Interferon-ß acts directly on T cells to prolong allograft survival by enhancing regulatory T cell induction through Foxp3 acetylation.

Type I interferons (IFNs) are pleiotropic cytokines with potent antiviral properties that also promote protective T cell and humoral immunity. Paradoxically, type I IFNs, including the widely expressed IFNß, also have immunosuppressive properties, including promoting persistent viral infections and treating T-cell-driven, remitting-relapsing multiple sclerosis. Although associative evidence suggests that IFNß mediates these immunosuppressive effects by impacting regulatory T (Treg) cells, mechanistic links remain elusive. Here, we found that IFNß enhanced graft survival in a Treg-cell-dependent murine transplant model. Genetic conditional deletion models revealed that the extended allograft survival was Treg cell-mediated and required IFNß signaling on T cells. Using an in silico computational model and analysis of human immune cells, we found that IFNß directly promoted Treg cell induction via STAT1- and P300-dependent Foxp3 acetylation. These findings identify a mechanistic connection between the immunosuppressive effects of IFNß and Treg cells, with therapeutic implications for transplantation, autoimmunity, and malignancy.

Impact of Olive Oil Supplement Intake on Dendritic Cell Maturation after Strenuous Physical Exercise: A Preliminary Study.

Physical exercise is known to have a dose-dependent effect on the immune system and can result in an inflammatory process in athletes that is proportional to the intensity and duration of exertion. This inflammatory process can be measured by cell markers such as dendritic cells (DCs), which, in humans, consist of the myeloid DC (mDCs) and plasmacytoid DC (pDCs) subpopulations. The aim of this study was to measure DC differentiation to determine the possible anti-inflammatory effects, after intense aerobic effort, of the intake of a 25 mL extra-virgin olive oil supplement. Three healthy sports-trained subjects went through resistance exercise loads on two days separated by a week: on one day after active supplement intake and on the other day after placebo supplement intake. The results show that the highest increase (77%) in the percentage of mDCs as a proportion of pDCs was immediately after testing. Independently of the supplement taken, mature mDCs showed a decreasing trend between the test one hour after and 24 h after testing ended. Nevertheless, measured in terms of the coefficient of variation, only the decrease (46%) for extra-virgin olive oil supplementation was statistically significant (95% CI: 30-62%; p = 0.05). In conclusion, an extra-virgin olive oil supplement could reduce the inflammatory impact of intense aerobic effort and improve recovery at 24 h.

Mycophenolic acid interferes the transcriptional regulation and protein trafficking of maturation surface markers in dendritic cells.

BACKGROUND: The ability of dendritic cells (DCs) to regulate adaptive immunity makes them interesting cells to be used as therapeutic targets modulating alloimmune responses. Mycophenolic acid (MPA) is an immunosuppressor commonly used in transplantation, and its effect on DCs has not been fully investigated. METHODS: Monocyte-derived DCs were obtained from healthy volunteers and cultured for 7 days. Cells were treated with MPA on day 2 and matured by lipopolysaccharide (LPS) stimulation. Functionality of mature DC (mDCs) was evaluated by allogeneic mixed lymphocytes reaction. Surface expression of maturation markers (CD40, CD83, CD86, and ICAM-1) was analyzed in both immature DCs (iDCs) and mDCs by flow cytometry. To assess transcriptional regulation and protein subcellular location, RT-PCR and confocal microscopy were used, respectively. RESULTS: MPA decreased surface expression of all maturation markers in mDCs and significantly abrogated DCs-induced allogeneic T-cell proliferation after MPA pre-treatment. In iDCs, the reduced surface protein expression after MPA paralleled with mRNA downregulation of their genes. In mDCs, the mRNA levels of ICAM-1, CD40 and CD83 were enhanced in MPA-treated mDCs with an increase in the expression of CD83 and ICAM-1 near the Golgi compared to non-treated mDCs. In contrast, mRNA levels of CD86 were diminished after MPA treatment. CONCLUSIONS: The reduced surface markers expression in mDCs exerted by MPA produced a decline in their capacity to activate immune responses. Moreover, the inhibition of guanosine-derived nucleotide biosynthesis by MPA treatment leads to DC maturation interference by two mechanisms depending on the marker, transcriptional downregulation or disrupted intracellular protein trafficking.

C5aR1 regulates migration of suppressive myeloid cells required for costimulatory blockade-induced murine allograft survival.

Costimulatory blockade-induced murine cardiac allograft survival requires intragraft accumulation of CD11b+ Ly6Clo Ly6G- regulatory myeloid cells (Mregs) that expand regulatory T cells (Tregs) and suppress effector T cells (Teffs). We previously showed that C5a receptor (C5aR1) signaling on T cells activates Teffs and inhibits Tregs, but whether and/or how C5aR1 affects Mregs required for transplant survival is unknown. Although BALB/c hearts survived >60 days in anti-CD154 (MR1)-treated or cytotoxic T-lymphocyte associated protein 4 (CTLA4)-Ig-treated wild-type (WT) recipients, they were rejected at ~30 days in MR1-treated or CTLA4-Ig-treated recipients selectively deficient in C5aR1 restricted to myeloid cells (C5ar1fl/fl xLysM-Cre). This accelerated rejection was associated with ~2-fold more donor-reactive T cells and ~40% less expansion of donor-reactive Tregs. Analysis of graft-infiltrating mononuclear cells on posttransplant day 6 revealed fewer Ly6Clo monocytes in C5ar1fl/fl xLysM-Cre recipients. Expression profiling of intragraft Ly6Clo monocytes showed that C5aR1 deficiency downregulated genes related to migration/locomotion without changes in genes associated with suppressive function. Cotransfer of C5ar1fl/fl and C5ar1fl/fl xLysM-Cre myeloid cells into MR1-treated allograft recipients resulted in less accumulation of C5ar1-/- cells within the allografts, and in vitro assays confirmed that Ly6Chi myeloid cells migrate to C5a/C5aR1-initiated signals. Together, our results newly link myeloid cell-expressed C5aR1 to intragraft accumulation of myeloid cells required for prolongation of heart transplant survival induced by costimulatory blockade.

T Cell Expression of C5a Receptor 2 Augments Murine Regulatory T Cell (TREG) Generation and TREG-Dependent Cardiac Allograft Survival.

C5aR2 (C5L2/gp77) is a seven-transmembrane spanning receptor that binds to C5a but lacks motifs essential for G protein coupling and associated signal transduction. C5aR2 is expressed on immune cells, modulates various inflammatory diseases in mice, and has been shown to facilitate murine and human regulatory T cell (TREG) generation in vitro. Whether and how C5aR2 impacts in vivo TREG generation and pathogenic T cell-dependent disease models have not been established. In this article, we show that murine T cells express and upregulate C5aR2 during induced TREG (iTREG) generation and that the absence of T cell-expressed C5aR2 limits in vivo iTREG generation following adoptive transfer of naive CD4+ T cells into Rag1-/- recipients. Using newly generated C5aR2-transgenic mice, we show that overexpression of C5aR2 in naive CD4+ T cells augments in vivo iTREG generation. In a model of TREG-dependent cardiac allograft survival, recipient C5aR2 deficiency accelerates graft rejection associated with lower TREG/effector T cell ratios, whereas overexpression of C5aR2 in immune cells prolongs graft survival associated with an increase in TREG/effector T cell ratios. T cell-expressed C5aR2 modulates TREG induction without altering effector T cell proliferation or cytokine production. Distinct from reported findings in neutrophils and macrophages, TREG-expressed C5aR2 does not interact with ß-arrestin or inhibit ERK1/2 signaling. Rather, cumulative evidence supports the conclusion that C5aR2 limits C5aR1-initiated signals known to inhibit TREG induction. Together, the data expand the role of C5aR2 in adaptive immunity by providing in vivo evidence that T cell-expressed C5aR2 physiologically modulates iTREG generation and iTREG-dependent allograft survival.

The combination of CYP3A4*22 and CYP3A5*3 single-nucleotide polymorphisms determines tacrolimus dose requirement after kidney transplantation.

INTRODUCTION: Tacrolimus (Tac) has a narrow therapeutic window and shows large between-patient pharmacokinetic variability. As a result, over-immunosuppression and under-immunosuppression are frequently encountered in daily clinical practice. Unraveling the impact of genetic polymorphisms on Tac pharmacokinetics may help to refine therapy. In this study, the associations of single-nucleotide polymorphisms (SNPs) in drug-metabolizing enzymes (CYP3A) with Tac pharmacokinetics were investigated in renal transplant recipients. PARTICIPANTS AND METHODS: In a cohort of 272 kidney transplant recipients, associations between functional genetic variants (CYP3A4*22 and CYP3A5*3) and dose-adjusted predose Tac concentrations (C0) and daily doses of Tac at days 5-7 and 15 and 1, 3, 6 and 12 months after renal transplantation were evaluated. Patients were genotyped and clustered according to both CYP3A4*22 and CYP3A5*3 allelic status: poor (PM) (CYP3A4*22 carriers with CYP3A5*3/*3), intermediate (IM) (CYP3A4*1/*1 with CYP3A5*3/*3 or CYP3A4*22 carriers with CYP3A5*1 carriers) and extensive CYP3A-metabolizers (EM) (CYP3A4*1/*1 and CYP3A5*1 carriers). RESULTS: EM had an 88% lower dose-adjusted C0 compared with IM. PM had a 26% higher dose-adjusted C0 compared with IM. The percentage of patients with supratherapeutic Tac exposure (C0>15 ng/ml) was significantly higher in PM (43.5%) compared with EM (0%) at days 5-7 after transplantation (P=0.01). About 30% of EM had subtherapeutic exposure (C0<5 ng/ml) at days 5-7 after transplantation (P=0.001). CONCLUSION: The combined CYP3A4 and CYP3A5 genotype of renal transplant recipients has a major influence on the Tac dose required to reach the target exposure.

Online Haemodiafiltration Improves Inflammatory State in Dialysis Patients: A Longitudinal Study.

BACKGROUND: Patients undergoing conventional hemodialysis (C-HD) present a greater immuno-inflammatory state probably related to uremia, sympathetic nervous system (SNS) activation and /or membrane bioincompatibility, which could improve with a technique-switching to online hemodiafiltration (OL-HD). The antigen-independent pathway activation of this modified immunologic state turns dendritic cells (DC) into an accurate cell model to study these patients. The aim of this study is to further evaluate the immune-inflammatory state of patients in C-HD assessed by DC maturation. METHODS: 31 patients were submitted to C-HD and after 4 months switched to the OL-HD technique. Monocytes-derived DCs from HD patients were cultured in the presence of IL-4/GM-CSF. DC-maturation was evaluated by assessing the maturation phenotype by flow cytometry (FACs). DCs-functional capacity to elicit T-cell alloresponse was studied by mixed leucocyte reaction. Cytokine release was assessed by FACs and SNS was evaluated measuring renalase levels by ELISA. RESULTS: An up-regulation of maturation markers was observed in C-HD DCs which induced two fold more T cells proliferation than OL-HD DCs. Also, C-HD-mDCs presented with over-production of pro-inflammatory cytokines (IL-6, IL-1ß, IL-8, IL-10 and TNF-α) compared with OL-HD-mDC (P<0·05). Results were correlated with clinical data. When SNS was evaluated, hypotension events and blood pressure were significantly lower and renalase levels were significantly higher after conversion to OL-HD. Diabetes mellitus type 2 patients also found beneficial reduction of mDC when converted to OL-HD compared to non-diabetics. CONCLUSIONS: OL-HD could interfere with immuno-inflammatory state in HD patients with an improvement of renalase levels as potential key mediators in the mechanistic pathway of down-regulation of DC maturation.

Induction of suppressive allogeneic regulatory T cells via rabbit antithymocyte polyclonal globulin during homeostatic proliferation in rat kidney transplantation.

Experimental studies have shown that rabbit antithymocyte polyclonal globulin (ATG) can expand human CD4+CD25++Foxp3+ cells (Tregs). We investigated the major biological effects of a self-manufactured rabbit polyclonal anti-rat thymoglobulin (rATG) in vitro, as well as its effects on different peripheral T-cell subsets. Moreover, we evaluated the allogeneic suppressive capacity of rATG-induced Tregs in an experimental rat renal transplant model. Our results show that rATG has the capacity to induce apoptosis in T lymphocyte lymphocytes as a primary mechanism of T-cell depletion. Our in vivo studies demonstrated a rapid but transient cellular depletion of the main T cell subsets, directly proportional to the rATG dose used, but not of the effector memory T cells, which required significantly higher rATG doses. After rATG administration, we observed a significant proliferation of Tregs in the peripheral blood of transplanted rats, leading to an increase in the Treg/T effector ratio. Importantly, rATG-induced Tregs displayed a strong donor-specific suppressive capacity when assessed in an antigen-specific allogeneic co-culture. All of these results were associated with better renal graft function in rats that received rATG. Our study shows that rATG has the biological capacity immunomodulatory to promote a regulatory alloimmune milieu during post-transplant homeostatic proliferation.

Developments in renal pharmacogenomics and applications in chronic kidney disease.

Chronic kidney disease (CKD) has shown an increasing prevalence in the last century. CKD encompasses a poor prognosis related to a remarkable number of comorbidities, and many patients suffer from this disease progression. Once the factors linked with CKD evolution are distinguished, it will be possible to provide and enhance a more intensive treatment to high-risk patients. In this review, we focus on the emerging markers that might be predictive or related to CKD progression physiopathology as well as those related to a different pattern of response to treatment, such as inhibitors of the renin-angiotensin system (including angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers; the vitamin D receptor agonist; salt sensitivity hypertension; and progressive kidney-disease markers with identified genetic polymorphisms). Candidate-gene association studies and genome-wide association studies have analyzed the genetic basis for common renal diseases, including CKD and related factors such as diabetes and hypertension. This review will, in brief, consider genotype-based pharmacotherapy, risk prediction, drug target recognition, and personalized treatments, and will mainly focus on findings in CKD patients. An improved understanding will smooth the progress of switching from classical clinical medicine to gene-based medicine.

Preformed frequencies of cytomegalovirus (CMV)-specific memory T and B cells identify protected CMV-sensitized individuals among seronegative kidney transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) infection remains a major complication after kidney transplantation. Baseline CMV risk is typically determined by the serological presence of preformed CMV-specific immunoglobulin (Ig) G antibodies, even though T-cell responses to major viral antigens are crucial when controlling viral replication. Some IgG-seronegative patients who receive an IgG-seropositive allograft do not develop CMV infection despite not receiving prophylaxis. We hypothesized that a more precise evaluation of pretransplant CMV-specific immune-sensitization using the B and T-cell enzyme-linked immunospot assays may identify CMV-sensitized individuals more accurately, regardless of serological evidence of CMV-specific IgG titers. METHODS: We compared the presence of preformed CMV-specific memory B and T cells in kidney transplant recipients between 43 CMV IgG-seronegative (sR(-)) and 86 CMV IgG-seropositive (sR(+)) patients. Clinical outcome was evaluated in both groups. RESULTS: All sR(+) patients showed a wide range of CMV-specific memory T- and B-cell responses. High memory T- and B-cell frequencies were also clearly detected in 30% of sR(-) patients, and those with high CMV-specific T-cell frequencies had a significantly lower incidence of late CMV infection after prophylactic therapy. Receiver operating characteristic curve analysis for predicting CMV viremia and disease showed a high area under the receiver operating characteristic curve (>0.8), which translated into a high sensitivity and negative predictive value of the test. CONCLUSIONS: Assessment of CMV-specific memory T- and B-cell responses before kidney transplantation among sR(-) recipients may help identify immunized individuals more precisely, being ultimately at lower risk for CMV infection.

Do drug transporter (ABCB1) SNPs and P-glycoprotein function influence cyclosporine and macrolides exposure in renal transplant patients? Results of the pharmacogenomic substudy within the symphony study.

The function of the efflux pump P-glycoprotein (Pgp) and ABCB1 single nucleotide polymorphisms (SNPs) should be considered as important tools to deepen knowledge of drug nephrotoxicity and disposition mechanisms. The aim of this study is to investigate the association of C3435T, G2677T, C1236T, and T129C ABCB1 SNPs with Pgp activity and exposure to different immunosuppressive drugs in renal transplant patients. Patients included in the Symphony Pharmacogenomic substudy were genotyped for ABCB1 SNPs. According to the design, patients were randomized into four immunosuppressive regimens: low and standard dose of cyclosporine (n = 30), tacrolimus (n = 13), and sirolimus (n = 23) concomitantly with mycophenolate and steroids. Pgp activity was evaluated in PBMC using the Rhodamine 123 efflux assay. TT carrier patients on C3435T, G2677T, and C1236T SNPs (Pgp-low pumpers) showed lower Pgp activity than noncarriers. Pgp-high pumpers treated with cyclosporine showed lower values of Pgp function than macrolides. There was a negative correlation between cyclosporine AUC and Pgp activity at 3 months. Results did not show any correlation between tacrolimus and sirolimus AUC and Pgp activity at 3 months. We found an important role of the ABCB1 SNPs Pgp function in CD3(+) peripheral blood lymphocytes from renal transplant recipients. Pgp activity was influenced by cyclosporine but not macrolides exposure.

Impact of small molecules immunosuppressants on P-glycoprotein activity and T-cell function.

PURPOSE: P-glycoprotein (Pgp) is a member of the ABC-transporter family that transports substances across cellular membranes acting as an efflux pump extruding drugs out of the cells. Pgp plays a key role on the pharmacokinetics of several drugs. Herein, we have studied the effects of immunosuppressants on Pgp function, assessing rhodamine-123 (Rho123) uptake and efflux in different T-cell subsets. METHODS: Different immunosuppressants such as Cyclosporine (CsA), Rapamycin (Rapa) and Tacrolimus (Tac) were used to assess the in vitro effect on Pgp function of main T-cell subsets among healthy volunteers. We measured Rho123 uptake, efflux and kinetic of extrusion in CD4+ and CD8+ subsets by flow cytometry. Antigen-specific memory T-cell responses were assessed by measuring T-cell proliferation and cytokine secretion using an allogeneic mixed lymphocyte reaction. RESULTS: Rho123 uptake in groups treated with CsA and CsA+Rapa was significantly decreased compared to non-treated group and the other immunosupressants in both T cells subsets. Pgp activity was also reduced in CsA and CsA+Rapa compared to the other immunosupressants but it was only significant in the CsA group for CD8+ subset. Kinetic extrusion of Rho123 by Pgp in all groups was faster in CD8+ T cells. All immunosuppressants and the specific Pgp inhibitor PSC833 diminished antigen-primed T-cell proliferation, especially CD8+ T-cell subset. CONCLUSIONS: Our data indicate that small molecules immunosuppressants, especially CsA, inhibit Pgp activity and T-cell function being the CD8+ T cells more susceptible to this effect. These findings support the importance of Pgp when designing combined immunosuppressive regimens.

Different storing and processing conditions of human lymphocytes do not alter P-glycoprotein rhodamine 123 efflux.

P-glycoprotein (Pgp), a protein codified by Multi Drug Resistance (MDR1) gene, has a detoxifying function and might influence the toxicity and pharmacokinetics and pharmacodynamics of drugs. Sampling strategies to improve Pgp studies could be useful to optimize the sensitivity and the reproducibility of efflux assays. This study aimed to compare Pgp expression and efflux activity by measuring Rhodamine123 (Rh123) retention in lymphocytes stored under different conditions, in order to evaluate the potential utility of any of the storing conditions in Pgp functionality. Our results show no change in protein expression of Pgp by confocal studies and Western blotting, nor changes at the mRNA level (qRT-PCR). No differences in Rh123 efflux by Pgp activity assays were found between fresh and frozen lymphocytes after 24 hours of blood extraction, using either of the two Pgp specific inhibitors (VP and PSC833). Different working conditions in the 24 hours post blood extraction do not affect Rh123 efflux. These results allow standardization of Pgp activity measurement in different individuals with different timing of blood sampling and in different geographic areas.