Gene Expression Profiles of Fenofibrate Effects in mouse grafts and spleens: significant repression of IL17 and Th1 genes in vivo in spleens and grafts and formation of a single network of direct interactions.

<p>PCR of RNA from mice cardiac allografts (5a) and recipient spleens (b) corroborated in vitro findings and further characterized the mechanism of Fenofibrate to regulate the IL17 pathway and Th1 response (c). Gene expression results in mice recipient grafts (a,c) and spleens (b,c) at POD7 are displayed as box and whisker plots of mean relative fold changes with 10^th and 90^th percentile to universal RNA after 18S normalization using the ΔΔCt method. Significance between Fenofibrate (FF) treatment over no treatment (NT) and between Cyclosporine (Cys) treatment over no treatment were calculated by a 2-sided Student t-test and a p-value of p<0.05 considered as significant. Results of our additional network analyses in MetaCore revealed a central role for the transcription factor c-jun (c). Subsequent PCR for c-jun and its associated cytokine receptor IL7R showed decreased expression by Fenofibrate and suggested different sites of actions in spleen and grafts (c).</p

Significance and Suppression of Redundant IL17 Responses in Acute Allograft Rejection by Bioinformatics Based Drug Repositioning of Fenofibrate

<div><p>Despite advanced immunosuppression, redundancy in the molecular diversity of acute rejection (AR) often results in incomplete resolution of the injury response. We present a bioinformatics based approach for identification of these redundant molecular pathways in AR and a drug repositioning approach to suppress these using FDA approved drugs currently available for non-transplant indications. Two independent microarray data-sets from human renal allograft biopsies (n = 101) from patients on majorly Th1/IFN-y immune response targeted immunosuppression, with and without AR, were profiled. Using gene-set analysis across 3305 biological pathways, significant enrichment was found for the IL17 pathway in AR in both data-sets. Recent evidence suggests IL17 pathway as an important escape mechanism when Th1/IFN-y mediated responses are suppressed. As current immunosuppressions do not specifically target the IL17 axis, 7200 molecular compounds were interrogated for FDA approved drugs with specific inhibition of this axis. A combined IL17/IFN-y suppressive role was predicted for the antilipidemic drug Fenofibrate. To assess the immunregulatory action of Fenofibrate, we conducted <i>in-vitro</i> treatment of anti-CD3/CD28 stimulated human peripheral blood cells (PBMC), and, as predicted, Fenofibrate reduced IL17 and IFN-γ gene expression in stimulated PMBC. <i>In-vivo</i> Fenofibrate treatment of an experimental rodent model of cardiac AR reduced infiltration of total leukocytes, reduced expression of IL17/IFN-y and their pathway related genes in allografts and recipients’ spleens, and extended graft survival by 21 days (p<0.007). In conclusion, this study provides important proof of concept that meta-analyses of genomic data and drug databases can provide new insights into the redundancy of the rejection response and presents an economic methodology to reposition FDA approved drugs in organ transplantation.</p> </div

Fenofibrate gene expression in human PBMC: Fenofibrate regulates IL17 and IFN-γ gene expression in CD3/CD28 stimulated human PBMC.

<p>PBMC from healthy individuals (n = 5) were stimulated with CD3/CD28 antibodies (S) leading to significant upregulation of IL17 and IFN-γ which was inhibited by Fenofibrate (S+FF). Values represent mean fold changes versus non-stimulated (NS) cells plus Standard error of mean calculated using ΔΔCt method and 18S as endogenous control gene; experiments were performed in triplicates. Student T-test for paired data: * p-value <0.05. Individual p-values are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056657#pone-0056657-t003" target="_blank">Table 3</a>.</p

IL17 pathway- and IL17⁺ Th- gene sets in human AR: Segregation of AR and STA.

<p>Genes from the IL17 pathway gene-set (2a) and from the Th17 gene-set (2b) were used for hierarchical clustering of post-transplant biopsies and resulted in clear separation of AR and STA after mean centering arrays and genes (Euclidean Distance, Cosine Dissimilarity). Unsupervised principal component analysis (PCA) of the same samples using the same genes confirmed the separation of AR and STA (2c, d). A separation of the AR group into C4d+ and C4d- cases was not seen.</p

GSA identifies increasing enrichment of IL-17 gene-sets in human renal allograft acute rejection across independent Patient Data-Sets.

<p>There were total 140 gene-sets significantly enriched in AR in both Data-Sets (FDR = 0.5), the IL-17 pathway and Th17 gene-sets are listed above. Other significant gene-sets that reached the threshold of FDR = 0.5 included gene-sets associated with innate immune cells (Dendritic Cell, Natural Killer Cell, Granulocyte, Monocyte), and innate immune responses (CTLA4 pathway, Toll-like receptor pathway, NFKB targets, PD1 signaling), with Cytokine Signaling (IL-10, IL-2, IL-5, IL-22BP, IL-12) as well as with gene-sets related to Th-differentiation and activation (CD40 signaling, Costimulation, Th1/Th2, Th-Differentiation). Th1 (FDR = 0.3, p = 0.007) and IL-12 (FDR = 0.3, p = 0.011) had higher FDR and p-values compared to the Th17 and IL-17 gene-sets in our data-set and similar values compared to GSE 9493. Other gene-sets were Graft versus Host Disease, Autoimmune Thyroiditis, and Antigen processing.</p

Study Design.

<p>Whole genome microarrays from 66 pre- and post-transplant kidney graft biopsies and from 35 post-transplant kidney graft biopsies were analyzed for rejection specific injury pathways using 3 computational databases comprising 3305 independent gene-sets. AR specific IL17 pathway (FDR = 0.3, p = 0.011) and activated IL17⁺ T-helper cells cell (FDR = 0.5; p = 0.008) gene-sets were aligned to a database of 7200 compounds with proven interactions with the input genes (MetaCore™, GeneGo, Thomson Reuters) resulting in the identification of Fenofibrate for drug repositioning in AR. Efficacy of Fenofibrate was tested in vitro using human PBMC and in vivo in mouse total allo-mismatch cardiac rejection for anti-inflammatory and for its potency to prolong graft survival.</p

Patient Demographics.

a<p>HLA MM = human leukocyte antigen mismatch;</p>b<p>Sir = Sirolimus; SB = steroid based; Aza = Azathioprine;</p

Effects of Fenofibrate on IFNG and IL17A expression in human PBMC: Fold changes (Fc) comparing stimulated (S) to no-stimulated (NS) PBMC (upper part) and comparing stimulated (S) to stimulated and Fenofibrate treated (S+FF) PBMC (lower part); direction of the gene expression change and p-values calculated by two-sided Student T-test.

<p>Effects of Fenofibrate on IFNG and IL17A expression in human PBMC: Fold changes (Fc) comparing stimulated (S) to no-stimulated (NS) PBMC (upper part) and comparing stimulated (S) to stimulated and Fenofibrate treated (S+FF) PBMC (lower part); direction of the gene expression change and p-values calculated by two-sided Student T-test.</p

Graft Survival with Fenofibrate: Fenofibrate treatment alone prolonged cardiac graft survival in transplanted mice.

<p>Kaplan Meyer curve for graft survival data after total allo-mismatch murine heart transplantation was assessed by determining the number of post operational days (POD) on which transplanted mice showed a palpatable graft beating score (BS). The median number of days grafts of Fenofibrate treated animals (FF, n = 6) showed beating was 25 compared to 9.5 days in non-treated animals (NT, n = 6). Significance of graft survival was assessed by Wilcoxon log-rank test (p = 0.0007).</p

tCRM score correlates with AR lesions and chronic allograft nephropathy.

<p>[A] The tCRM score positively correlates significantly (p = 0.001) with the degree of infiltrates found on biopsy for the t-score = 0.722 and [B] i score = 0.736. [C] A tCRM score across a subset of 7 of the 11 genes differentiated most samples with pIFTA or progressors (3.29 ± 0.93) from no-AR patients (1.2 ± 0.18; p = 0.037). Stable/non-progressors (NP) and AR were highly distinguishable (1.198±0.1801 versus 5.582±0.8651; p = 0.0063). pIFTA/Progressors and AR were not different with regards to their tCRM scores (p = 0.16).</p