Immunomodulatory therapy for ischemic heart disease

Abstract

Ischemic heart disease is a leading cause of death worldwide, manifested clinically as myocardial infarction (MI) and ischemic cardiomyopathy. Presently, there exists a notable scarcity of efficient interventions to restore cardiac function following MI. Cumulative evidence suggests that impaired tissue immunity within the ischemic microenvironment aggravates cardiac dysfunction, contributing to progressive heart failure. Recent research breakthroughs propose immunotherapy as a potential approach by leveraging immune and stroma cells to recalibrate the immune microenvironment, holding significant promise for the treatment of ischemic heart disease. In this Primer, we highlight three emerging strategies for immunomodulatory therapy in managing ischemic cardiomyopathy: targeting vascular endothelial cells to rewire tissue immunity, reprogramming myeloid cells to bolster their reparative function, and utilizing adoptive T cell therapy to ameliorate fibrosis. We anticipate that immunomodulatory therapy will offer exciting opportunities for ischemic heart disease treatment.

Ischemic heart disease (IHD) is one of the most important risk factors of heart failure. It is often attributed to coronary artery disease, a condition characterized by narrowing or obstruction of coronary arteries responsible for delivering nutrient- and oxygen-rich blood to the myocardium. A significant reduction in blood supply and sustained ischemia can cause irreversible damage to the cardiac tissue, leading to potential myocardial infarction (MI), commonly referred to as heart attack. The immune system plays a pivotal role in the aftermath of MI. Restoration of cardiac function is mediated by a complex and highly coordinated immune cascade. Following MI, a substantial amount of T lymphocytes and myeloid cells including neutrophils, monocytes, macrophages, and dendritic cells, infiltrate into the damaged myocardial region. These cells contribute to the repair process, which involves reabsorption of cell debris in the necrotic area, promotion of myofibroblast proliferation, collagen deposition, scar tissue formation, and neovascularization essential for restoring tissue perfusion.

Critical to this reparative process is the regulation of inflammation. The rate and extent of cardiac recovery is largely affected by the extent of inflammation and its subsequent resolution. Early inflammation facilitates the recruitment of key myeloid effectors, enabling their extravasation into the infarct site for debris clearance. Whereas this is indispensable, the progression to effective cardiac healing hinges on timely resolution of this acute response. In general, the acute inflammatory phase lasts 3-7 days after MI with variable rates of resolution and chronicity. The transition from acute to chronic phases is characterized by changes in immune cell populations and polarity. Notably, driven by the activation of specific endogenous inhibitory pathways, a relative increase in anti-inflammatory macrophages and monocytes fine-tunes the tissue microenvironment to favor immunomodulation and cardiac repair. Conversely, persistent pro-inflammatory cell populations and cytokines are thought to promote ongoing tissue damage and fibrosis, which can exacerbate cardiac dysfunction, leading to adverse remodeling and progressive heart failure.

Current standards in the treatment of IHD and MI predominantly rely on revascularization procedures (e.g., percutaneous coronary intervention and coronary artery bypass grafting) along with pharmacological interventions (e.g., anticoagulants, beta-blockers, angiotensin-converting enzyme inhibitors). Despite these interventions, the progression toward adverse remodeling and eventual heart failure remains a significant clinical challenge. The transition from acute to chronic phases post-MI involves complex interactions between vascular, myeloid, myocardial, and fibroblast lineages, which offers novel therapeutic opportunities. To achieve optimal repair, immunomodulatory therapies that target these cell types may improve ischemic cardiomyopathy and ameliorate adverse remodeling by timely attenuation of the acute inflammatory response and enhancement of reparative mechanisms post-MI.

Vascular-based immunomodulatory therapies

Angiogenesis, the formation of new blood vessels, is fundamental to post-MI cardiac repair and function recovery. Therapeutic neovascularization that primarily targets pro-angiogenic pathways to promote tissue vascularization has been exploited in recent decades for ischemic cardiovascular disease; however, the outcomes have been modest and short-lived^1,2. Recent work indicates that ischemia-inducible transcriptomic plasticity in endothelial cells (ECs) drives vascular abnormalities, charactered by disorganized vessel morphology and suboptimal blood perfusion³. This finding suggests that therapies targeting vessel normalization to enhance vascular function may join pro-angiogenesis as important strategies for IHD. Consistent with this notion, genetic and pharmacological inhibition of EC plasticity, particularly PDGF-mediated mesenchymal-like transcriptional activation, enhances cardiac repair and functional recovery after MI in mouse models³. Interestingly, the therapeutic effects appear to extend beyond restoration of tissue perfusion and could be linked to the emerging role of ECs as regulators of tissue immune cell homeostasis.

Mounting evidence suggests that ECs act as a regulatory node of innate and adaptive immunity in cancer⁴, which shares a hypoxic and inflammatory microenvironment similar to that observed in IHD. The role of ECs as modulators of tissue immunity in solid tumors is multifaceted: blood vessels deliver anti-cancer lymphocytes and immunosuppressive myeloid cells to the tumor; ECs modulate T cell adhesion and infiltration via the endothelial adhesome, i.e., the complement of cell-cell and cell-matrix adhesion receptors and its regulatory network; and ECs can modulate macrophage polarization and suppress T cell activity through EC-derived cytokines and metabolites. Likewise, ECs in the infarcted myocardium may perform functions beyond their conventional role in facilitating gas and nutrient diffusion to satisfy heightened metabolic demands of damaged myocytes. ECs may participate in orchestrating the immune response after MI by recruiting immunocytes from the circulation, affecting the differentiation and polarization of infiltrating effector cells, and modulating the immune microenvironment.

The evolving understanding of tumor microvasculature in mediating immunomodulation may serve as a paradigm for understanding EC functions in the ischemic myocardium, although many differences exist. In cancer, tumor ECs function to prevent infiltration of cytotoxic T cells. This observation has led to the exploration of novel strategies that directly target tumor ECs to enhance anti-tumor immune cell infiltration, thereby sensitizing tumors to T cell-based immunotherapies. For example, genetic and pharmacological ablation of PAK4, a key regulator of mesenchymal transition of ECs, reprograms the endothelial transcriptome and normalizes tumor vasculature. This stimulates T cell adhesion and infiltration, overcoming tumor resistance to chimeric antigen receptor (CAR) T cell therapy⁵. Likewise, it may be possible to target ECs after MI to modulate the inflammatory milieu and the transition from acute to chronic phases of recovery. For example, a recent study suggests that the transcription factor Tbx1 can function in post-MI lymphatic ECs to bolster immunosuppression and promote cardiac repair⁶. The infiltrating lymphatic ECs serve as intramyocardial immune hubs, engaging tolerogenic dendritic cells and regulatory T cells to mitigate excessive inflammation and promoting the expansion of reparative macrophages to enhance healing⁶. Furthermore, ECs play a critical role for regulation of myeloid cell-mediated tissue immunity, modulating macrophage function via released angiocrines such as IL-6 and osteopontin^7,8.

EC-targeted immunomodulatory therapeutic strategies may include: 1) genetic reprogramming of ECs, targeting EC plasticity and tailoring the expression of EC adhesion proteins (such as ICAM-1 and VCAM) and cytokines (such as IL-6, TGF-β and IL-10) using targeted lipid nanoparticles (tLNPs) to deliver modulatory mRNA or siRNA; and 2) metabolic modulation of ECs, focusing on glycolysis and serine biosynthesis that drive EC outgrowth and sprouting and control EC release of immune-modulatory metabolites, such as lactate and 2-hydroxyglutarate⁹ (Figure 1). Collectively, these approaches may foster favorable reparative immune responses to enhance recovery from ischemic cardiac events.

Figure. 1. Endothelial cell-targeting immunomodulatory therapy.

Neovascularization proceeds by endothelial cell (EC) sprouting and overgrowth, contributing to cardiac repair. Aberrant vascularization compromises blood perfusion and impedes infiltration and activation of reparative immunocytes: EC adhesion is disrupted, inducing dysfunctional interaction between ECs and immunocytes, and dysfunctional EC-derived cytokine and metabolites form an immune-hostile milieu that inhibits repair-favorable immunity. Immunomodulatory therapy by genetic and metabolic approaches to target ECs may promote vessel functions and rewire the tissue microenvironment to foster reparative immunity, facilitating cardiac repair after MI. tLNP, targeted lipid nanoparticles; 2-HG, 2-hydroxyglutarate.

Myeloid cell immunotherapy

Myeloid cells, especially neutrophils, monocytes, and macrophages, are instrumental in the immune response after MI. Myocardial injury mobilizes tissue-resident and bone marrow-derived immune cells to infiltrate the infarct site, where they actively participate in the inflammatory cascade by scavenging debris and amplifying the immune response through the release of inflammatory cytokines. As the tissue microenvironment transitions into the reparative phase, the neutrophil population begins to diminish, while monocytes and macrophages persist and polarize towards a pro-resolving phenotype to dampen inflammation and reinforce tissue repair. Macrophages exhibit dynamic adaptation to microenvironmental stimuli by reshaping their transcriptional landscape to undertake reparative tasks, stimulating myofibroblasts or directly contributing collagens and other components of the extracellular matrix to promote fibrosis, and enhancing EC proliferation to expedite angiogenesis^10,11. The inherent plasticity and phenotypic diversity make macrophages an attractive candidate for treatment strategies aiming to normalize dysregulated immune responses.

Recent advances in our understanding of macrophage heterogeneity within the human heart have established a foundational basis for therapeutic interventions targeting myeloid cells in cardiovascular disease. The human myocardium harbors a diverse population of macrophages with distinct origins and divergent reparative and inflammatory functions, categorized based on the presence or absence of C-C chemokine receptor 2 (CCR2)¹². CCR2⁻ macrophages represent a tissue-resident population that is replenished through local proliferation, while CCR2⁺ macrophages arise from adult hematopoietic progenitors, i.e., bone marrow-derived monocytes recruited from the circulation. Consistent with an early study showing that blockade of CCR2-mediated macrophage recruitment attenuates adverse left ventricular remodeling after MI¹³, CCR2⁺ macrophages represent an inflammatory subset, and their presence in the post-infarct myocardium is clinically associated with adverse cardiac remodeling and persistent left ventricular systolic dysfunction in heart failure patients¹². Conversely, CCR2⁻ macrophages promote immune-favorable tissue remodeling to preserve cardiac function during the chronic phase of recovery from MI¹⁴. The regulation of macrophage-mediated inflammation is a critical determinant of optimal cardiac repair post MI. Macrophages could undergo classic pro-inflammatory to stimulating phagocytosis and inflammation, or alternative anti-inflammatory activation to induce immune suppression and tissue repair¹⁵. Pro-inflammatory macrophages secrete interleukin-1β (IL-1β), a cytokine that induces vascular inflammation in atherosclerosis and recurrent cardiovascular events in patients with MI^16,17. Consistent with their reparative role, CCR2⁻ macrophages express higher levels of alternative activation markers such as CD206 and CD163, compared to their CCR2⁺ counterparts¹².

Limiting the inflammatory functions of certain macrophage subtypes or harnessing the immunomodulatory capacities of others may represent a novel strategy for future therapeutic developments in IHD. The potential approaches may include: 1) genetic or epigenetic modulation of macrophage function by targeting specific subtypes, such as pro-inflammatory activation through Toll-like receptors (TLRs) or B-cell adapter for PI3K (BCAP)¹⁸; 2) modulation of metabolic pathways that support pro-inflammatory or anti-inflammatory activities, such as from glycolysis and fatty acid synthesis to oxidative phosphorylation and fatty acid oxidation, respectively¹⁹ and by targeting serine metabolism-mediated IL-1β production²⁰; 3) cell immunotherapy with enforced regenerative polarization of macrophage subtypes via gene editing; and 4) stem cell therapy with selected subpopulations of hematopoietic stem cells and myeloid progenitors that can favorably differentiate into immunologically reparative macrophages (Figure 2).

Figure 2. Immunomodulatory therapy by targeting macrophages.

Pro-inflammatory and pro-resolving macrophages have different roles for cardiac repair after MI, inducing prolong inflammation and inflammation resolution, respectively. Immunomodulatory therapy by genetic or epigenetic reprogramming, metabolic rewiring, or gene editing to modulate polarization and using regenerative myeloid progenitor cells may promote macrophage-mediated cardiac repair after MI. OXPHOS, oxidative phosphorylation; FA, fatty acid.

Activated fibroblasts as a target for immunotherapy

In the heart, stromal cells, particularly fibroblasts, are necessary for structural integrity and the acute response to injury, yet they also contribute to adverse outcomes in chronic heart disease due to persistent activation and extracellular matrix deposition²¹. Damage to the myocardium causes cardiac fibroblasts to activate, proliferate, and secrete increased levels of extracellular matrix proteins and pro-inflammatory cytokines. In the setting of MI, this activation process helps to protect the heart from rupture and maintains the integrity of the injured myocardium²². Over time, however, the production of pro-inflammatory cytokines triggers an immune response that can lead to ongoing inflammation and progressive cardiac dysfunction. As a result, investigators have begun targeting activated fibroblasts with the goal of dampening the pro-inflammatory environment and reducing extracellular matrix accumulation in the injured myocardium (Figure 3).

Figure 3. Immunomodulatory approaches to target activated fibroblasts.

Activated fibroblasts, i.e., myofibroblasts, exacerbate post-MI cardiac fibrosis through pro-inflammatory response. Immunomodulatory therapy using bi-specific antibodies, CAR-T cells, or tLNP to eliminate FAP⁺ fibroblasts or modulating their cytokine expression profile may recondition the immune microenvironment to improve cardiac repair after MI.

Activated cardiac fibroblasts express cell surface proteins that are normally not found on quiescent cardiac fibroblasts. For example, fibroblast-activation protein (FAP) is not detected in normal human hearts²³ but is robustly expressed by cardiac fibroblasts after ischemic injury. FAP is also expressed by activated fibroblasts in other organs and many tumors. CAR-T cells targeting FAP have been shown to improve cardiac function in a murine model of heart failure and cardiac fibrosis²⁴. This therapeutic strategy was subsequently improved by treating mice with tLNPs to generate FAP CAR-T cells in vivo. LNPs were decorated with an anti-CD5 antibody which resulted in specific uptake by T cells after intravenous injection, with subsequent expression of the CAR encoded by the encapsulated mRNA. Thus, ex vivo manufacturing of CAR-T cells was obviated, greatly reducing the cost and technical difficulty of CAR-T cell production²⁵.

In vivo T cell engineering is not restricted to LNP-based approaches. Studies utilizing viral vectors and polymer-based nanoparticles (PNPs) have also shown promise for in vivo delivery of therapeutic nucleic acids to immune cells²⁶. Lentivirus and AAV have been used to successfully engineer T cells in vivo, and both lead to long-term expression of target genes when compared to the transient expression from mRNA delivery^27,28. Long-term expression of the CAR is especially beneficial in cancer where complete elimination of tumor cells and surveillance for recurrence is desired, but this may not be as critical in the context of reducing the burden of fibrosis in ischemic cardiac injury. In fact, long-term persistence of anti-fibrotic CAR-T cells would likely elevate the risk of cardiac rupture in the event of a subsequent MI. Viral integration into the genome carries the risk of mutagenesis which could limit the patient population who qualify for viral-mediated in vivo CAR-T therapy. LNP and PNP approaches are not without risks either. Both approaches are well suited for mRNA delivery and transient CAR expression but could necessitate multiple doses for sufficient therapeutic efficacy which could elicit immune reactions or cumulative dose-dependent toxicities. Moreover, most LNP compositions have significant adjuvant qualities²⁹ which can lead to adverse drug reactions, potentially limiting their use in some patients. However, the potential ability to titrate the number of CAR-T cells present in patients as well as the amount of CAR-induced cell lysis could reduce the onset of cytokine release syndrome, a common side effect of CAR-T therapy³⁰.

The use of a CAR to guide therapeutic cells to the source of inflammation is not limited to cytotoxic T cells. Regulatory T cells (Tregs) represent another valuable cell type that could be amenable to CAR-based targeting. Tregs can exhibit anti-inflammatory activity in the absence of cytotoxicity, allowing for the repression of pro-inflammatory, activated cardiac fibroblasts without cell lysis³¹. Engineering Tregs with FAP CAR could target them to accumulate in areas of active fibrosis where they could function to reduce inflammation. This approach might prove to be beneficial following acute cardiac injury such as MI with less potential for toxicity than traditional CAR-T approaches since lysis of activated fibroblasts is likely to increase the risk of cardiac rupture in the setting of infarcted myocardium³². Targeted Treg therapies have also shown promise in other inflammatory diseases, particularly type 1 diabetes. In a recent study, Tregs engineered with a CAR against insulin-producing beta cells were able to prevent the onset of diabetes and prevent its progression in a murine model of the disease³³. Notably, CAR Tregs performed better than untargeted Tregs. It may also be possible to engineer CAR Tregs in vivo with tLNPs and mRNA, eliminating the need to expand the small number of donor-derived Tregs ex vivo before treatment. Another potential solution to the problem of the low number of donor-derived Tregs is to deliver a FoxP3 expression cassette along with the CAR. In this case, expression of the CAR would drive expression of FoxP3, a master regulator of the Treg phenotype, to convert cytotoxic T cells into an anti-inflammatory Tregs. A similar approach has been explored for type 1 diabetes, in which a type of islet-specific T cell receptor (TCR) was expressed in conjunction with editing of the FoxP3 locus to induce expression in CD4⁺ T cells ex vivo. Delivery of the engineered cells prevented the onset of type 1 diabetes in a mouse model and was more effective than engineered Tregs without the islet-specific TCR³⁴.

CAR-based therapies for cardiac injury could be further enhanced with a fourth-generation CAR, or a so-called CAR TRUCK (T cells redirected for antigen unrestricted cytokine-initiated killing)³⁵. In these approaches, the targeting and directed killing from CAR is modified to drive expression of a therapeutic immunomodulatory factor such as a cytokine or neutralizing antibody. When these T cells are activated through CAR binding, expression of the desired cytokine is also induced, leading to local and targeted delivery. This strategy could be used to recruit additional T cells to the injured tissue through expression of IL-2³⁶ or to inhibit pro-inflammatory signaling though expression of IL-10³⁷ or IL-1β neutralizing antibodies³⁸.

The targeting of T cells to activated fibroblasts and areas of inflammation is not limited to CAR-based approaches. Bi-specific antibodies have also been utilized in which an antibody is engineered to express multiple unique targeting domains to bring two cell types into close proximity³⁹. For example, one arm of the antibody may be specific to a T cell receptor complex subunit (e.g., CD3) while the other arm binds to an extracellular antigen on the target cells. Bi-specific antibodies induce the formation of an immune synapse between the bound T cells and the target cells, allowing for T cell-mediated cytotoxicity without the need of TCR antigen specificity. Bi-specific antibodies have been used primarily in cancer (for example, to target T cells to BCMA and CD20⁴⁰) but are now being explored for other indications. Bi-specific antibodies are “off the shelf” and eliminate the requirement for ex vivo T cell manufacturing and therefore reduce the cost and time of treatment. Furthermore, early studies in cancer suggest bi-specifics antibodies have lower rates of cytokine release syndrome than CAR-T cell therapies, potentially making these treatments safer for high risk patients⁴¹.

Finally, a distinct approach for modulating fibroblast activity could be to directly target tLNPs to activated fibroblasts to deliver mRNA payloads that affect fibroblast survival or function. This could be achieved, for example, by coupling an anti-FAP antibody to the surface of LNP. Possible therapeutic mRNAs include suicide genes such as PUMA⁴² which has previously been shown to deplete hematopoietic stem cells after tLNP delivery, inhibitors of known activation pathways such as siRNA against Meox1⁴³ or TGFβ-receptor signaling pathway members^44,45, or genes encoding anti-inflammatory cytokines such as IL-10³⁷ or IL-35⁴⁶. However, it remains unknown if activated fibroblasts in poorly vascularized regions can be effectively and efficiently targeted⁴⁷. While there have been examples of successfully targeting hepatocytes⁴⁸, endothelial cells⁴⁹ or immune cells²⁵, fewer examples exist for reliable delivery to less well vascularized tissue. It may be necessary to target high percentage of pathologic fibroblasts to achieve therapeutic benefit. Studies to date suggest that ~25% of T cells can be reproducibly targeted with anti-CD5 decorated LNPs, but this level of efficiency for targeting activated fibroblasts in the damaged heart may not be sufficient.

Conclusions and future perspectives

By targeting key components of the immune response following cardiac injury, immunomodulatory strategies seek to recalibrate the tissue microenvironment toward a less inflammatory immune state (Figure 4). In navigating the intricate landscape of post-MI reparative processes, combined therapeutic approaches targeting diverse cellular pathways and multiple cell types are essential for effective treatment during the transition from acute to chronic phases of MI. Recognizing the pivotal role of therapeutic timing, an optimized strategy is imperative, tailoring interventions to the distinctive temporal dynamics of various cell types involved: macrophages typically targeted within 3-7 days post-MI, endothelial cells (ECs) within 7-10 days, and fibroblasts within 1-2 weeks. However, this temporal schema may necessitate adaptation according to disease severity, individual medical contexts, and inflammatory responses, underscoring the potential of precision medicine to refine treatment modalities. The integration of precise diagnostic modalities into therapeutic paradigms holds promise for enhancing treatment outcomes. Advances in non-invasive imaging techniques, such as magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), offer avenues for comprehensive assessment of cardiac function, local inflammation, tissue fibrosis, and blood perfusion. The use of FAP inhibitor (FAPi) tracers for PET imaging is currently being explored in patients with ischemic injury⁵⁰. These studies aim to identify patients with significant interstitial fibrosis and a large burden of FAP⁺ cells who may be best suited for anti-FAP cellular therapies to gently reduce fibrosis, although clinical trials will be required to determine the best timing for treatment and when such therapies are beneficial. Understanding of spatiotemporal disease progression will facilitate the development of tailored treatment regimens, optimizing patient care and therapeutic efficacy. Particularly under the conditions of systemic chronic inflammation, such as in type 2 diabetes, cell type-specific approaches may need to be developed to target immunomodulatory cells within ischemic hearts.

Figure 4. Immunomodulatory therapy by targeting endothelial cells, macrophages, and fibroblasts for ischemic heart disease.

Tissue immunity is fundamental for cardiac repair in ischemic heart disease, which is subjected to spatiotemporal regulation by ECs, macrophages, and fibroblasts in the injured heart. These cells have spatiotemporal roles for tissue injury and repair after MI. Immunomodulatory therapy by genetic, epigenetic, or metabolic approaches to target these cells may offer exciting opportunities to rewire the tissue microenvironment and foster reparative immunity, facilitating cardiac repair after MI.

Despite the therapeutic potential of immunomodulatory interventions, vigilant monitoring is obligatory to mitigate associated risks. Immunomodulatory therapies, while promising, harbor potential complications including infusion-related infections, myocardial rupture stemming from excessive fibrosis clearance, and immune-related adverse events (irAEs) encompassing a spectrum of manifestations such as optic neuritis, endocrine dysfunction, psoriasis, anaemia, peripheral neuropathy, myocarditis, nephritis, and colitis. Given the variable onset of irAEs, i.e., happening at any time but most often occurring within the first few weeks or months after starting treatment, a vigilant post-therapy surveillance protocol is necessary to promptly identify and manage adverse events.

Further refinement and validation of immunomodulatory therapies necessitate comprehensive evaluation of off-target effects, pharmacokinetic/pharmacodynamic (PK/PD) profiles, and toxicity assessments. Large animal models serve as invaluable tools for elucidating therapeutic efficacy and safety profiles, while production standards of good manufacturing practice (GMP) ensure the reliability and consistency of LNP and therapeutic cell preparations. Further elucidation of the intricate mechanisms that orchestrate tissue immunity mediated through ECs, myeloid cells, and fibroblasts holds promise for identifying novel therapeutic targets. Future investigations aimed at identifying these underlying mechanisms herald promise to improve therapeutic approaches, fostering the development of targeted interventions tailored to individual patient needs. As ongoing research continues to unravel the complex pathology, these therapies hold the potential to transform clinical management, paving the way for more innovative, targeted treatments that would significantly improve patient outcomes in IHD.

Non-standard Abbreviations and Acronyms

MI

myocardial infarction

IHD

ischemic heart disease

ECs

endothelial cells

CAR

chimeric antigen receptor

tLNPs

targeted lipid nanoparticles

FAP

fibroblast-activation protein