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CD4+T-cells facilitate wound healing post-myocardial infarction (MI) but promote left-ventricular (LV) remodeling during ischemic heart failure (HF; 8 weeks post-MI). Therefore, it is critical to understand if sustained CD4+T-cell activation leads to this pathological response, or if phenotypically different T-cells are activated during MI vs. HF. Using flow cytometry, we found that cardiac CD4+T-cells exhibit two distinct patterns of transmigration. First pattern consisted of a rapid CD4+T-cell response with maximal levels seen at 3 days post-MI which return to baseline by 14 days. However, during HF we observed a 2nd phase of activation and CD4+T-cells were ∼20-fold higher in HF as compared to sham-operated mice. Importantly, these biphasic kinetics were observed with all major T-cell subsets such as Th1, Th2, Th17, and regulatory T-cells suggesting a global change. To determine the role of this 2nd peak of T-cell activation, CD4-iDTR mice were generated and treated with DT every 10 from 28 days post-MI to deplete CD4+T-cells during chronic HF. While littermate control mice showed increased end-systolic and end-diastolic volumes (ESV and EDV) and decreased ejection fraction (EF) from 4 to 8 weeks post-MI, depletion of CD4+T-cells in Cre + mice significantly blunted LV remodeling and inhibited progressive increases in the EDV and ESV, and reduction in EF. This suggests that CD4+T-cell responses occurring during HF are different than those occurring during MI and promote LV remodeling and progressive cardiac dysfunction. Temporal immunomodulation of CD4+T-cells could be a translatable modality for ischemic HF.

Ischemic heart failure (HF) is associated with elevated inflammatory responses including pro-inflammatory cytokines, such as Tumor necrosis factor (TNF) α [and its cognate receptors, TNF receptor (TNFR) 1 and TNFR2] and pathological switching of CD4 + T-cells. TNF-TNFR2 signaling is known to promote CD4 + T-cell activation and proliferation. However, it is unknown if CD4 + T-cells also express TNFR1, and if elevated TNFα-TNFR1 signaling during HF mediates pathological effects of CD4 + T-cells. Using flow cytometry, we show that TNFR1 + CD4 + T-cells are amplified in the hearts at 3 days (d) and 8 weeks (wks) post-MI (HF) in mice. As compared to sham, while TNFα expression (and %) is blunted in CD4 + T-cells at 3 d post-MI, it is increased during HF with significantly higher expression in TNFR1 + T-cells as compared to TNFR1 - T-cells. Splenic CD4 + T-cells activated using anti-CD3/CD28 antibodies and treated with neutralizing anti-TNFR1 antibody showed increased T-cell proliferation, and Bcl-xL (pro-survival factor) and TNFα expression without affecting activation marker (CD69) suggesting a role of TNFR1 in mediating pro-apoptotic signaling without affecting activation. To confirm these results, we adoptively transferred (AT) HF-activated splenic WT and TNFR1 -/- T-cells (CD45.2) into CD45.1 naïve mice, and measured changes in cardiac function of recipient mice by echocardiography (as an indicator of pathological activity), and donor T-cells (as a measure of cell survival) in the blood, heart, spleen and mediastinal lymph nodes (mLNs) at 14 wks post-AT. Compared to WT, we observed amplified levels of donor TNFR1 -/- T-cells in the blood, spleen, and mLNs which were not different in the hearts. Consistently, recipient mice injected either with WT and TNFR1 -/- T-cells showed similar degrees of cardiac dysfunction. Interestingly, at 14 wks post-AT the expression of ki67, a proliferation marker, was magnified in HF-activated TNFR1 -/- T-cells (as compared to WT) infiltrated into the myocardium suggesting that TNF-TNFR1 mediates pro-survival and proliferative signaling in T-cells during HF, and not their pathogenicity. Our results also provide deeper insights into the paradoxical outcomes observed in the clinical trials antagonizing TNFα in human HF patients.

Background: CD4 + T cells temporally transition from protective to pathological during ischemic heart failure (HF; 8 weeks postmyocardial infarction). Cellular mechanisms mediating this shift are unknown. Methods: RNA-sequencing of cardiac CD4 + T cells and flow cytometric analysis of immune cells was conducted. Results: RNA-sequencing of CD4 + T cells from the failing hearts of male mice indicated activation of ER (estrogen receptor)-α signaling. Flow cytometric analysis showed that ERα in CD4 + T cells decreases significantly at 3-day postmyocardial infarction but increases during HF. To antagonize ERα, we tested a novel ERβ agonist (OSU-ERb-012) to inhibit T cells and blunt left ventricular remodeling. Proliferation assays showed that OSU-ERb-012 dose-dependently inhibited proliferation and proinflammatory cytokine expression in anti-CD3/CD28 stimulated splenic T cells isolated from both the sexes. For in vivo efficacy, 10- to 12-week-old male and ovariectomized female mice were randomized at 4 weeks postmyocardial infarction and treated with either vehicle or drug (60 mg/kg per day; oral). While vehicle-treated HF mice displayed progressive left ventricular dilatation with significantly increased end-systolic and end-diastolic volumes from 4 to 8 weeks postmyocardial infarction, treatment with OSU-ERb-012 significantly blunted these changes and stopped left ventricular remodeling in both the sexes. Reduction in tibia-normalized heart and left ventricular weights, cardiomyocyte hypertrophy and interstitial fibrosis further supported these results. Additionally, OSU-ERb-012 treatment selectively inhibited cardiac, splenic, and circulating CD4 + T cells without affecting other myeloid and lymphoid cells in the HF mice. Conclusions: Our studies indicate that ERβ agonists and OSU-ERb-012, in particular, could be used as selective immunomodulatory drugs to inhibit CD4 + T cells during chronic HF.

Immune activation post-myocardial infarction is an orchestrated sequence of cellular responses to effect tissue repair and healing. However, excessive and dysregulated inflammation can result in left ventricular remodeling and pathological alterations in the structural and mechanical attributes of the heart. Identification of key pathways and critical cellular mediators of inflammation is thus essential to design immunomodulatory therapies for myocardial infarction and ischemic heart failure. Despite this, the experimental approaches to isolate mononuclear cells from the heart are diverse, and detailed protocols to enable maximum yield of live cells in the shortest time possible are not readily available. Here, we describe optimized protocols for the isolation, fixation, and flow cytometric characterization of cardiac CD45+ leukocytes. These protocols circumvent time-consuming coronary perfusion and density-mediated cell-separation steps, resulting in high cellular yields from cardiac digests devoid of contaminating intravascular cells. Moreover, in contrast to methanol and acetone, we show that cell fixation using 1% paraformaldehyde is most optimal as it does not affect antibody binding or cellular morphology, thereby providing a considerable advantage to study activation/infiltration-associated changes in cellular granularity and size. These are highly versatile methods that can easily be streamlined for studies requiring simultaneous isolation of immune cells from different tissues or deployment in studies containing a large cohort of samples with time-sensitive constraints. NEW & NOTEWORTHY In this article, we describe optimized protocols for the isolation, fixation, and flow cytometric analysis of immune cells from the ischemic/nonischemic hearts. These protocols are optimized to process several samples/tissues, simultaneously enabling maximal yield of immune cells in the shortest time possible. We show that the low-speed centrifugation can be used as an effective alternative to lengthy coronary perfusion to remove intravascular cells, and sieving through 40-μm filter can replace density-mediated mononuclear cell separation which usually results in 50–70% cell loss in the sedimented pellets. We also show that cell fixation using 1% paraformaldehyde is better than the organic solvents such as methanol and acetone for flow cytometric analysis.

CD4 + T-cells turn pathological during chronic heart failure (HF). Phenotypic changes that mediate this transition are unknown. Thus, at 8 weeks post-infarction we conducted limited cell RNA-sequencing on 150 CD4 + T-cells isolated from rodent failing hearts and using ingenuity pathway analysis (z score>2) compared them either with CD4 + T-cells isolated from the mediastinal lymph nodes (mLNs) of the same mice or cardiac CD4 + T-cells from sham mice. T-cells isolated from the mLNs of HF mice showed enhanced TCR signaling (p-4 ) with 3-6 fold increase in several downstream mediators such as ITK, MAPK, LCK and Zap70. We also observed heightened leukocyte extravasation signaling (p-5 ) with significant increase in PKCθ, and Rho GTPase activating and Ras association domain proteins. These are consistent with enhanced antigen-presentation, and TCR activation during HF. Moreover, we observed significant upregulation of oxidative phosphorylation (p-15 ) in cardiac CD4 + T-cells as compared to mLN T-cells with 2-3 fold increase in the gene expression of ATP synthase subunits and cytochrome C indicative of their increased metabolic activity upon infiltration into the failing hearts. Compared to CD4 + T-cells from sham hearts, significant upregulation of inflammatory genes (p-5 ), chemotactic factors (p-4 ), and several pro-inflammatory cytokine-mediated pathways such as IL-6 (p-4 ), IL-1a (p-3 ) and TNFα (p-3 ) was observed in cardiac CD4 + T-cells from HF mice. Interestingly, we also observed a significant upregulation of homing (p-4 ) and connective tissue remodeling genes (p-3 ) with >8-fold increase in genes like LCN2, SLPI, IGF1, FGFR1, LTF, and TIMP2 required for transmigration and homing of CD4 + T-cells into the ischemic hearts. Our studies show enhanced antigenic CD4 + T-cell activation in the mLNs supporting enhanced pro-inflammatory signaling, metabolic activation, and extravasation into the ischemic hearts.

Fidelity of wound healing after myocardial infarction (MI) is an important determinant of subsequent adverse cardiac remodeling and failure. Infiltrating Ly6C hi blood monocytes are a key component of this healing response; however, the importance of other monocyte/macrophage populations is unclear. Here, we demonstrate in mice an essential role for splenic CD169 + Tim4 + marginal zone metallophilic macrophages (MMMs) in post-MI wound healing. CD169 + Tim4 + MMMs circulate as Ly6C low monocytes, and dynamically populate the naïve heart. After acute MI, mobilization robustly increases to the heart, where these macrophages are obligatory for apoptotic neutrophil clearance and the induction of pro-resolving and reparative tissue macrophages. Splenic MMMs are both necessary and sufficient forpost-MI wound healing, and limit late pathological remodeling.Pharmacological expansion of the splenic marginal zone during acute MI alleviates inflammation and cardiac remodeling. Finally, humans with acute MI also exhibit expansion of blood CD169 + monocytes. Hence, splenic CD169 + Tim4 + MMMs are required for pro-resolving and reparative responses after MI, and can be manipulated for therapeutic benefit to limit long-term heart failure.

Ischemic heart failure (HF) results in the activation of self-reactive CD4+ T-lymphocytes against unknown cardiac antigens. The mechanisms that mediate the survival of self-reactive T-cells during ischemic HF are not known. TNF mediated NF-kB signaling regulates cellular apoptotic pathways and T cells express high levels of TNF receptor-1 (TNFR1) during chronic heart failure. Thus, we hypothesized that TNFR1 regulates pro-apoptotic signaling through NF-kB and is critical for the clearance of self-reactive T-cells during HF. To test this, male WT or TNFR1 -/- mice underwent coronary artery ligation to induce HF (10 wks post-ligation), followed by isolation and adoptive-transfer (AT) of splenic CD4+ T-cells (9X10 6 cells/mouse) into the tail-veins of naïve CD45.1 mice. Flow cytometric analysis showed ~7-fold higher levels of HF-activated TNFR1 -/- T cells in recipient mice when compared with WT T-cells at 2d post-AT, and were not different between the 2-groups at 1 wk. Interestingly, TNFR1 -/- T cells were significantly higher again at 2 wks (as compared to WT T-cells) and the levels were sustained for up to 14 wks post-AT. In contrast, donor WT T-cells were slowly cleared from the recipient mice. In addition, as compared to WT T-cells, ~11-fold higher levels of adoptively transferred TNFR1 -/- T cells were observed in the spleens of recipient mice at 14 wks suggesting that TNFR1 -/- T cells had higher propensity to seed into the lymphoid tissues. This data also suggests that TNFR1 -/- T cells either have enhanced pro-survival pathways or were less prone to activation-induced cell death. In-vitro T-cell proliferation assays using anti-CD3/CD28 antibodies showed enhanced proliferation and expression of anti-apoptotic Bcl-xL upon treatment with a TNFR1 neutralization antibody corroborating in-vivo results. Intriguingly, as opposed to WT T-cells, adoptive transfer of HF-activated TNFR1 -/- T-cells induced significant cardiac dysfunction and the ejection fraction (EF) of naïve recipient mice was significantly decreased from 63±6% to 45±6% at 14 weeks post-AT. Our data show that TNFR1 signaling regulates pro-vs anti-apoptotic pathways in a context-dependent manner and potentiate pathological phenotype of HF activated T-cells by enhancing their life-span.

CD4+ T-cells mediate wound-healing post-myocardial infarction (MI) but exacerbate left-ventricular (LV) remodeling during chronic heart failure (HF). Mechanisms that lead to this transition are unknown. Therefore, we hypothesized that as opposed to MI, T-cells activate specific pathological signals to promote LV-remodeling during chronic HF. To identify such signals, we conducted limited cell RNA-sequencing of CD4+ T-cells sorted from the failing hearts (8 weeks post-MI) of male mice and, surprisingly, found activation of estrogen receptor (ER)-α signaling. As ERα effects are antagonized by ERβ, we tested a novel ERβ agonist (OSU-ERB-012) to modulate T-cell activity and LV remodeling. In-vitro assays showed that OSU-ERB-012 dose dependently inhibit activation and proliferation of T-cells isolated from male mice (IC 50 3.4 μM). In-vivo assays (60 mg/kg/day; oral) showed no overt toxicity and a significant reduction in circulating T cells without affecting neutrophils, monocytes, or B-cells indicating specificity against T-cells. Furthermore, this effect was specific to TCR-mediated T-cell activation and the drug did not affect T-cells stimulated with PMA/Ionomycin suggesting preferential inhibition of antigenically-activated T-cells. To test therapeutic efficacy, male 10-12 week old mice underwent coronary ligation or sham operation and at 4 weeks post-MI randomized according to their cardiac function to receive either the vehicle or OSU-ERB-012 (60 mg/kg/day, oral) for the next 4 weeks. Consistently, at 8 weeks we observed a significant reduction in T-cells in the circulation and the spleens of drug-treated mice when compared with the vehicle treatment. As expected, vehicle treated HF mice showed progressive LV dilatation with significantly increased end-diastolic and end-systolic volumes (EDV and ESV, respectively) from 4-8 weeks post-MI. Importantly, treatment with OSU-ERB-012 significantly inhibited these changes and blunted LV remodeling from 4-8 weeks post-MI. Significant reduction in tibia-normalized heart weights supported these results. Our studies indicate that OSU-ERB-012 is a novel orally-active drug molecule that selectively inhibits T-cell activation and blunts pathological LV-remodeling during chronic HF.

Visual Abstract, Highlights • Hypothesis: CCR2+ monocyte-derived cardiac macrophages are required for adverse LV remodeling, cardiac T-cell expansion, and the transition to HF following pressure overload. • The imposition of pressure overload via TAC resulted in the early up-regulation of CCL2, CCL7, and CCL12 chemokines in the LV, increased Ly6ChiCCR2+ monocytes in the blood, and augmented CCR2+ infiltrating macrophages in the heart. • Specific and circumscribed inhibition of CCR2+ monocytes and macrophages early during pressure overload reduced pathological hypertrophy, fibrosis, and systolic dysfunction during the late phase of pressure overload. • The early expansion of CCR2+ macrophages after pressure overload was required for long-term cardiac T-cell expansion. • CCR2+ monocytes/macrophages may represent key targets for immunomodulation to delay or prevent HF in pressure-overload states., Summary Although chronic inflammation is a central feature of heart failure (HF), the immune cell profiles differ with different underlying causes. This suggests that for immunomodulatory therapy in HF to be successful, it needs to be tailored to the specific etiology. Here, the authors demonstrate that monocyte-derived C-C chemokine receptor 2 (CCR2)+ macrophages infiltrate the heart early during pressure overload in mice, and that blocking this response either pharmacologically or with antibody-mediated CCR2+ monocyte depletion alleviates late pathological left ventricular remodeling and dysfunction, T-cell expansion, and cardiac fibrosis. Hence, suppression of CCR2+ monocytes/macrophages may be an important immunomodulatory therapeutic target to ameliorate pressure-overload HF.

Inflammation has recently gained tremendous attention as a key contributor in several chronic diseases. While physiological inflammation is essential to counter a wide variety of damaging stimuli and to improve wound healing, dysregulated inflammation such as in the myocardium and vasculature can promote cardiovascular diseases. Given the high severity, prevalence, and economic burden of these diseases, understanding the factors involved in the regulation of physiological inflammation is essential. Like other complex biological phenomena, RNA-based processes are emerging as major regulators of inflammatory responses. Among such processes are cis-regulatory elements in the mRNA of inflammatory genes, noncoding RNAs directing the production or localization of inflammatory cytokines/chemokines, or pathogenic RNA driving inflammatory responses. In this review, we describe several specific RNA-based molecular mechanisms by which physiological inflammation pertaining to cardiovascular diseases is regulated. These include the role of AU-rich element-containing mRNAs, long non-coding RNAs, microRNAs, and viral RNAs.

Background: Heart failure (HF) is a state of inappropriately sustained inflammation, suggesting the loss of normal immunosuppressive mechanisms. Regulatory T-lymphocytes (Tregs) are considered key suppressors of immune responses; however, their role in HF is unknown. We hypothesized that Tregs are dysfunctional in ischemic cardiomyopathy and HF, and they promote immune activation and left ventricular (LV) remodeling. Methods: Adult male wild-type C57BL/6 mice, Foxp3-diphtheria toxin receptor transgenic mice, and tumor necrosis factor (TNF) α receptor-1 (TNFR1) − /− mice underwent nonreperfused myocardial infarction to induce HF or sham operation. LV remodeling was assessed by echocardiography as well as histological and molecular phenotyping. Alterations in Treg profile and function were examined by flow cytometry, immunostaining, and in vitro cell assays. Results: Compared with wild-type sham mice, CD4 + Foxp3 + Tregs in wild-type HF mice robustly expanded in the heart, circulation, spleen, and lymph nodes in a phasic manner after myocardial infarction, beyond the early phase of wound healing, and exhibited proinflammatory T helper 1-type features with interferon-γ, TNFα, and TNFR1 expression, loss of immunomodulatory capacity, heightened proliferation, and potentiated antiangiogenic and profibrotic properties. Selective Treg ablation in Foxp3-diphtheria toxin receptor mice with ischemic cardiomyopathy reversed LV remodeling and dysfunction, alleviating hypertrophy and fibrosis, while suppressing circulating CD4 + T cells and systemic inflammation and enhancing tissue neovascularization. Tregs reconstituted after ablation exhibited restoration of immunosuppressive capacity and normalized TNFR1 expression. Treg dysfunction was also tightly coupled to Treg-endothelial cell contact- and TNFR1-dependent inhibition of angiogenesis and the mobilization and tissue infiltration of CD34 + Flk1 + circulating angiogenic cells in a C-C chemokine ligand 5/C-C chemokine receptor 5-dependent manner. Anti-CD25-mediated Treg depletion in wild-type mice imparted similar benefits on LV remodeling, circulating angiogenic cells, and tissue neovascularization. Conclusions: Proinflammatory and antiangiogenic Tregs play an essential pathogenetic role in chronic ischemic HF to promote immune activation and pathological LV remodeling. The restoration of normal Treg function may be a viable approach to therapeutic immunomodulation in this disease.

Background— Inappropriately sustained inflammation is a hallmark of chronic ischemic heart failure (HF); however, the pathophysiological role of T lymphocytes is unclear. Methods and Results— Permanent coronary ligation was performed in adult C57BL/6 mice. When compared with sham-operated mice, mice with HF (8 weeks after ligation) exhibited the following features: (1) significant ( P + CD8 + cytotoxic and CD3 + CD4 + helper (Th) T lymphocytes, together with increased Th1, Th2, Th17, and regulatory T-cell (Treg) CD4 + subsets; (2) significant expansion of CD8 + and CD4 + T cells in failing myocardium, with increased Th1, Th2, Th17, and Treg CD4 + subsets, marked reduction of the Th1/Th2 ratio, augmentation of the Th17/Treg ratio, and upregulation of Th2 cytokines; and (3) significantly increased Th1, Th2, Th17 cells, and Tregs, in the spleen and mediastinal lymph nodes, with expansion of splenic antigen-experienced effector and memory CD4 + T cells. Antibody-mediated CD4 + T-cell depletion in HF mice (starting 4 weeks after ligation) reduced cardiac infiltration of CD4 + T cells and prevented progressive left ventricular dilatation and hypertrophy, whereas adoptive transfer of splenic CD4 + T cells (and, to a lesser extent, cardiac CD3 + T cells) from donor mice with HF induced long-term left ventricular dysfunction, fibrosis, and hypertrophy in naive recipient mice. Conclusions— CD4 + T lymphocytes are globally expanded and activated in chronic ischemic HF, with Th2 (versus Th1) and Th17 (versus Treg) predominance in failing hearts, and with expansion of memory T cells in the spleen. Cardiac and splenic T cells in HF are primed to induce cardiac injury and remodeling, and retain this memory on adoptive transfer.

In the failing heart, iNOS is expressed by both macrophages and cardiomyocytes. We hypothesized that inflammatory cell-localized iNOS exacerbates left ventricular (LV) remodeling. Wild-type (WT) C57BL/6 mice underwent total body irradiation and reconstitution with bone marrow from iNOS−/− mice (iNOS−/−c) or WT mice (WTc). Chi-meric mice underwent coronary ligation to induce large infarction and ischemic heart failure (HF), or sham surgery. After 28 days, as compared with WTc sham mice, WTc HF mice exhibited significant (p < 0.05) mortality, LV dysfunction, hypertrophy, fibrosis, oxidative/nitrative stress, inflammatory activation, and iNOS upregulation. These mice also exhibited a ∼twofold increase in circulating Ly6Chi pro-inflammatory monocytes, and ∼sevenfold higher cardiac M1 macrophages, which were primarily CCR2– cells. In contrast, as compared with WTc HF mice, iNOS−/− c HF mice exhibited significantly improved survival, LV function, hypertrophy, fibrosis, oxidative/nitrative stress, and inflammatory activation, without differences in overall cardiac iNOS expression. Moreover, iNOS−/−c HF mice exhibited lower circulating Ly6Chi monocytes, and augmented cardiac M2 macrophages, but with greater infiltrating monocyte-derived CCR2+ macrophages vs. WTc HF mice. Lastly, upon cell-to-cell contact with naïve cardiomyocytes, peritoneal macrophages from WT HF mice depressed contraction, and augmented cardiomy-ocyte oxygen free radicals and peroxynitrite. These effects were not observed upon contact with macrophages from iNOS−/− HF mice. We conclude that leukocyte iNOS is obligatory for local and systemic inflammatory activation and cardiac remodeling in ischemic HF. Activated macrophages in HF may directly induce cardiomyocyte contractile dysfunction and oxidant stress upon cell-to-cell contact; this juxtacrine response requires macrophage-localized iNOS.

Heart failure (HF) is characterized by exaggerated systemic and myocardial pro-inflammatory responses, which impede myocardial healing and promote pathological remodeling and dysfunction. Currently, there are no strategies available to restrain and/or modulate immune cell responses triggered by myocardial injury. 4F, an apolipoprotein A-I mimetic, exhibits anti-inflammatory properties and influences macrophage polarity. We hypothesized that 4F treatment following reperfused myocardial infarction (MI) in mice would attenuate post-MI cardiac remodeling by inducing an anti-inflammatory and tissue healing monocyte polarity. C57BL/6 mice (6-8/group) were intravenously injected with either PBS or L-4F (100 μg/day) for 5 d starting at day 3 after reperfused MI (60 min ischemia). Flow cytometry revealed that compared to PBS injected MI mice, 4F significantly (p < 0.05) decreased levels of pro-inflammatory monocytes (CD45+Cd11b+Ly6Chigh) in blood (20.4 ± 4.6 vs 9.4 ± 1.7%), spleen (25.5 ± 7.4 vs 8.0 ± 6.0%), and heart (0.9 ± 0.4 vs 0.2 ± 0.1%); and decreased myocardial macrophage (CD45+CD11b+F4/80+) infiltration (1.6 ± 0.8 vs 0.28 ± 0.2%). The levels of anti-inflammatory monocytes, however, were unchanged. Echocardiography revealed that these changes in immune cell profiles in 4F-injected mice were also accompanied by improvements in cardiac function. Compared to PBS injected MI mice, 4F treatment attenuated post-MI LV chamber dilatation (84.1 ± 16 vs 65.0 ± 8.5 uL and 59.15 ± 16.6 vs 38.4 ± 11.8 uL; end-diastolic and systolic volumes, respectively), and improved LV ejection fraction (28.4 ± 10.8 vs 41.7 ± 12.8%). In addition, in vitro studies using mouse peritoneal macrophages indicated that 4F promotes an anti-inflammatory macrophage phenotype potentially by inhibiting the glycolytic metabolic profile typical of pro-inflammatory macrophages. These effects on macrophage polarity by 4F were associated with parallel alterations in the expression of hypoxia inducible factor isoforms 1 and 2α, known markers of macrophage polarity. We conclude that 4F could be used therapeutically after MI to augment reparative immune responses and thereby limit the long-term maladaptive effects of exaggerated inflammation on adverse post-MI cardiac remodeling.

The functional role of regulatory T-cells (Tregs) in chronic heart failure (HF) is unknown. Using flow cytometry, we assessed Tregs during the progression of ischemic HF in male C57Bl/6 mice after coronary ligation and myocardial infarction (MI). At 7 d post-MI, circulating Tregs (% CD4+ T-cells) were comparable to sham-operated mice. Thereafter, blood Tregs significantly (p < 0.05 vs sham) increased ~1.5 fold at 2, 4, 6, and 8 w, and exhibited increased levels of pro-inflammatory intracellular IFNγ (40.5 ± 5.9 vs 28.4 ± 5.1%) and TNFα (34.6 ± 8.8 vs 21.7 ± 6.9%). Moreover, at 8 w post-MI, Tregs were increased in the spleen (24.1 ± 3.9 vs 18.3 ± 1.4%) and failing heart (0.95 ± 0.42 vs 0.33 ± 0.23%) as compared with sham mice. In contrast to splenic Tregs from sham mice, Tregs isolated from HF mice (8 w post-MI) failed to suppress effector T-cell proliferation, and were robustly antiangiogenic as indexed by mouse coronary endothelial cell tube formation assays. Transgenic mice expressing diphtheria toxin (DT) receptor (DTR) under control of the Foxp3 promoter underwent coronary ligation and DT-induced selective Treg ablation starting at 4 w post-MI (PBS control). Echocardiography before and after (8 w post-MI) PBS or DT injection revealed that in contrast to progressive LV dysfunction and remodeling after PBS, DT-mediated Treg ablation and subsequent reconstitution reversed pathological LV remodeling (decreased EDV and ESV, and increased EF). As compared with PBS injection, DT-induced Treg ablation also significantly (p < 0.05) reduced LV hypertrophy; enhanced circulating CD34+Flk1+ cells, cardiac CCR5 gene expression, and myocardial capillary density; reduced border zone fibrosis; and decreased pro-inflammatory (IL-17, IL-2 and IL-6) and pro-fibrotic (IL-4 and IL-10) serum cytokines. Similar results were obtained upon Treg depletion with anti-CD25 antibody in HF. We conclude that Tregs in chronic HF acquire pathological pro-inflammatory, profibrotic, and antiangiogenic properties, and lose their immunosuppressive capacity. Dysfunctional Tregs promote inflammation and adverse LV remodeling in HF; depletion of dysfunctional Tregs and subsequent restoration to a normal Treg phenotype may represent a novel approach to therapeutic immunomodulation in HF.

As opposed to chronic ischemic heart failure (HF), spleen-derived macrophages (Mf) are not prominent disease mediators during the chronic stages of pressure-overload (PO) HF. Increases in Mf during the early stages of PO, however, may serve as important drivers of both remodeling and activation of T-cell responses during later stages. Additionally, recent evidence indicates distinct groups of resident and circulating monocyte-derived infiltrating Mf that have different reparative vis-à-vis pro-inflammatory capacities. We hypothesized that cardiac Mf are expanded during the early stages of PO, primarily due to an increase in infiltrating Mf, and contribute to long-term T-cell activation in the PO heart. Cardiac Mf were characterized by flow cytometry as MerTK + CD64 + F480 + MHCII + cells, with infiltrating Mf populations identified as expressing chemokine receptor 2 (CCR2). One week after transverse aortic constriction (TAC), C57Bl/6 mice exhibited significant (p < 0.05) hypertrophy (heart weight/body weight 7.7 ± 0.5 vs 5.7 ± 0.2 mg/g; LV wall thickness 0.84 ± 0.03 vs 0.74 ± 0.02), without change in LVEF as compared with sham. There were robust increases in cardiac macrophages (3.15 ± 0.64 vs 1.09 ± 0.24%), specifically in the infiltrating CCR2 + MHCII hi population (1.6 ± 0.5 vs 0.5 ± 0.1%). TAC mice also exhibited higher circulating Gr1 – CD11b + Ly6C hi monocytes (2.6 ± 0.5 vs 1.3 ± 0.2%) that are recruited to tissue via CCR2. To determine activation of the adaptive immune response, we analyzed the mediastinal heart draining lymph nodes (MLN) for T-cells and antigen presenting cells. As compared with sham mice, the MLN of TAC mice were larger with greater cellularity (10.8 x 10 5 vs 4.2 x 10 5 ), especially related to MerTK + CD169 + Mf and CD3 + CD8 + T cells. We conclude that there is both an organ-specific and systemic immune response during compensatory PO hypertrophy, despite the absence of ischemic tissue injury. As infiltrating Mf are known to be less reparative, potently pro-inflammatory, and robust activators of adaptive immune cells, specifically targeting Ly6C hi monocyte recruitment and CCR2 + infiltrating Mf during the early compensated stages of pressure-overload may prevent subsequent inflammatory activation, adverse remodeling, and the transition to HF.