CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury
Authors: Laura de Ramon, Elia Ripoll, Ana Merino, Marc Lúcia, Josep M. Aran, S. Pérez-Rentero, Nuria Lloberas, Josep M. Cruzado, Josep M. Grinyó, Juan Torras
Journal: Kidney International (2015)

Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte–endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing. In the warm ischemia model different doses were used, resulting in clear renal function improvement and a structural renoprotective effect. Renal ischemia activated the CD40 gene and protein expression, which was inhibited by intravenous siRNA administration. CD40 gene silencing improved renal inflammatory status, as seen by the reduction of CD68 and CD3 T-cell infiltrates, attenuated pro-inflammatory, and enhanced anti-inflammatory mediators. Furthermore, siRNA administration decreased a spleen pro-inflammatory monocyte subset and reduced TNFα secretion by splenic T cells. In the cold ischemia model with syngeneic and allogeneic renal transplantation, the most effective dose induced similar functional and structural renoprotective effects. Our data show the efficacy of our siRNA in modulating both the local and the systemic inflammatory milieu after an ischemic insult. Thus, CD40 silencing could emerge as a novel therapeutic strategy in solid organ transplantation. Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte–endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing. In the warm ischemia model different doses were used, resulting in clear renal function improvement and a structural renoprotective effect. Renal ischemia activated the CD40 gene and protein expression, which was inhibited by intravenous siRNA administration. CD40 gene silencing improved renal inflammatory status, as seen by the reduction of CD68 and CD3 T-cell infiltrates, attenuated pro-inflammatory, and enhanced anti-inflammatory mediators. Furthermore, siRNA administration decreased a spleen pro-inflammatory monocyte subset and reduced TNFα secretion by splenic T cells. In the cold ischemia model with syngeneic and allogeneic renal transplantation, the most effective dose induced similar functional and structural renoprotective effects. Our data show the efficacy of our siRNA in modulating both the local and the systemic inflammatory milieu after an ischemic insult. Thus, CD40 silencing could emerge as a novel therapeutic strategy in solid organ transplantation. Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury, contributing significantly to delayed graft function and acute rejection after transplantation.1.Loverre A. Capobianco C. Stallone G. et al.Ischemia-reperfusion injury-induced abnormal dendritic cell traffic in the transplanted kidney with delayed graft function.Kidney Int. 2007; 72: 994-1003Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 2.Fuquay R. Renner B. Kulik L. et al.Renal ischemia-reperfusion injury amplifies the humoral immune response.J Am Soc Nephrol. 2013; 24: 1063-1072Crossref PubMed Scopus (72) Google Scholar, 3.Siedlecki A. Irish W. Brennan D.C. Delayed graft function in the kidney transplant.Am J Transplant. 2011; 11: 2279-2296Crossref PubMed Scopus (512) Google Scholar The inflammatory response has a critical role in the outcome of IRI4.Bonventre J.V. Zuk A. 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De Beuf A. et al.T cells as mediators in renal ischemia/reperfusion injury.Kidney Int. 2004; 66: 491-496Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar Multiple roles of T cells have been described in several studies of ischemia, using T cell–deficient mice with either T cell–specific depleting monoclonal antibodies or knockout animals, combined with adaptive transfer of purified T-cell subsets.7.Rabb H. The T cell as a bridge between innate and adaptive immune systems: implications for the kidney.Kidney Int. 2002; 61: 1935-1946Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 8.Burne M.J. Daniels F. El Ghandour A. et al.Identification of the CD4(+) T cell as a major pathogenic factor in ischemic acute renal failure.J Clin Invest. 2001; 108: 1283-1290Crossref PubMed Scopus (385) Google Scholar, 9.Burne-Taney M.J. Yokota-Ikeda N. Rabb H. 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Torras J. et al.In vivo therapeutic efficacy of intra-renal CD40 silencing in a model of humoral acute rejection.Gene Ther. 2011; 18: 945-952Crossref PubMed Scopus (27) Google Scholar However, translation of CD40L blockade into non-human primate and human studies revealed thromboembolic complications.18.Koyama I. Kawai T. Andrews D. et al.Thrombophilia associated with anti-CD154 monoclonal antibody treatment and its prophylaxis in nonhuman primates.Transplantation. 2004; 77: 460-462Crossref PubMed Scopus (96) Google Scholar Thus, CD40 has become a new emerging target. Although several anti-CD40 monoclonal antibodies (mAbs) have been tested as alternatives to anti-CD40L mAbs,19.Oura T. Yamashita K. Suzuki T. et al.Long-term hepatic allograft acceptance based on CD40 blockade by ASKP1240 in nonhuman primates.Am J Transplant. 2012; 12: 1740-1754Crossref PubMed Scopus (56) Google Scholar other mechanisms can be used to block the pathway. The RNA interference phenomenon (RNAi) is an innate gene silencing mechanism that acts at the post-transcriptional level and has emerged as a potent mechanism to specifically knockdown mRNA transcripts.20.McManus M.T. Sharp P.A. Gene silencing in mammals by small interfering RNAs.Nat Rev Genet. 2002; 3: 737-747Crossref PubMed Scopus (1215) Google Scholar In previous studies, our group developed a small inhibitory RNA (siRNA) against CD40-mRNA that effectively inhibits the translation to CD40 mRNA and protein. This siRNA was used in a model of humoral acute rejection.17.Ripoll E. Pluvinet R. Torras J. et al.In vivo therapeutic efficacy of intra-renal CD40 silencing in a model of humoral acute rejection.Gene Ther. 2011; 18: 945-952Crossref PubMed Scopus (27) Google Scholar Thus, local CD40 silencing proved to be a useful strategy to prevent Ab-mediated rejection. Rapid degradation by exo/endonucleases constitutes a serious challenge to the successful intracellular delivery of siRNAs in vivo and their ultimate biological activity.21.Shim M.S. Kwon Y.J. Efficient and targeted delivery of siRNA in vivo.FEBS J. 2010; 277: 4814-4827Crossref PubMed Scopus (253) Google Scholar,22.Corey D.R. Chemical modification: the key to clinical application of RNA interference?.J Clin Invest. 2007; 117: 3615-3622Crossref PubMed Scopus (247) Google Scholar siRNA through cholesterol conjugation has been demonstrated to improve its distribution and cellular uptake.23.Soutschek J. Akinc A. Bramlage B. et al.Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.Nature. 2004; 432: 173-178Crossref PubMed Scopus (1920) Google Scholar Moreover, chemical modifications introducing phosphorothioate linkages and O-methyl greatly prolong half-life in plasma and increase resistance to nucleases. We used a murine-specific siRNA anti-CD40, chemically stabilized and cholesterol conjugated, in a model of autoimmune nephritis and was demonstrated that systemic CD40 blockade induced immune deactivation of the inflamed kidneys.24.Ripoll È Merino A. Goma M. et al.CD40 gene silencing reduces the progression of experimental lupus nephritis modulating local milieu and systemic mechanisms.PLoS One. 2013; 8: e65068Crossref PubMed Scopus (27) Google Scholar In the present study, the effect of CD40 blockade with specific siRNAs was analyzed in warm and cold ischemia models in rodents. We hypothesized that blocking the co-stimulatory CD40-CD40L dyad would reduce the inflammatory response developing from IRI. We aimed to evaluate whether the CD40 blockade interfered locally in chemokine-attracting molecules, transduction signals, or cell recruitment. Moreover, we assessed whether systemic siRNA administration affected distant spleen cell populations. After confirming the efficacy in mice and the greater efficiency of siRNA conjugated with cholesterol (Supplementary data), we further analyzed different doses to find an optimal dosage for the transplant model. As seen in Figure 1a, the scrambled (SC) and vehicle (Veh) groups showed worse renal function compared with all the treated groups. The higher doses were the most effective. Urea data showed parallel results. Again, the higher doses were the most effective (Figure 1b). Download .doc (.03 MB) Help with doc files Supplementary Information Examination of renal tissue revealed that Veh and SC groups had severe kidney injury, as evaluated by ischemic damage overall scores of 8.3 and 9.0, respectively (Figure 1c). In contrast, this ischemic injury was less severe in all treated groups in a dose-dependent way. Conventional histology showed well-preserved renal architecture in the group treated with 500 μg siRNA (Figure 2). To assess the activation of the CD40 co-stimulatory pathway, kidney RNA expression was measured. CD40 in Veh and SC groups was overexpressed 2.2-fold and 1.8-fold, respectively, compared with healthy kidneys. CD40 gene silencing effectively reduced CD40 expression in all treated groups (Table 1).Table 1Kidney gene expression and inflammatory cell infiltration characterizationCells/hpfShamVehSCCD40-15CD40-50CD40-150CD40-500P-valueCD6810.2±0.820.6±4.916.3±3.412.2±1.66aP<0.05 vs. Veh15.9±2.65.2±1.2a,P<0.05 vs. VehbP<0.05 vs. SC.8.8±1.9aP<0.05 vs. Veh0.010CD30.9±0.24.8±0.73.3±1.11 ±0.3a,P<0.05 vs. VehbP<0.05 vs. SC.0.8±0.1a,P<0.05 vs. VehbP<0.05 vs. SC.0.5±0.2a,P<0.05 vs. VehbP<0.05 vs. SC.0.6±0.2a,P<0.05 vs. VehbP<0.05 vs. SC.0.0001Folds/18s CD401.2±0.22.2±0.61.8±0.20.8±0.2aP<0.05 vs. Veh0.9±0.3aP<0.05 vs. Veh1.0±0.1aP<0.05 vs. Veh0.9±0.2aP<0.05 vs. Veh0.060 TLR30.6±0.11.1±0.10.4±0.10.7±0.20.7±0.80.6±0.80.8±0.10.007 TLR20.7±0.53.6±1.13.7±0.62.6±0.72.3±0.62.6±1.31.8±0.30.280 NF-Kβ1.7±0.32.4±0.42.5±0.41.8±0.31.1±0.2aP<0.05 vs. Veh1.6±0.31.7±0.3NS IL-42.6±0.92.1±0.94.0±0.73.9±0.93.2±0.75.0±0.8aP<0.05 vs. Veh5.8±1.2aP<0.05 vs. Veh0.101 CCL20.4±0.13.5±0.93.0±0.41.6±0.7aP<0.05 vs. Veh2.6±0.72.7±0.42.6±0.50.064 CCL30.6±0.11.5±0.31.2±0.50.5±0.1aP<0.05 vs. Veh0.9±0.30.8±0.10.9±0.1NS CCL41.2±0.11.9±0.41.7±0.40.6±0.2a,P<0.05 vs. VehbP<0.05 vs. SC.1.1±0.3aP<0.05 vs. Veh0.8±0.1aP<0.05 vs. Veh0.8±0.2aP<0.05 vs. Veh0.025 CCL50.8±0.11.0±0.21.3±0.20.5±0.1a,P<0.05 vs. VehbP<0.05 vs. SC.0.4±0.1a,P<0.05 vs. VehbP<0.05 vs. SC.0.5±0.1a,P<0.05 vs. VehbP<0.05 vs. SC.0.7±0.1aP<0.05 vs. Veh0.001 CXCL90.5±0.10.7±0.20.8±0.20.3±0.1a,P<0.05 vs. VehbP<0.05 vs. SC.0.4±0.10.6±0.10.5±0.1NS CXCL101.8±0.23.7±0.73.2±0.71.1±0.4a,P<0.05 vs. VehbP<0.05 vs. SC.1.2±0.3a,P<0.05 vs. VehbP<0.05 vs. SC.1.9±0.6aP<0.05 vs. Veh1.7±0.5aP<0.05 vs. Veh0.014 CXCL110.8±0.12.8±0.62.5±0.70.9±0.3aP<0.05 vs. Veh1.3±0.3aP<0.05 vs. Veh2.2±0.42.1±0.20.037Abbreviations: SC, scrambled; Veh, vehicle. siRNA anti-CD40, especially knocked-down CD40 expression, as well as other pro-inflammatory and chemokine-related genes. CD68 and CD3 were also reduced by CD40 silencing. Data are expressed as mean±s.e.m.a P<0.05 vs. Vehb P<0.05 vs. SC. Open table in a new tab Abbreviations: SC, scrambled; Veh, vehicle. siRNA anti-CD40, especially knocked-down CD40 expression, as well as other pro-inflammatory and chemokine-related genes. CD68 and CD3 were also reduced by CD40 silencing. Data are expressed as mean±s.e.m. CD40 protein expression and location in renal tissue were analyzed by immunostaining. The CD40 overexpression pattern differed in the diverse compartments of the warm ischemic kidneys (tubuli, glomeruli, and vessels). A reduction in CD40 expression was seen in all compartments in the treated groups, with the highest dose being the most effective (Figure 1). To better characterize the inflammatory cell infiltration observed by conventional histology, CD68 (macrophages) and CD3 (T cells) surface markers were analyzed (Figure 2). CD68 immunostaining revealed a lower degree of macrophage infiltration in siRNA CD40–treated groups, which was significant in those treated with higher siRNA doses. Moreover, CD3 immunostaining showed a significant reduction in CD3+ T cells in all treated groups (Table 1). Given the innate immune response, gene expression of both pro-inflammatory and anti-inflammatory renal mediators was measured. For Toll-like receptor (TLR)-4, classically related to ischemia damage, there were no differences between groups (not shown). Conversely, TLR2 was reduced in all treated animals. Furthermore, systemic injection of siRNA did not appear to activate the local innate immune response as TLR3 expression was not overexpressed. The downstream nuclear factor, NF-KB expression, had a similar outcome to TLR2 and CD40. Thus, there was a 2.4-fold overexpression in Veh-treated animals, with a clear reduction in treated kidneys. CD40 silencing induced a significant overexpression of the anti-inflammatory cytokine IL-4 only in animals receiving the highest siRNA dose (Table 1). We also assessed the expression of chemokines involved in ischemic damage. CXCL9, CXCL10, and CXCL11 were significantly lower after siRNA-CD40 treatment than in the Veh and SC groups. After reperfusion, Veh and SC groups expressed higher levels of CCL2, CCL3, CCL4, and CCL5, which were clearly reduced by CD40 gene silencing (Table 1). The previous PROLIGEN study (Supplementary data) defined a gene list relating regeneration versus injury after ischemia insults. There, we validated the renal expression profile of a subset of genes involving defense, immune response, inflammation, and chemotaxis after rat warm ischemic insult. In present study, we found overexpression of Socs3––a Jak-Stat pathway component––and of several genes related to cell cycle, growth, and cell communication, such as Emp1, Emp3, Fcnb, Reln, and Tnfrsf12a in Veh and SC animals compared with siRNA CD40–treated animals (Figure 3). In particular, the Ca2+-binding S100 protein family members (S100a6, S100a8, and S100a9), involved in the regulation of neutrophil chemotaxis and inflammation, were overexpressed due to ischemia but were significantly downregulated by CD40 treatment. Moreover, the expression of galectin family genes (Lgals1, Lgals3), with immune regulatory functions, was also significantly reduced by siRNA CD40 administration. Neither the S100 nor the galectin gene families had been previously related to CD40 signaling. We were interested in substantiate changes in the Galectin family, and thus we analyzed protein expression. As shown in Figure 4, galectin 1 and 3 were reduced by CD40 gene silencing in the western blot analysis, with no significant differences. Interestingly, a reduction in cells expressing galectin was seen, which was clearly significant at higher doses. Analysis of the B- and T-cell phenotype in splenocytes population by flow cytometry did not reveal differences in SC or gene silencing–treated rats, 48 h after ischemia (not shown). In treated animals, there were fewer monocytes (ED9) than in Veh and SC groups. Moreover, a positive double-stained CD43high/CD161 monocyte population was overexpressed in Veh and SC rats. Interestingly, this cell population clearly decreased in rats after CD40 gene silencing (Figure 5). To further analyze the systemic inhibitory effect of gene silencing, CD40 expression in spleen B cells (CD45+) was measured. Renal ischemia in SC-treated rats induced CD40 overexpression in non-circulating spleen B cells. Those rats suffering from warm ischemia and treated with siRNA-CD40 had lower CD40 expression, reaching similar values to the healthy group (not shown). Although no differences in spleen T-cell frequency were observed, intracellular cytokine production was analyzed by flow cytometry. SC-treated splenocytes, obtained 48 h after ischemia, revealed TNFα overproduction in CD3+ cells than splenocytes from healthy rats. In CD3+ T-cell subpopulations, the TNFα secretion levels doubled in the CD4+ cells, whereas the increase in CD8+ cells was lower. Gene silencing caused a decrease in TNFα production in CD3+ T cells, reaching similar levels to that in healthy rats. Apparently, CD4+ T cells were less responsive to gene silencing, as the reduction was modest. Instead, in CD8+ T cells TNFα levels returned to healthy values (Figure 6). The effect of CD40 silencing was also analyzed in our well-established model of renal cold ischemia, with renal transplantation. As shown in Figure 7a, in the syngeneic Lew-Lew studies, the group treated with our siRNA-CD40 improved renal function, as seen by the reduction in creatinine values. In contrast, Veh and SC groups had worse renal function. According to the histopathological evaluation, all ischemic parameters, which were increased in Veh or SC groups, were improved by CD40 gene silencing. Although only acute tubular necrosis was strongly reduced in the treated group, the overall score denoted improvement in the ischemic injury (Figure 7b). In the allogeneic Wist-Lew studies, there was an initial renal dysfunction peaking on day 1 and decreasing on day 3, indicating the cold ischemic damage. As shown in Figure 8, creatinine values in siRNA-CD40-treated animals were lower than the other groups. From day 5, a further decrease in renal function occurred indicating the acute renal rejection. Thus, in Veh and SC groups, the mean survival time was 12 and 15days due to severe renal dysfunction. In contrast, the renal dysfunction in siRNA-CD40 treated group was less intense with somewhat prolonged survival to 17 days. Animals receiving cyclosporine survived the 21 days of follow-up, with an almost normal renal function. The histological analysis showed severe Banff inflammatory parameters in Veh and SC groups. Those animals that were treated with siRNA-CD40 and survived more than 18 days presented a less severe inflammatory pattern. The cyclosporine animals presented a conserved histological pattern. In vivo protection by co-stimulatory molecule blockade in IRI models has scarcely been reported,25.Takada M. Chandraker A. Nadeau K.C. et al.The role of the B7 costimulatory pathway in experimental cold ischemia/reperfusion injury.J Clin Invest. 1997; 100: 1199-1203Crossref PubMed Scopus (199) Google Scholar,26.Chandraker A. Takada M. 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Torras J. et al.In vivo therapeutic efficacy of intra-renal CD40 silencing in a model of humoral acute rejection.Gene Ther. 2011; 18: 945-952Crossref PubMed Scopus (27) Google Scholar to maximize donor organ use and function in humans. We first began studies using mouse warm ischemia models to prove the concept that silencing the CD40 costimulatory pathway was effective in kidney function and morphology protection. In the nineties, Sayegh's group showed that the B7 costimulatory pathway has a critical role in organ dysfunction after renal cold IRI.26.Chandraker A. Takada M. Nadeau K.C. et al.CD28-b7 blockade in organ dysfunction secondary to cold ischemia/reperfusion injury.Kidney Int. 1997; 52: 1678-1684Abstract Full Text PDF PubMed Scopus (91) Google Scholar Later, Kupiec-Weglinski's group13.Ke B. Shen X.-D. Gao F. et al.Gene therapy for liver transplantation using adenoviral vectors: CD40-CD154 blockade by gene transfer of CD40Ig protects rat livers from cold ischemia and reperfusion injury.Mol Ther. 2004; 9: 38-45Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar modulated cold liver IRI by targeted gene therapy with a CD40Ig-hybrid molecule. Although the effectiveness of the CD40-ligand blockade using monoclonal antibodies has been confirmed on preclinical models, thromboembolic events obscured its translation to the clinic.27.Lievens D. Zernecke A. Seijkens T. et al.Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis.Blood. 2010; 116: 4317-4327Crossref PubMed Scopus (226) Google Scholar Our group has recently reported encouraging experimental results that showed that CD40 silencing reduces the progression of lupus nephritis by modulating both local milieu and systemic mechanisms.24.Ripoll È Merino A. Goma M. et al.CD40 gene silencing reduces the progression of experimental lupus nephritis modulating local milieu and systemic mechanisms.PLoS One. 2013; 8: e65068Crossref PubMed Scopus (27) Google Scholar We later moved to the rat warm ischemia model aiming to confirm the efficacy and dose dependency of gene silencing in another rodent species, using a different siRNA sequence. As stated in the Results section, the highest doses of siRNA exerted the most robust protection in all analyzed functional, morphological, and inflammatory parameters. As the doses were reduced, there was a gradual loss in their beneficial effects, also indicating that gene inhibition completely covered the critical period of post-ischemic inflammatory response. In liver ischemia, CD40-CD40L signaling, but not IFN-γ signaling, has been reported as important for T-CD4+ cell function without the requirement of de novo Ag-specific activation.28.Shen X. Wang Y. Gao F. et al.CD4 T cells promote tissue inflammation via CD40 signaling without de novo activation in a murine model of liver ischemia/reperfusion injury.Hepatology. 2009; 50: 1537-1546Crossref PubMed Scopus (76) Google Scholar Our group previously described how CD40 silencing in vitro modulates several genes involved in different processes such as innate immune response, signal transduction, and apoptosis.29.Pluvinet R. Olivar R. Krupinski J. et al.CD40: an upstream master switch for endothelial cell activation uncovered by RNAi-coupled transcriptional profiling.Blood. 2008; 112: 3624-3637Crossref PubMed Scopus (35) Google Scholar In the present in vivo study, we confirmed that CD40 silencing modulated the downstream signal transduction cascade in the ischemic-inflammatory environment. In particular, CD40 silencing modulated TLR expression in kidney tissue. It is known that TLRs participate in IRI, especially TLR 2 and 4, acting as tissue damage sensors, activating innate immunity through pro-inflammatory transcription factors such as NF-kB.30.Leemans J.C. Stokman G. Claessen N. et al.Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney.J Clin Invest. 2005; 115: 2894-2903Crossref PubMed Scopus (472) Google Scholar, 31.Chen J. John R. Richardson J.A. et al.Toll-like receptor 4 regulates early endothelial activation during ischemic acute kidney injury.Kidney Int. 2011; 79: 288-299Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 32.Arslan F. Keogh B. Mcguirk P. et al.TLR2 and TLR4 in ischemia reperfusion injury.Mediators Inflamm. 2010; 2010: 704202Crossref PubMed Scopus (142) Google Scholar CD40 silencing was shown to inhibit TLR2 activation selectively and therefore to reduce NF-kB expression. Chemokines act as potent attractants of inflammatory cells in ischemia injury.33.Frangogiannis N.G. Chemokines in ischemia and reperfusion.Thromb Haemost. 2007; 97: 738-747PubMed Google Scholar CCL2, CCL3, and CCL4 enhance the recruitment of macrophages and neutrophils within the tissue. CCL5 is involved in the migration of T cells, monocytes, natural killer, and dendritic cells. CXCL9, CXCL10, and CXCL11 elicit T-cell chemoattraction by interaction with the chemokine receptor CXCR3. Here we showed that all these chemokines were downregulated by CD40 gene silencing. Therefore, the CD40 signaling pathway proved a key contributor to the inflammatory molecular mechanisms induced by ischemia. In particular, the above chemokine modulation could be instrumental in the reduction of infiltrating T cells and macrophages seen in the CD40 siRNA–treated groups. Furthermore, we report for the first time two new downstream CD40 signaling target families, which might be critical effectors of the pro-inflammatory response to the ischemic insult. Thus, CD40 gen silencing downregulated the expression of S100 and galectin protein families. The Ca2+-binding S100 proteins participate in innate and adaptive immune responses to cell migration, chemotaxis, and inflammation.34.Donato R. Cannon B.R. Sorci G. et al.Functions of S100 proteins.Curr Mol Med. 2013; 13: 24-57Crossref PubMed Scopus (880) Google Scholar CD40 silencing knocked down the expression of calcyclin (S100a6) known for its potential role in cell responses to various stress stimuli and apoptosis. Moreover, a recent study has shown its involvement in the regulation of endothelial cell cycle progression and senescence.35.Bao L. Odell A.F. Stephen S.L. et al.The S100A6 calcium-binding protein regulates endothelial cell-cycle progression and senescence.FEBS J. 2012; 279: 4576-4588Crossref PubMed Scopus (33) Google Scholar The downregulation of S100a8 and S100a9 also contributes to inflammatory modulation through reduction in oxidative stress and neutrophil chemotaxis.36.Tong L. Lan W. Lim R.R. et al.S100A proteins as molecular targets in the ocular surface inflammatory diseases.Ocul Surf. 2014; 12: 23-31Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Galectins are a group of lectins that act as potent immune regulators in inflammatory diseases or acute renal failure.37.Seropian I.M. Cerliani J.P. Toldo S. et al.Galectin-1 controls cardiac inflammation and ventricular remodeling during acute myocardial infarction.Am J Pathol. 2013; 182: 29-40Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 38.Li S. Yu Y. Koehn C.D. et al.Galectins in the pathogenesis of rheumatoid arthritis.J Clin Cell Immunol. 2013; 4Google Scholar, 39.Fernandes Bertocchi A.P. Campanhole G. Wang P.H.M. et al.A Role for