The role of GILZ in modulation of adaptive immunity in a murine model of myocardial infarction

Experimental and Molecular Pathology 102 (2017) 408–414

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The role of GILZ in modulation of adaptive immunity in a murine model of myocardial infarction

MARK

Babak Babana,⁎, Lin Yina, Xu Qina, Jun Yao Liua, Xingming Shib, Mahmood S. Mozaffaria a b

Department of Oral Biology, Augusta University, Augusta, GA 30912-1128, United States Department of Neuroscience and Regenerative Medicine, Augusta University, Augusta, GA 30912-1128, United States

A R T I C L E I N F O

A B S T R A C T

Keywords: Myocardial infarction GILZ T cells Cytokines Mitochondria Cell death

Myocardial infarction (MI) is associated with intense immune and inflammatory responses which contribute to tissue injury. Increasing evidence indicates that the glucocorticoid-induced leucine zipper (GILZ) protein suppresses immune and inflammatory responses. However, the status of and the role of GILZ in MI are not known. We tested the hypotheses that a) MI reduces cardiac GILZ associated with intense inflammation and cell death and b) intramyocardial GILZ delivery confers cardioprotection in association with increased Tregs and suppression of inflammation. Male Balb/C mice were subjected to MI or sham operation; the infarcted animals were subdivided to receive intramyocardial injections of PBS, GILZ overexpressing cells (GILZ) or their controls expressing the green fluorescent protein (GFP). Three hours after the procedures, hearts were procured for subsequent analyses. MI markedly reduced cardiac GILZ expression accompanied with a) increase in Th-17 cells (i.e., CD3+ CD4+ IL-17+ BNP−) but decrease in Tregs (i.e., CD3+ CD4+ FoxP3+ BNP−), and b) disruption of mitochondrial membrane potential (ψm) associated with significant increases in apoptotic and necrotic cell death. While both GILZ and GFP returned the aforementioned parameters towards those of sham controls, these effects were most marked for mice receiving GILZ. Thus, GILZ markedly reduced Th-17 cells but increased Tregs and the anti-inflammatory cytokine, IL-10 positive cells accompanied with preservation of ψm and prevention of cell death. To our knowledge, this is the first report indicating an important role for GILZ in MI, in part via modulation of adaptive immune response, which raises the prospect of exogenous GILZ delivery as a novel cardioprotective modality.

1. Introduction Myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide. In the United States, an MI occurs every 43 s with an annual incidence of 525,000 and 210,000 first and recurrent attacks, respectively; an estimated 155,000 silent MIs also occur annually (Mozaffarian et al., 2016). Advances in the management of MI have reduced pre- and in-hospital mortality to about 15% (Mozaffarian et al., 2016). Despite such success, MI has emerged as a leading cause of heart failure (van Hout et al., 2016; Duncker et al., 2014). Multiple and diverse pathogenic mechanisms contribute to MI and its sequel of adverse cardiac remodeling. Prominent among them is dysregulation of the immune system which is increasingly recognized as both a triggering and an essential amplifying factor leading to an excessive inflammatory response thereby exacerbating tissue injury. The role of innate immunity in MI has been extensively investigated (Fang et al., 2015; Yan et al., 2013; Bodi et al., 2008). More recently, ⁎

the role of adaptive immunity, which plays pivotal roles in regulation of inflammatory mechanisms, in the pathogenesis of MI has come into focus (Ramos et al., 2016; Hofmann and Frantz, 2016; del Rosario Espinoza Mora et al., 2014). In this context, while intramyocardial injection of bone marrow-derived B lymphocytes, in the left anterior descending artery (LAD)-ligated rats, improved myocardial salvage and ventricular function (Goodchild et al., 2009), depletion of B lymphocytes also limited injury after MI and promoted recovery of the heart (Zouggari et al., 2013). Compared to B cells, T lymphocytes consist of multiple subsets whose functional phenotypes and cytokine profile are determined by expression of signature molecular markers (Ramos et al., 2016; Hofmann and Frantz, 2016). Accordingly, several subsets of effector T cells (Teffs) are described, including Th-17 cells, which can be differentiated from other Teffs by their expression of interleukin (IL17). On the other hand, regulatory T cells (Tregs) express the signature transcription factor forkhead box P3 (FoxP3) (Ramos et al., 2016; Hofmann and Frantz, 2016; Weirather et al., 2014; Yang et al., 2006; Mor et al., 2006). Recent studies suggest that Tregs are critical players

Corresponding author at: Department of Oral Biology, Dental College of Georgia, Augusta University, CL-2140, Augusta, GA 30912-1128, United States. E-mail address: [email protected] (B. Baban).

http://dx.doi.org/10.1016/j.yexmp.2017.05.002 Received 29 March 2017; Accepted 8 May 2017 Available online 10 May 2017 0014-4800/ © 2017 Published by Elsevier Inc.

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and is well-suited for cell transplantation studies as it produces welldefined infarct zones into which cells of interest can be injected (Baban et al., 2015; van Amerongen et al., 2008; Strungs et al., 2013; Costa et al., 2012; van den Bos et al., 2005). Immediately following induction of MI, hearts were injected, in the infarcted zone, with 50 μl of PBS or with a similar volume of PBS which contained 5 × 105 of mesenchymal stem cells (MSCs) which overexpress GILZ or express green fluorescent protein (GFP); these cells have been developed and characterized using Western blotting and immunofluorescent studies and utilized in a variety of studies (e.g., Yang et al., 2015; Zhang et al., 2008; Pan et al., 2014). Briefly, for this study, murine bone marrow-derived MSCs were initially isolated using a negative immune-depletion followed by a positive immune-selection procedure and characterized for their multilineage differentiation capacity. Thereafter, retroviral transfection protocols were carried out to generate GILZ/MSCs and GFP/MSCs (Yang et al., 2015; Zhang et al., 2008) which will be referred to as GILZ and GFP, respectively. At the conclusion of the protocols for induction of infarction and treatment protocols, surgical site on the thorax was closed by placement of 4.0 silk sutures to approximate the ribs, muscle layer and the skin; chest air was removed during this process by application of a 20G ×1″ Terumo SURFLO I.V. catheter connected to a 1 ml syringe. This was followed by removal of the tracheal tube, application of prolene suture to close off the tracheostomy and closure of the fascia and skin layers using 4.0 silk suture. Three hours after the surgical procedure, the animals were sacrificed and hearts procured.

in curtailing the intense pro-inflammatory changes that accompany a number of conditions including MI (Goodchild et al., 2009; Zouggari et al., 2013; Weirather et al., 2014; Yang et al., 2006; Mor et al., 2006). Indeed, the “inverted pyramid” model proposes that a few Tregs, at the bottom of the imaginary inverted pyramid, control the upper parts containing neutrophils, monocytes and effector T lymphocytes, among others (Bodi et al., 2008). Thus, accentuating the contribution of Tregs following MI should curtail inflammation and be conducive to repair and recovery of the damaged heart. The glucocorticoid-induced leucine zipper (GILZ) protein, also known as the tuberous sclerosis complex 22 (TSC22), is a recentlydescribed glucocorticoids (GCs)-induced transcriptional regulatory protein (Fan and Morand, 2012). As a member of the leucine zipper protein family, GILZ has emerged as a pivotal mediator of the anti-inflammatory effects of GCs (Fan and Morand, 2012; Ayroldi and Riccardi, 2009; Beaulieu et al., 2010; Lekva et al., 2010; Yang et al., 2015). The profound impact of GCs on GILZ expression is well-established and relates to the direct binding of GC/glucocorticoid receptor complex to the six glucocorticoid-responsive elements located in the promoter region of the GILZgene (Fan and Morand, 2012). Importantly, GILZ promotes the development of Tregs whereas GILZ deficiency causes impaired generation of peripheral Tregs (Yang et al., 2015). However, the status of GILZ and its role in regulation of Tregs and inflammation in MI are not known. Importantly, the advent of GILZ overexpressing mesenchymal stem cells offers a unique opportunity to explore the potential impact of intramyocardial GILZ delivery in the infarcted heart. Indeed, it is believed that beneficial effects of stem cells in injured tissues (e.g., heart) relate primarily to their release of a whole host of soluble factors and their subsequent paracrine effects, rather than stem cells trans-differentiation, thereby promoting tissue healing and repair (Hodgkinson et al., 2016; Yeghiazarians et al., 2009). We conjectured that GILZ delivery immediately post-MI effectively curtails the intense immune and inflammatory responses of the infarcted heart that occur during the initial phase post-injury. Accordingly, we tested the hypotheses that a) MI reduces cardiac GILZ associated with intense inflammation and cell death and b) intramyocardial GILZ delivery confers cardioprotection in association with increased Tregs and suppression of inflammation.

2.3. Assessment of GILZ mRNA Total RNA was isolated using TRIzol reagent (Invitrogen Corp., Grand Island, NY, USA). Thereafter, 1 μg of RNA was reverse transcribed using iScript cDNA synthesis kit (Bio-Rad, Richmond, CA). Realtime polymerase chain reaction (PCR) was performed on the StepOnePlus real-time PCR System (Applied Biosystems, CA); analyses were carried out in triplicates using appropriate primer and the Universal SYBR Green Supermix (Bio-Rad, Richmond, CA; n = 3hearts/group); β-actin mRNA was assessed as control and gene expression was expressed as fold change (2–ΔΔCt method) (Zhang et al., 2008). The PCR primer sequences used were as follow:

2. Methods 2.1. Animals Male Balb/C mice (Harlan Laboratories; 10–11 weeks of age) were obtained and housed in the institutional laboratory animal facilities and maintained at constant humidity (60 ± 5%), temperature (24 ± 1 °C) and light cycle (0600–1800 h) with free access to food and water. The use of animals for this study was in accordance with established institutional guidelines for care and use of animals in research.

2.4. Immunofluorescent imaging To confirm GILZ overexpression, cells expressing GFP or GILZ (25–50 × 103) were applied to slide using a cytospin (Shannon IV, USA) and then fixed and stained using primary antibody against GILZ (eBioScience USA) and the nuclear stain 4′,6-Diamidino-2-Phenylindole (DAPI; Invitrogen, USA) as described previously (Baban et al., 2015, 2013, 2014a, 2014b).

2.2. Surgical and treatment protocols One week after arrival, each animal was anesthetized with an intraperitoneal injection of ketamine/xylazine (120/15 mg/kg). Thereafter, surgical sites for tracheotomy and thoracotomy were shaved followed by application of Betadine and alcohol for topical disinfection. Intratracheal intubation with a Terumo SURFLO I.V. 18G × 2″ catheter was achieved and animals ventilated using a CWE SAR-830P ventilator. Thereafter, a 2.5 cm incision was made on the chest, corresponding to the third intercostal space and exposure of the heart following opening of the pericardium. Cryoinjury was affected by application of a 4 mm stainless probe, which was pre-cooled in liquid nitrogen, to the anterior surface of the left ventricle, midway between the left atrium and the apex of the heart; for sham-operated animals, the probe at room temperature, was applied to the anterior surface of the heart (Baban et al., 2015). The cryoinfarction model produces standardized infarcts

2.5. Analytical flow cytometry Hearts of other animals in each experimental group was used for preparation of cardiac cells for flow cytometry-based studies as described previously (n = 4–5 hearts/group; Baban et al., 2015, 2013, 2014a, 2014b). Flow cytometry offers the distinct advantage of determination of multiple parameters using the same pool of cells from a given tissue. Accordingly, commercially available antibodies against each protein of interest were used coupled with the use of a FACSCalibur flow cytometer (BD BioSciences, San Diego, CA; Baban et al., 2015, 2013, 2014a, 2014b). Briefly, the primary antibodies against brain natriuretic (BNP; abcam USA) as well as GILZ, IL-10, CD3, CD4 and isotype controls (eBioScience USA) were used. On the other hand, 409

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10+ and IL-17 + cells in cardiac cell preparations of experimental groups because other cardiac cells (e.g., cardiomyocytes) are known to also generate inflammatory cytokines (Baban et al., 2013, 2014a, 2014b; Wang et al., 2005; Poynter et al., 2011; Herrmann et al., 2010). Thus, utilizing the BNP and CD3, we initially identified fraction of T lymphocytes in cardiac cell preparations of whole ventricles of experimental groups. This was followed by subsequent analyses using CD4, FoxP3 and IL-17 markers. The results show that, indeed, GILZ cells are more effective than GFP cells in increasing Tregs (i.e., CD3+ CD4+ FoxP3+ BNP−)but reducing Th-17 cells (i.e., CD3+ CD4+ IL-17+ BNP−) in infarcted hearts (Fig. 5). We also assessed ψm in the context of determination of cell death using the same pool of cardiac cells of experimental groups for which other determinations were made. Fig. 6 shows representative histograms for JC-1 monomers and aggregates (panel A) as well as aggregates/monomers ratio (panel B) for experimental groups. Dominance of JC-1 aggregates, over monomers, was a feature of shamoperated hearts suggesting preservation of functional integrity of mitochondria. Cells prepared from infarcted hearts displayed marked reduction in JC-1 aggregates but prominent increase in JC-1 monomers suggestive of disruption of ψm (Baban et al., 2013, 2014a, 2014b). As a result, the ratio of aggregates to monomers was significantly reduced in infarcted hearts. While intramyocardial injection of GFP was associated with a modest increase in JC aggregates/monomers ratio, GILZ essentially restored this ratio to normal suggestive of marked preservation of ψm in infarcted hearts. These observations are consistent with results of cell death analyses which was achieved using caspase 3 (a marker of apoptosis) and 7-AAD (a marker of necrosis). Fig. 7 shows representative scatter plots (panel A) and percent of apoptotic and necrotic cell death are shown under panel B. As expected, MI was associated with marked increase in apoptosis/necrosis compared to sham group. While, intramyocardial injection of GFP caused a moderate reduction in apoptosis/necrosis, injection of GILZ was associated with a marked reduction in cell death, essentially resembling that of the shamoperated group.

intracellular staining for IL-17 and FoxP3 were also performed after fixing and permeablization of cells using Fix/Perm kit from eBioScience for 15 min on ice in dark according to manufacturer's instruction. Thereafter, cells were incubated with IL-17 and FoxP3 antibodies for 15 min before final wash with PBS. All samples were run using four color FACS Caliber flow cytometer (BD Biosciences). As gating strategy, for each sample, isotype-matched controls were analyzed to set the appropriate gates. For each marker, samples were analyzed in duplicate measurements. To minimize false-positive events, the number of double-positive events detected with the isotype controls was subtracted from the number of double-positive cells stained with corresponding antibodies (not isotype control), respectively. Cells expressing a specific marker were reported as a percentage of the number of gated events. For assessment of mitochondrial membrane potential (ψm), the JC-1 assay was used while the caspase 3/7-amino actinomycin D (7AAD) protocol was utilized used for determination of necrotic and apoptotic cell death (Baban et al, 2015, 2013, 2014a, 2014b). 2.6. Statistics Student t-test was used for comparison between sham and infarcted groups (Fig. 1). All other data were analyzed using analysis of variance followed by Newman-Keuls' post hoc test to establish significance (p < 0.05) among experimental groups. Data are expressed as means ± SEM. 3. Results Cryoinjury is associated with significant reduction in GILZ + cells in cardiac cells prepared from infarcted than sham-operated hearts (Fig. 1A). Similarly, GILZ mRNA was reduced in the infarct zone compared to cardiac tissue of the sham group (Fig. 1B). We conjectured that intramyocardial GILZ delivery should exert cardioprotection in association with suppression of inflammation. Thus, we confirmed that, indeed, cytospin-prepared GILZ cells display marked expression of GILZ compared to GFP cells (Fig. 2). Fig. 3A shows that the percent of CD3 + cells were markedly increased in infarcted hearts. Intramyocardial injection of GFP or GILZ reduced the percent of CD3 + cells, with the effect greater for the latter. On the other hand, FoxP3 + cells were similar between sham and infarcted hearts (Fig. 3B). By contrast, intramyocardial injection of GFP or GILZ increased the percent of FoxP3 + cells, with the effects significantly greater for GILZ than GFP group. Fig. 4 shows the percent of IL-10 + and IL-17 + cells for experimental groups. While IL-10 + cells were similar between sham and infarcted hearts, the percent of IL17 + cells were markedly increased in infarcted than sham hearts. Intramyocardial injections of GFP and GILZ increased IL-10 + but decreased IL-17 + cells, with the effects more marked for the latter. Indeed, the percent of IL-17 + cells were similar between GILZ and the sham groups (Fig. 4). We next determined whether T cells are potential sources for the IL-

4. Discussion The present study shows marked reduction in cardiac GILZ in association with increased Th-17 cells accompanied with marked disruption of ψm and increased apoptotic/necrotic cell death in hearts subjected to MI. Importantly, intramyocardial injection of either GFP or GILZ reversed these effects but the impact was more marked for the latter group. Particularly prominent effects of GILZ delivery were significant increases in Tregs and IL-10 + cells but decreases in Th17cells accompanied with preservation of ψm and decreases in apoptotic and necrotic cell death. Collectively, these observations indicate that GILZ curtails the robust immune and inflammatory responses of infarcted heart culminating in cardioprotection. GILZ is known to cause a myriad of effects including immunosuppressive and anti-inflammatory outcomes, among others. The recogni-

*

Sham

* Sham

Infarcted

Infarcted

Fig. 1. Bar graph shows percent of cardiac cells which are positive for GILZ in hearts subjected to infarction compared to their sham controls (A). Panel B shows reduced GILZ mRNA expression for infarcted than sham hearts. Data are average ± SEM.*p < 0.05 compared to the sham.

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DAPI

Merged

GILZ Cells

GFP Cells

GILZ

Fig. 2. Panels show immunofluorescent images for GILZ, the nuclear marker, DAPI, and their merged images of cytospin-prepared GFP and GILZ cells. DAPI: 4′,6-diamidino-2phenylindole.

cells but reduced Th-17 cells. Our collective observations are consistent with the robust immune and inflammatory responses of the infarcted heart, during the immediate phase post-injury, and the role of GILZ in curtailing immune and inflammatory processes. With respect to cardiac injury (e.g., caused by ischemia-reperfusion), there is convincing evidence that pro-inflammatory CD4 + T lymphocytes contribute to tissue damage while the role of Tregs remains to be better established (Hofmann and Frantz, 2016). In the non-reperfused heart, conventional effector CD4 + T cells, with a shift towards Th-1 cytokine profile, infiltrate the myocardium within a few days after MI (Hofmann and Frantz, 2016; Hofmann et al., 2012). Indeed, a Th-1/Th-2 functional imbalance exists in both coronary arterial and myocardial inflammatory processes, contributing to adverse ventricular remodeling post-MI (Cheng et al., 2005). Importantly, activation and proliferation of FoxP3 + Tregs occur in heart-draining mediastinal lymph nodes as early as 3 days post-MI as well as infiltration of Tregs in infarct zone in coronary artery-ligated mice 7-days post-MI (Hofmann and Frantz, 2016). These observations coupled with the pivotal role of Tregs in resolution of inflammation has led to intense interest in their role in MI. Accordingly, it is shown that Tregs depletion (e.g., genetic Tregs ablation via use of FoxP3DTR mice or anti-CD25 antibody) aggravates cardiac inflammation associated with deterioration of functional outcomes in the LAD-ligated murine model of MI. On the other hand, Tregs activation (e.g., by superagonistic anti-CD28 monoclonal antibody 2 days post-MI) or adoptive transfer of Tregs improves healing and survival (Weirather et al., 2014; Sharir et al., 2014). Consistent with the ability of Tregs to influence innate immune response, Tregs depletion promoted inflammatory M1-macrophage polarization while Tregs

tion of the role GILZ as a pivotal mediator of anti-inflammatory effects of GCs has led to intense research to unravel its mechanism(s) of actions. Accordingly, it is believed that anti-inflammatory effects of GILZ are likely independent of the glucocorticoid receptors thereby avoiding metabolic effects of GCs. Rather, a pivotal mechanism of action for GILZ relates to complex formations with other proteins and transcription factors thereby preventing their effects. For example, GILZ directly interacts with nuclear factor-κB causing inhibition of its nuclear translocation and DNA interaction as well as binding to two components of activator protein-1, namely c-Fos and c-Jun, culminating in anti-inflammatory and immunosuppressive effects in lymphoid and myeloid compartments (Fan and Morand, 2012; Ayroldi and Riccardi, 2009). With respect to T lymphocytes, GILZ mimics the effects of GCs and exiting evidence suggest a crucial role for it in regulation of T cells functions. In this context, GILZ in dendritic cells causes phenotypic changes that are conducive to Tregs development and suppression of T cell activation (Fan and Morand, 2012; Ayroldi and Riccardi, 2009; Bereshchenko et al., 2014; Jones et al., 2015). Consistent with these observations, we recently showed that expression of GILZ in bone marrow mesenchymal lineage cells or bone marrow-derived MSCs increases the production of Tregs coupled with increased production of IL-10 but decreased generation of pro-inflammatory cytokines, IL-6 and IL-12 (Yang et al., 2015). A major finding of the present study relates to significant reduction of GILZ in the infarcted heart associated with increase in Th-17 cells with a tendency for reduced Tregs 3 h. post-injury. On the other hand, intramyocardial delivery of either GFP or GILZ reversed these effects; however, GILZ delivery more markedly increased Tregs and IL-10 +

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Sham PBS

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Fig. 3. Panels show percent of CD3 + (panel A) or FoxP3 + (panel B) cells in cardiac cell preparations of hearts of mice subjected to sham surgery or those subjected to infarction and receiving intramyocardial injections of PBS, GPF cells or GILZ cells (105 cells/heart). Data are average ± SEM.*p < 0.05 compared to sham group.#p < 0.05 compared to infarcted/ PBS.†p < 0.05 compared to other groups.

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Fig. 4. Panels show percent of IL-10 + (panel A) or IL-17 + (panel B) cells in cardiac cell preparations of hearts of mice subjected to sham surgery or those subjected to infarction and receiving intramyocardial injections of PBS, GPF cells or GILZ cells (105 cells/heart). Data are average ± SEM.*p < 0.05 compared to sham or infarcted/PBS group.#p < 0.05 compared to infarcted/PBS or infarcted-GFP group.

Fig. 5. Representative dot matrices identifying CD3+ cells are shown for cardiac cells prepared from experimental groups; CD3 + cells were further resolved using CD, FoxP3, and IL-17 and the brain natriuretic peptide (BNP). MI increased Th-17 cells (i.e., CD3+ CD4+ IL-17+ BNP−) but decreased in Tregs (i.e., CD3+ CD4+ FoxP3+ BNP−), effects which were more markedly reversed by GILZ than GFP.

The profound impact of GILZ delivery to suppress IL-17 expression is consistent with the observation that it also curtails IL-17-mediated skin inflammation. On the other hand, among other effects, IL-10 may exert matrix-stabilizing effects via induction of tissue inhibitors of metalloproteinases by mononuclear cells thereby contributing to matrix stabilization. Thus, GILZ-induced upregulation of IL-10 in the infarcted heart could beneficially influence tissue preservation and integrity. Another major finding of the present study relates to the ability of GILZ delivery to prevent disruption of ψm and exert significant cytoprotection in the infarcted heart. From a mechanistic viewpoint, GILZ is known to modulate mitochondria-dependent apoptotic pathways and influence pro- and anti-apoptotic proteins of Bcl-2 family (e.g., physical interaction and complex formation; Fan and Morand, 2012; Ayroldi and Riccardi, 2009). Thus, GILZ is shown to exhibit cell-

activation induced regulatory/anti-inflammatory M2 macrophage differentiation within the healing myocardium (Wang et al., 2016). In light of these observations, the emerging role of GILZ in regulation of the relative proportion of Tregs in general, and in the infarcted heart in particular, adds to a complex list of factors that are known to regulate functional phenotypes of T lymphocytes and offers the possibility of modulating Tregs, early post-MI, to beneficially affect the outcome of an injurious stimulus to the heart. This is an important consideration given that an imbalance in Th-17/Tregs favoring Th-17 cells is a major feature of plaque destabilization, acute myocardial infarction and infarction-related cardiogenic shock (del Rosario Espinoza Mora et al., 2014). Indeed, the imbalance among Th-17 cells and Tregs, as well as between IL-17 and IL-10 was a hallmark feature of infracted hearts which were markedly reversed by intramyocardial GILZ delivery. 412

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Fig. 6. Panels under A show representative histograms for JC-1 aggregates (red) and monomers (green) in cardiac cells prepared from hearts of experimental groups. On the other hand, average ± SEM values for the ratio of aggregates to monomers are shown under B.*p < 0.05 compared to other groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

survival by upregulating GRP78 protein level while reducing CHOP, ATF4 and XBPIs expression (André et al., 2016). Collectively, these observations point to the remarkable ability of GILZ to exert cytoprotective effects under a variety of conditions, an aspect particularly beneficial to infarcted heart since reduction of cardiomyocyte injury is known to limit adverse cardiac remodeling, a sequel of MI (Gorman et al., 2011). In conclusion, the present study established that reduction in cardiac GILZ accompanies the robust immune and inflammatory responses of the infarcted heart. On the other hand, intramyocardial GILZ delivery markedly upregulates Tregs and increases IL-10 + cells but reduces Th-17 cells accompanied by marked cardioprotection. These novel observations lay the foundation for subsequent investigation of many relevant aspects including long-term effects of GILZ delivery and associated mechanisms including the impact of GILZ

context dependent modulation of proliferation and cell death depending on the nature of stimulus and cell type (Fan and Morand, 2012). Our observation that GILZ delivery exerts prominent cardioprotection is consistent with a report indicating that GILZ overexpression protects against doxorubicin-induced cardiomyopathy as exemplified by induction of pro-survival protein Bcl-xL, prevention of mitochondrial release of cytochrome c and cleavage of caspase-3 (Aguilar et al., 2014). Similarly, GILZ overexpression protects against endoplasmic reticulum (ER) stress-mediated cell death likely via stimulation of mitochondrial oxidative phosphorylation (André et al., 2016). Utilizing several cancer cell lines, it is shown that GCs protect against tunicamycin-induced cell death, an effect altered by silencing of endogenous GILZ. On the other hand, under ER stress conditions, GILZ overexpression significantly reduced activation of mitochondrial pathway of apoptosis by maintaining Bcl-xL level. Importantly, GILZ shifted the balance in favor of cell

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Fig. 7. Panels under A show representative dot matrices for apoptosis/necrosis in cardiac cells prepared from hearts of experimental groups. On the other hand, average ± SEM values for percent of cell death are shown under B. a: necrosis; b: late apoptosis; c: early apoptosis.*p < 0.05 compared to other groups.#p < 0.05 compared to infarcted/PBS or infarcted-GFP group.

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