 signalling complex. APN inhibited the expression of a TLR4-mediated inflammatory phenotype induced by exogenous and endogenous TLR4 ligands as assessed by attenuated nuclear factor (NF)-κB activation and reduced expression of TNFα, IL-6, CCL2, and ICAM-1. Accordingly, following TLR4 ligation, splenocytes from APN-KO mice showed enhanced expression of TNFα, IL-6, IL-12, CCL2, and ICAM-1, whereas dendritic cells (DCs) from APN-KO mice demonstrated increased activation and T-cell priming capacity. Moreover, APN diminished TLR4-mediated splenocyte migration towards cardiac cells as well as cardiomyocyte apoptosis after co-cultivation with splenocytes. Mechanistically, APN inhibited TLR4 signalling through cyclooxygenase (COX)-2, protein kinase A (PKA), and meiosis-specific serine/threonine kinase (MEK)1. CONCLUSION: Our observations indicate that APN protects against inflammation and injury in autoimmune myocarditis by diminishing TLR4 signalling thereby attenuating inflammatory activation and interaction of cardiac and immune cells.

Adiponectin modulates NK-cell function.

Adiponectin (APN) has been shown to exert antiinflammatory effects in various disease models but little is known concerning its regulation of NK-cell function. Here, we show that the majority of human CD56(dim) NK cells express surface Adiponectin receptor (AdipoR) 1 and 2 while most CD56(high) NK cells are AdipoR-negative. Toll-like receptor (TLR) ligand-induced IFN-γ production was diminished by APN while it had no influence on NK-cell cytotoxicity. In contrast only a small subpopulation of murine NK cells expresses surface AdipoRs, but about 90% store them intracellularly. APN-deficient knockout (KO) mice had elevated frequencies of NK cells. However, cytotoxic degranulation of NK cells was decreased in APN knockout (APN-KO) animals. Accordingly, frequencies of CD11b(high) CD27(high) and CD94(high) effector NK cells and expression of NKG2D were lower in APN-KO mice. Upon CVB3 infection NK-cell function was restored in APN-KO mice. Our data suggest that in addition to its antiinflammatory effects APN also influences the numerical and differentiation status of NK cells, which may further impact the outcome of immune-mediated diseases in APN-KO mice.

How do I begin? Sensing extracellular stress to maintain yeast cell wall integrity.

The cell wall integrity (CWI) signalling pathway is necessary to remodel the yeast cell wall during normal morphogenesis and in response to cell surface stress. In the Baker's yeast Saccharomyces cerevisiae, a set of five membrane-spanning sensors, namely Wsc1, Wsc2, Wsc3, Mid2 and Mtl1, detect perturbations in the cell wall and/or the plasma membrane and activate a downstream signal transduction pathway with a central MAP kinase module. As a consequence, the expression of genes whose products are involved in cell wall structure and remodelling is induced. This review summarises our recent results on sensor structure and function, as well as the advances made regarding sensor mechanics.

Adiponectin is a negative regulator of antigen-activated T cells.

Adiponectin (APN), a cytokine constitutively produced in fat tissue, has been shown to exert anti-inflammatory effects in various disease models. While the influence of APN on monocytic cells has been extensively studied in vitro, little is known about its role in T cells. In this study, we show that while <10% of human peripheral blood T cells express adiponectin receptors (AdipoRs) on their surface, most T cells store AdipoRs in intracellular compartments. AdipoRs colocalized with immune regulatory molecules CTLA-4 and TIRC7 within clathrin-coated vesicles. After stimulation, the expression of adiponectin receptor 1 (AdipoR1) and AdipoR2 was upregulated on the surface of antigen-specific T cells, as determined by tetramer or CD137 staining, and AdipoR1 and AdipoR2 coexpressed with CTLA-4. Addition of APN resulted in a significant diminution of antigen-specific T-cell expansion. Mechanistically, APN enhanced apoptosis and inhibited proliferation of antigen-specific T-cell lines. Further, APN directly inhibited cytokine production in response to antigen stimulation. In line with the in vitro data, APN-deficient (knockout, KO) mice had higher frequencies of CD137(+) T cells upon Coxsackie B virus infection. Altogether, our data suggest that APN is a novel negative T-cell regulator. In contrast to the CTLA-4 ligand B7 only expressed on APCs, APN is abundant in human plasma.

Adiponectin expression in patients with inflammatory cardiomyopathy indicates favourable outcome and inflammation control.

AIMS: Circulating adiponectin (APN) is an immunomodulatory, pro-angiogenic, and anti-apoptotic adipocytokine protecting against acute viral heart disease and preventing pathological remodelling after cardiac injury. The purpose of this study was to describe the regulation and effects of APN in patients with inflammatory cardiomyopathy (DCMi). METHODS AND RESULTS: Adiponectin expression and outcome were assessed in 173 patients with DCMi, 30 patients with non-inflammatory DCM, and 30 controls. Mechanistic background of these findings was addressed in murine experimental autoimmune myocarditis (EAM), a model of human DCMi, and further elucidated in vitro. Adiponectin plasma concentrations were significantly higher in DCMi compared with DCM or controls, i.e. 6.8 ± 3.9 µg/mL vs. 5.4 ± 3.6 vs. 4.76 ± 2.5 µg/mL (P< 0.05, respectively) and correlated significantly with cardiac mononuclear infiltrates (CD3+: r(2)= 0.025, P= 0.038; CD45R0+: r(2)= 0.058, P= 0.018). At follow-up, DCMi patients with high APN levels showed significantly increased left ventricular ejection fraction improvement, decreased left ventricular end-diastolic diameter, and reduced cardiac inflammatory infiltrates compared with patients with low APN levels. A multivariate linear regression analysis implicated APN as an independent prognostic factor for inhibition of cardiac inflammation. In accordance with these findings in human DCMi, EAM mice exhibited elevated plasma APN. Adiponectin gene transfer led to significant downregulation of key inflammatory mediators promoting disease. Mechanistically, APN acted as a negative regulator of T cells by reducing antigen specific expansion (P< 0.01) and suppressed TNFα-mediated NFκB activation (P< 0.01) as well as release of reactive oxygen species in cardiomyocytes. CONCLUSION: Our results implicate that APN acts as endogenously upregulated anti-inflammatory cytokine confining cardiac inflammation and progression in DCMi.

A block of endocytosis of the yeast cell wall integrity sensors Wsc1 and Wsc2 results in reduced fitness in vivo.

The response to cell surface stress in yeast is mediated by a set of five plasma membrane sensors. We here address the relation of intracellular localization of the sensors Wsc1, Wsc2, and Mid2 to their turnover and signaling function. Growth competition experiments indicate that Wsc2 plays an important role in addition to Wsc1 and Mid2. The two Wsc sensors appear at the bud neck during cytokinesis and employ different routes of endocytosis, which govern their turnover. Whereas Wsc1 uses a clathrin-dependent NPFDD signal, Wsc2 relies on a specific lysine residue (K495). In end3 and doa4 endocytosis mutants, both sensors accumulate at the plasma membrane, and a hypersensitivity to cell wall-specific drugs and to treatment with zymolyase is observed. A haploid strain in which endocytosis of the two sensors is specifically blocked displays a reduced fitness in growth competition experiments. If the Mid2 sensor is mobilized by the addition of an endocytosis signal, it mimics the dynamic distribution of the Wsc sensors, but is unable to complement the specific growth defects of a wsc1 deletion. These data suggest that sensor distribution is not the major determinant for its specificity.

Single-molecule atomic force microscopy reveals clustering of the yeast plasma-membrane sensor Wsc1.

Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains ("rafts") is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of approximately 200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches.

The yeast Wsc1 cell surface sensor behaves like a nanospring in vivo.

Here we report on in vivo measurement of the mechanical behavior of a cell surface sensor using single-molecule atomic force microscopy. We focus on the yeast wall stress component sensor Wsc1, a plasma membrane protein that is thought to function as a rigid probe of the cell wall status. We first map the distribution of individual histidine-tagged sensors on living yeast cells by scanning the cell surface with atomic force microscopy tips carrying nitrilotriacetate groups. We then show that Wsc1 behaves like a linear nanospring that is capable of resisting high mechanical force and of responding to cell surface stress. Both a genomic pmt4 deletion and the insertion of a stretch of glycines in Wsc1 result in substantial alterations in protein spring properties, supporting the important role of glycosylation at the extracellular serine/threonine-rich region.