Adding Insult to Injury - Inflammation at the Heart of Cardiac Fibrosis

Abstract

The fibrotic response has evolutionary worked in tandem with the inflammatory response to facilitate healing following injury or tissue destruction as a result of pathogen clearance. However, excessive inflammation and fibrosis are key pathological drivers of organ tissue damage. Moreover, fibrosis can occur in several conditions associated with chronic inflammation that are not directly caused by overt tissue injury or infection. In the heart, in particular, fibrotic adverse cardiac remodeling is a key pathological driver of cardiac dysfunction in heart failure. Cardiac fibroblast activation and immune cell activation are two mechanistic domains necessary for fibrotic remodeling in the heart, and, independently, their contributions to cardiac fibrosis and cardiac inflammation have been studied and reviewed thoroughly. The interdependence of these two processes, and how their cellular components modulate each other’s actions in response to different cardiac insults, is only recently emerging. Here, we review recent literature in cardiac fibrosis and inflammation and discuss the mechanisms involved in the fibrosis-inflammation axis in the context of specific cardiac stresses, such as myocardial ischemia, and in nonischemic heart conditions. We discuss how the search for anti-inflammatory and anti-fibrotic therapies, so far unsuccessful to date, needs to be based on our understanding of the interdependence of immune cell and fibroblast activities. We highlight that in addition to the extensively reviewed role of immune cells modulating fibroblast function, cardiac fibroblasts are central participants in inflammation that may acquire immune like cell functions. Lastly, we review the gut-heart axis as an example of a novel perspective that may contribute to our understanding of how immune and fibrotic modulation may be indirectly modulated as a potential area for therapeutic research.

2. Introduction

Cardiac fibrosis, defined as the excessive deposition of collagen in the myocardium, is a major cause of impaired cardiac function, consequently preventing the heart from efficiently contracting and relaxing in each beat and meeting the metabolic demands of the body. This chronic state, broadly referred to as heart failure (HF), remains an enormous global burden, affecting ~6.2 million people in the United States [1]. Cardiac stress imposed in the heart by different insults results in adverse cardiac remodeling, characterized by fibrosis, cardiac hypertrophy, and inflammation due, in part, to immune cell infiltration. Fibrotic remodeling contributes to myocardial stiffness, which ultimately impacts cardiac function (specifically, the contractility, conduction, and/or relaxation of the myocardium), resulting in HF [2]. Indeed, fibrosis is a key predictor of fatal ventricular arrhythmias arising in conditions including coronary artery disease, hypertrophic cardiomyopathy, and myocarditis, and improvements in cardiac magnetic resonance imaging have allowed for accurate mapping and tracking of fibrotic remodeling [3]. It is now appreciated that a key driver of excessive fibrosis is prolonged activity of the immune system. Moreover, fibroblasts express pattern recognition receptors (PRR) that can be engaged, induce secretion of pro-inflammatory cytokines and chemokines, and actively participate in the recruitment of immune cells to sustain inflammation. These properties, in fact, suggest that fibroblasts may themselves be regarded as semi-professional inflammatory cells. Thus, while many experimental and therapeutic approaches focus on them separately, the activity of immune cells and fibroblasts cannot be delineated from one other to comprehensively understand adverse cardiac remodeling in HF.

The nature of the cardiac insult elicits a particular inflammatory response that often determines the fibrotic response necessary to adapt, and consequently, which element of cardiac function will be impaired. Some conditions result in massive damage or necrosis of the myocardium, while others expose the heart to constant pressure over a long period of time. Regardless, fibrosis and inflammation are common components of remodeling aiming to change the physical properties of the tissue during repair or in response to modified physiological pressures. Extensive reviews have described inflammation as a critical driver of several pathological responses driving HF, and significant work has demonstrated that not only cytokines, but immune cells themselves, play active pro-fibrotic roles, oftentimes through interactions with fibroblasts [4]. The central cell type in fibrosis is the cardiac fibroblast, which is one of four major cell types in the heart, and significantly participates in extracellular matrix (ECM) turnover from development to old age [5]. In response to pathological stress, cardiac fibroblasts transform into activated myofibroblasts, which rapidly turn over the ECM (degrade existing proteins and deposit new ones) [4].

The heart is seeded with populations of resident immune cells early in development. Indeed, spatial transcriptomics in the developing human heart reveal the presence of immune cells as early as 6 weeks post-conception [6]. Furthermore, it is becoming apparent and accepted that the activity of fibroblasts and the immune system are not two processes occurring in parallel – rather, they are two cellular components of one orchestrated response (Figure 1).

Figure 1: Fibroblasts and immune cells actively communicate and coordinate actions during cardiac homeostasis and disease.

In the resting heart, cardiac homeostasis involves cross-talk between fibroblasts and resident immune cells that facilitate myocardial upkeep through secretion of growth factors and baseline extracellular matrix (ECM) turnover. Stress sensed by the heart results in fibroblast responses that involve their transformation into myofibroblasts, and their ability to release inflammatory mediators such as cytokines and chemokines that attract immune cells from the periphery, actively participating this way in the immune response. These early fibroblast and immune cell actions are somehow interdependent, and when sustained over time, result in ongoing fibrosis and inflammation, with secondary waves of immune cells and continuous fibroblast transformation, processes in which immune cells and fibroblasts influence each other’s function. Cardiac fibrosis and cardiac inflammation ultimately result in cardiac dysfunction.

Many elegant reviews have focused on either inflammation or fibrosis in the context of cardiac repair or HF, but few that regard both as united processes driving cardiac pathology. Here, we will review the most recent literature detailing the intimate relationship between cardiac fibrosis and inflammation to specifically highlight how their interconnected activities change throughout course of disease, and strategies used to modulate one by targeting the other. We introduce the cellular and protein components of the myocardium during homeostasis, focusing on how the interactions between fibroblasts, the ECM, and the immune cell repertoire poise the heart for rapid response to stimulus, and review the specific contributions of immune cells to fibrosis, and the inflammatory actions of fibroblasts in ischemic and nonischemic heart disease in acute and chronic settings. We will delineate mechanistic conclusions in animal models and specify correlations and discrepancies with patient data, and propose areas with therapeutic potential, such as modulating fibrosis using immunotherapy or through targeting the gut microbiota.

3. Cardiac fibroblasts: key effectors of fibrosis and contributors to inflammation

3.1. Fibroblasts and immune cells in the healthy myocardium

Fibroblasts comprise <20% of the non-myocytes in the mouse heart and are central in maintaining cardiac health through interactions with ECM, endothelial cells and hematopoietic-derived cells, which comprise 60% and 5–10%, respectively [7]. They primarily function to support ECM remodeling in homeostasis and disease, which is absolutely necessary for cardiac health, as the composition and integrity of the ECM dictates both cardiac contractility and relaxation, both of which consequently affect cardiac output. Complex and dynamic, the ECM is composed of a mix of structural (e.g. collagen) and non-structural proteins (e.g. matricellular proteins), the latter of which can relay signals from secreted proteins and growth factors from resident cells to invoke changes to the ECM [8].

Along with fibroblasts, resident immune cells have recently emerged as cellular contributors to myocardial homeostasis and remodeling, and are well-positioned to rapidly respond to physiological triggers. Single cell sequencing of the murine heart reveals populations of macrophages, T cells (both CD8+ and CD4+), B cells, natural killer (NK) cells, dendritic cells, and mast cells in the healthy heart [9]. While all cardiac resident macrophages express CD45, F4/80, CD11b, and MerTK, a population of these cells (those that are CC-chemokine receptor 2 (CCR2) negative) are seeded during embryogenesis and are entirely self-renewing, while a minor population in the healthy heart (CCR2+ cells) are replenished by circulating monocytes [10]. CCR2- tissue resident macrophages are primarily responsible for maintaining myocardial homeostasis, with a range of functions including promoting cardiac conduction [11]. In contrast, CCR2+ macrophages are often major orchestrators early in the inflammatory response. In the context of cardiac damage or stress, the immune landscape of the heart dramatically changes, as pro-inflammatory cells (CCR2+ macrophages among them) infiltrate and promote the immune response by the continued recruitment of other leukocytes to the heart [12]. Their abundance in the left ventricle is often associated with worsened systolic function in patients [13]. Though less abundant in the myocardium, lymphocytes (B cells and T cells) are also part of the cardiac resident immune repertoire. In addition to being poised for rapid response to injury, studies using B cell deficient mice reveal the substantial population of myocardial B cells also plays a role in contractility and myocardial mass in homeostasis [14]. The immune cell landscape co-exists with resident fibroblasts, and the plasticity of both cell types in close proximity can have significant implications for how the heart adapts to disruptions to homeostasis. Endothelial cells, although not the focus of this review, also directly contribute to both inflammation and cardiac fibrosis following injury through mechanisms that involve transforming growth factor-β (TGFβ)-mediated endothelial to mesenchymal transition (EndoMT) [15], and are therefore intricately involved in immune-fibroblast crosstalk (Figure 2).

Figure 2: Fibroblasts and immune cells interdependent modulation of immunity and myocardial remodeling.

In addition to maintaining active crosstalk with both resting and activated fibroblasts, immune cells also facilitate the transition between those two phenotypes. A. Fibroblasts, in resting, transitional, or activated states, participate in the immune response by secreting chemokines and cytokines that promote endothelial cell activation and immune cell infiltration into the myocardium, sensing damage (damage associated molecular patterns, DAMPs) through pattern recognition receptors (PRRs) and clearing myocardial debris. B. Similarly, immune cells actively maintain myocardial homeostasis, activate fibroblasts through cytokine release and physical interactions, and secrete growth factors, high quantities of cytokines and chemokines that directly impact fibroblast phenotype and function. These independent actions facilitate rapid, coordinated responses to myocardial physiological disturbances.

3.2. Fibroblast to myofibroblast conversion and their participation in the inflammatory response

Cardiac stress results in inflammation and changes in the myocardial microenvironment that involve the generation of myofibroblasts, a subset of cells that are phenotypically distinct from other fibroblasts, and itself includes several different cell subtypes [16]. The mechanisms governing fibroblast-to-myofibroblast transition can differ depending on the nature of the myocardial insult and the specific inflammatory response invoked. In a way, cardiac fibroblasts share some characteristics with innate immune cells: they express receptors for damage- and pathogen-associated molecular patterns (DAMPs and PAMPs, respectively), receptors for accumulated advanced glycation end products (RAGEs), and chemokines and cytokines [17], [18], [19]. The main characteristic during this transition to myofibroblasts is the expression of α-smooth muscle actin (αSMA), which modulates myofibroblast contractility and proliferation [20], [21]. Myofibroblasts also express TGFβ receptors and make TGFβ, and thus signals invoke rapid dephosphorylation events in proteins downstream of TGFβ that modulate αSMA expression, cell alignment, and fibrosis [22], [23]. Taken together, intimate interaction with the local microenvironment initiates complex signaling events that promote the fibroblast transition to a myofibroblast phenotype. While these signaling events will not be reviewed in detail herein, here we review how cardiac fibroblasts transform to myofibroblasts in the context of cardiac inflammation, and discuss their ability to perform actions/roles similar to innate immune cells in this context (Figure 2).

4. Immune and fibrotic responses in ischemic disease

Ischemic heart disease is estimated to affect over 120 million people worldwide [1]. While inflammation and fibrosis are critical for reparation immediately after ischemia, the need for rapid repair results in a response that is ultimately excessive and detrimental for long-standing cardiac function. Inflammation and fibrosis are necessary for immediate physical stabilization of the infarct and removal of debris. However, the same fibrotic scar that is critical for survival early on to prevent cardiac rupture and death, impairs contractility and contributes to later systolic dysfunction and chronic HF. As such, the role of fibrosis and inflammation is complex and stage-specific throughout the cardiac response to myocardial infarction (MI).

Following occlusion of a coronary artery (such as due to rupture of an atherosclerotic plaque), the heart is deprived of oxygenated blood, resulting in cardiac tissue death in an event referred to as a MI. Necrosis and cell death, as well as the inability of the heart to regenerate viable myocardium to replace dead tissue, results in the initiation of a complex, multi-phasic immune response aiming to clear debris and promote the replacement of lost myocardium with a fibrotic scar [17]. The cellular responses to MI are often delineated into three separate phases in mouse pre-clinical experimental models: the inflammatory phase (hours to days post-MI), the proliferative phase (days to ~1 week post-MI), and the maturation phase (~4 weeks post-MI). Chronic ischemic heart disease in the mouse is considered to be 4–8 weeks post-MI. The inflammatory components of each one of these phases has been extensively reviewed in the recent years, and we have summarized it in Figure 3A–B [17], [10]. Thus, in this review, we briefly touch on the different types of fibrosis (acute repair in the infarct zone vs chronic repair in remote areas), how the immune response may influence fibrosis and how fibroblasts may actively contribute to immune cell recruitment at the different stages.

Figure 3: Fibrotic and immune responses in distinct cardiac diseases.

A. In response to myocardial ischemia, fibroblasts and immune cells need to rapidly coordinate a joint response to promote quick wound healing and fibrotic repair. A rapid immune and fibrotic response to necrotic tissue and damage-associated molecular pattern (DAMP) release, followed by debris clearance, characterizes the acute phase that results in the generation of a collagenous scar. The indicated molecular players have been extensively reviewed [10], [17], [34] B. In the chronic phase, fibrosis extends to remote zones in response to changed pressures in the heart, different immune cells are continuously recruited from peripheral lymphoid organs, and immune cell plasticity and cytokine release contributes to the fibrotic response. C. Long-standing pressures in the myocardium induce cytokine and chemokine release by cardiac resident cells, including fibroblasts, that help recruit monocytes/macrophages and specific T cells in a sequential and coordinated manner, perpetuating chronic, low-grade inflammation. IFNγ+ Th1 cells adhere to cardiac fibroblasts and further promote fibroblast activation and cardiac fibrosis. D. Infection of myocardial cells, or recognition of self proteins as strange, result in rapid immune responses that resemble, in magnitude, the responses to ischemia. Cytokines such as IL-17 and IFNγ have opposing roles in activating fibroblasts and promoting fibrosis. E. Age is associated with chronic dysregulated immune responses that impact many organs, including the heart. Macrophages, granulocytes, and CD4+ T cells infiltrate the heart in low numbers and contribute to low grade cardiac fibrosis through cytokine secretion, that is sufficient to induce cardiac dysfunction.

4.1. Acute immune and fibrotic response to myocardial infarction.

The immune system evolved in part to coordinate a fibrotic response that promoted quick repair in tissues in contact with the exterior and are consequently at risk of being damaged, such as the skin. This rapid and coordinated response leads to the formation of a scar. A similar response is observed in the initial phase of cardiac injury, in this case induced by ischemia. hypoxia results in cell death and releasing of DAMPs, which can signal in fibroblasts Fibroblasts acquire an “inflammatory state” and function as “semi-professional” inflammatory cells that make chemokines that attract immune cells that participate in healing [24]. Examples of this cardiac fibroblast-neutrophil cooperation have been recently delineated in in vitro experiments: cardiac fibroblasts cultured in the presence of neutrophil extracellular traps (NETs) increase their expression of TGFβ under hypoxic conditions, and murine models of MI following neutrophil depletion show less TFGβ expression in the myocardium, resulting in improper reparative fibrotic responses [25], [26]. Fibroblasts are also active participants in inflammation aside from their action on the ECM and roles in recruitment. Firstly, they assume a pro-inflammatory, leukocyte-recruiting phenotype, characterized by secretion of chemokines CCL5, colony stimulating factor 1 (CSF1), and CX3CL1, as well as reduced interleukin (IL)-10 expression [27]. Secondly, activated cardiac myofibroblasts are reported to engulf apoptotic cells in vivo 3 days after occlusion of the left anterior descending artery in mice, thereby actively participating in the clearance of debris [28]. Lastly, fibroblasts themselves directly interact with the alarmins in the pro-inflammatory cardiac milieu using their own PRRs, such as toll-like receptor 4 (TLR4) and RAGE. The binding of such receptors promotes fibroblast proliferation and fibrosis, necessary for consequent stages of wound healing [29].

While innate immune cells and fibroblasts are early responders in inflammation, the adaptive immune response is invoked soon after. Murine studies using transgenic mice with T cells autoreactive to cardiac antigens (specifically to α-myosin heavy chain; αMHC) demonstrate dendritic cell activation of T regulatory cells (Tregs) in steady state to maintain homeostasis. However, following MI, activated dendritic cells infiltrate local draining mediastinal lymph nodes and activate IL-17- and interferon-γ (IFNγ)-producing T cells, with no expansion of Tregs [30]. Activation of T cells is essential for the inflammatory phase and, importantly, its resolution, as deletion of CD28 co-stimulatory signals necessary for T cell activation results in worsened 5 day survival rates and increased cardiac rupture due to less fibrosis in mice post-MI with permanent ligation [31].

As is the case with other diseases, inflammation must be tightly controlled, and the acute phase must be resolved in order to proceed to the wound-healing phases post-MI. Continued action by innate immune cells prevents such resolution. Neutrophil-derived alarmins S100A8/A9, for example, are critical for promoting the initial immune response, as they prime nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, resulting in consequent release of interleukin-1β (IL-1β) (Figure 3A). However, released IL-1β also promotes granulopoiesis, so its presence in high abundance towards the end of the inflammatory phase prevents appropriate resolution [32]. This contributes to cardiac dysfunction, such that short-term blockade of S100A9 in a murine model of MI results in a cardiac output similar to sham control mice [33].

Following the acute inflammatory phase is the proliferation/maturation phase, in which fibroblasts and immune cells, mainly macrophages, both contribute to ongoing inflammation in the infarct area [34]. At this stage, the necrotic myocardium has been scavenged by immune cells, and fibroblasts, and must be replaced with a collagenous scar, with primary activity of cardiac fibroblasts that proliferate, and immune cells that adopt a pro-fibrotic phenotype, both working in tandem. The fibroblasts are polarized towards a highly-proliferative, pro-fibrotic, and pro-angiogenic profile (upregulation of Vegfa), with high expression of αSMA indicating an activated myofibroblast phenotype [27], [35]. A fibrin/fibronectin provisional matrix is generated as the ECM is turned over to facilitate the proliferation and migration of cells working to form the collagenous scar in the infarct area [17]. The secretomes of the activated fibroblasts and reparative immune cells allow for generation of this matrix, continued ECM remodeling, and the generation of a temporary microvasculature network serving to support the cells in this phase [36].

Key among these mediators to transition from pro-inflammation to repair are resolvins, lipid derivatives functioning to resolve inflammation through actions on immune cells, such as by modulating macrophage polarity and cardiac infiltration [37], [38]. Injection of Resolvin D1 (RvD1) has been reported to reduce excessive collagen deposition post-MI [39]. Moreover, pro-inflammatory Ly6C^hi monocytes undergo a phenotypic shift towards Ly6C^low CD11b^low F4/80^high pro-reparative macrophages that upregulate pathways involved in ECM remodeling and collagen fibril organization [40]. Recruited dendritic cells also support fibrosis and angiogenesis post-MI, as dendritic cell ablation in murine models results in increased inflammation, impaired angiogenesis, and less infiltration of LyC6^low monocytes [41]. Additionally, IL-35, a cytokine frequently secreted by Tregs, was reported to promote wound healing specifically by contributing to survival of Ly6C^low macrophages, and inhibition of IL-35 results in less fibrosis and increased incidences of cardiac rupture [42].

During the maturation phase, myofibroblasts deposit large amounts of collagen and crosslink the ECM to form a mature scar in the infarct area. After the scar has matured, fibroblasts receive anti-fibrotic cues from the microenvironment. Examples of this are chemokines such as CXCL10 (also referred to as interferon-γ-inducible protein-10, IP-10), which reduces migration of fibroblasts stimulated with growth factors in vitro [43]. Interestingly, while mice deficient in a CXCL10 receptor, CXCR3, experience delayed myofibroblast infiltration in the border zone following ischemia/reperfusion, this delay does not affect scar formation or collagen deposition, suggesting the anti-fibrotic actions of CXCL10 operate through CXCR3-independent processes [44]. In addition, factors promoting ECM turnover are depleted and fibroblast proliferation ceases, resulting in the return to basal numbers of fibroblasts [36]. Remaining fibroblasts transition to anti-angiogenic, homeostatic phenotypes, while the microvasculature previously supporting their proliferation and activity is disintegrated [27]. Furthermore, the transition of endothelial cells to fibroblasts reduces the microvasculature density and contributes to a pool of fibroblasts that may perpetuate long-standing reactive fibrosis in chronic HF (Figure 3A).

Taken together, the acute immune and fibrotic responses are critical for healing, and while scar formation is necessary for survival, the formation of the scar from a combination of pro-inflammatory actions of fibroblasts, and pro-fibrotic actions of immune cells, results in reductions in contractility and/or relaxation, and HF.

4.2. Chronic HF post-MI

This phase is characterized for remodeling in remote zones, not acutely affected by infarct, likely to adapt to the changing pressures in the post-MI heart [36]. This process known as reactive fibrosis, also contributes to impaired cardiac function observed in patients.

Given the intimate relationship between the immune response and fibrosis, it is unsurprising that overall unresolved inflammation plays critical, though not fully-understood, roles in maladaptive fibrotic remodeling long after scar maturation. While heightened inflammation can be attributed in part to the presence of infiltrating immune cells, fibroblasts also retain a pro-inflammatory state in later stages of chronic HF. MI induced in mice with fibroblast-specific knockouts of IL-1 receptor had improved systolic function 4 weeks post-MI compared to wild-type controls undergoing surgery, supporting a pro-inflammatory role for fibroblasts [45]. Furthermore, long-standing fibroblast activation promotes expression of both IL-11 and IL-11 receptor, creating an autocrine loop that perpetuates the expression of fibrinogenic proteins [46]. This contributes to ongoing fibrosis and consequent cardiac dysfunction (Figure 3B).

Interestingly, in chronic HF, splenic remodeling and trafficking persist as far as 8 weeks after MI. At this time, activated macrophages and dendritic cells continue to enter the heart, resulting in consistent low-grade inflammation [47]. Furthermore, at this time, CD4+ T cells continue to be activated in the spleen and mediastinal lymph nodes, and their depletion reduces adverse cardiac remodeling [48]. Additionally, contributing to a sustained pro-inflammatory state are a population of CD4+ FoxP3+ Treg cells expressing tumor necrosis factor α (TNFα) which are impaired in their ability to modulate the immune response and resolve inflammation [49]. Lastly, humoral immunity plays a role in chronic remodeling, as heart-reactive antibodies remain in circulation in the chronic stage post-MI in both humans and mice, and in the latter, are shown to directly target the remodeled myocardium [50].

Taken together, we know more about the acute phase post-MI and the immune cell contributions to fibrotic healing. However, we highlight the role of fibroblasts as “semi-professional immune like cells”, which are active contributors to immune responses in addition to playing vital roles in ECM remodeling. As reviewed here, in response to tissue damage, they are activated by and respond to DAMPs, assist in initiating inflammation through secretion of chemokines, and participate in the clearance of debris. Both the complex necessity of fibrosis and inflammation, as well as their interconnectedness, must be strongly considered when exploring potential therapeutic options, as the coordinated action of immune cells and fibroblasts changes across the post-MI temporal landscape.

5. Immune and fibrotic responses in nonischemic disease

Nonischemic disease is a consequence of a wide range of conditions, such as congenital heart disease, hemodynamic abnormalities, and aberrant immune activity. Changes in blood pressure (hypertension), vascular abnormalities (aortic stenosis), poor valvular function, or systemic inflammation (for example, due to obesity, age, infection, or autoimmune disease) impart stress on the myocardium. To compensate, the heart undergoes myocardial remodeling, which includes increase in cardiomyocyte size (hypertrophy), chamber dilation, and fibrosis to disperse pressure and strengthen the integrity of the chamber. As may be expected, the nature of the insult dictates the mechanisms of immune-mediated fibrosis, some of which have been well-characterized in mice and related to patient clinical data.

5.1. Pressure overload-induced HF

Cardiac fibrosis develops in effort to adapt to higher filling pressures in the left ventricle. Patients frequently experience such hemodynamic changes following aortic stenosis or valvular disease, which are commonly found in patients older than 65 years old [1]. Fibrosis and cardiac hypertrophy in the left ventricle worsen contractility and consequent systolic function, resulting in a reduced ejection fraction and chronic HF. To model this in mice, the transverse aortic constriction (TAC) model is employed, in which the aorta is partially ligated, resulting in mild cardiac inflammation, fibrosis, and impaired systolic function, all hallmarks of HF in patients. While the precise pathophysiology of HF in these patients remains unresolved, recent work has revealed mechanisms by which inflammation contributes and perpetuates fibrotic remodeling following TAC.

Fibrotic remodeling in response to TAC is primarily perpetuated by resident fibroblasts, which become activated and deposit collagen in a TGFβ and Smad2/3 dependent manner [23]. However, fibroblasts also participate in immune cell recruitment, as they actively recruit macrophage infiltration, such that when the secretion of chemokine ligands by fibroblasts is disrupted, cardiac dysfunction and fibrosis are reduced [51]. In addition, cardiomyocytes have been reported to initiate inflammation following TAC through calcium/calmodulin-regulated kinase δ signaling, which activates the NLRP3 inflammasome [52]. Lastly, tenascin-C, a matricellular protein produced by cardiac stromal cells after TAC, promotes pro-inflammatory macrophage polarization and CCL2 chemokine ligand expression by fibroblasts in mice [53]. Once in the myocardium, infiltrated macrophages promote inflammation and can directly contribute to ECM remodeling through the secretion of collagen-binding matricellular proteins like secreted protein acidic and rich in cysteine (SPARC) [54].

This infiltration of monocyte-derived CCR2+ macrophages at a stage preceding fibrosis and systolic dysfunction is the infiltration of monocyte-derived CCR2+ macrophages is required for consequent T cell expansion and chronic systolic dysfunction [55]. Recent studies, some published by our group, have demonstrated a critical role of T cells in mediating fibrosis following TAC. During pressure overload, infiltrated monocytes and cardiac fibroblasts secrete chemokine ligands CXCL9 and CXCL10, which recruit CXCR3+ T cells to the heart [56]. T cell recruitment is necessary for TAC-induced cardiac fibrosis, as T cell receptor knockout (TCRα−/−) mice, and mice deficient in T cells following antibody depletion, do not develop fibrosis or systolic dysfunction following TAC [57]. Moreover, TAC mice deficient in Intracellular Adhesion Molecule 1 (ICAM1), which mediates T cell and monocyte trafficking into the heart, do not develop fibrosis [58]. Similarly, inhibitors of CD40 signaling (which is necessary for activation of antigen-presenting cells) block T cell infiltration and activity, consequently preventing fibrotic remodeling following TAC [59].

CD4+ T helper 1 (Th1) cells in particular mediate cardiac fibrosis in TAC-induced HF in an IFNγ-dependent manner. Activation and expansion occur predominantly in the mediastinal lymph nodes, rather than in peripheral lymphoid organs like the spleen (as is the case for inflammation in ischemic HF). Th1 cells direct fibrosis by directly binding to cardiac fibroblasts and inducing their transition into αSMA-expressing myofibroblasts in an IFNγ-dependent manner, as T effector cells lacking IFNγ are unable to promote this transition [60]. Similar studies in rats undergoing abdominal aortic constriction (AAC) demonstrate deficiencies in T-bet (the master transcription factor for Th1 cells) alleviate cardiac fibrosis [61]. Unlike CD4+ T cells, CD8+ T cells do not seem to play significant roles in promoting HF, as CD8+ deficient mice have similar cardiac fibrosis as WT mice post-TAC [62]. Collectively, these studies support a pro-fibrotic role for CD4+ Th1 cells in TAC-induced HF.

5.2. Hypertension

Hypertension, defined as having systolic blood pressure higher than 130 mmHg or diastolic blood pressure higher than 80 mmHg, is a highly variable disease affecting 45.6% of adults in the United States and is a major risk factor for cardiovascular disease [1]. In response to elevated blood pressures, consequent changes in hemodynamic pressure in the heart prompt fibrotic and hypertrophic remodeling. Many risk factors for hypertension, which include age, diet (higher salt content increasing risk), lifestyle, smoking, and obesity, several of which can coexist together, have strong inflammation-associated pathologies, inviting several mechanisms through which inflammation can modulate fibrotic remodeling in chronic disease.

Fibroblasts play key roles in inflammation and ECM remodeling in the hypertensive heart. In response to cardiotrophin-1, a member of the IL-6 cytokine superfamily, cardiac fibroblasts increase expression of galectin-3, a notable mediator of myocardial fibrosis [63]. Galectin-3 expression consequently dampens the peroxiredoxin-4 antioxidant system, which increases oxidative stress and ROS production, resulting in a heightened pro-inflammatory environment [64]. As such, fibroblast and immunity coordinate to promote fibrotic cardiac remodeling.

In response to angiotensin-II (Ang-II)-induced hypertension in mice, expression of pro-inflammatory cytokines increase in the myocardium, most notably IL-6, a key orchestrator of immune-mediated fibrosis, as knockout studies report attenuated fibrosis in the absence of IL-6 [65]. Shortly after beginning Ang-II administration, monocytes and macrophages are recruited to the heart in a CXCL1-CXCR2-dependent manner, and inhibition of this recruitment is sufficient to significantly reduce fibrosis [66]. Importantly, studies in Sprague-Dawley rats characterize a critical trafficking route from the spleen to the heart, with splenectomies protecting against fibrotic remodeling [67]. In a spontaneous hypertensive rat model, inhibition of angiotensin-converting enzyme prior to inducing hypertension with a nitric oxide synthase inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME), suppresses fibroblast expression of macrophage-recruiting chemokines and overall macrophage infiltration, suggesting fibroblasts are key in relaying physiological changes and initiating innate immune responses [68], [69].

Adaptive immunity also plays a critical role in perpetuating cardiac pathology and ongoing inflammation. IFNγ, a Th1-derived cytokine, promotes pathological cardiac fibrosis in Ang-II mice, such that disruption of this axis using an IFNγ-receptor knockout results in reduced T cell infiltration and fibrosis [70]. Furthermore, in this model, T cell-derived IFNγ promotes fibrosis and macrophage infiltration, suggesting macrophages control an adaptive immune response that supports further monocyte infiltration and activity in a self-sustaining feedback loop [71]. Lastly, hypertension promotes the formation of γ-ketoaldehydes, referred to as isoketals, which activate dendritic cells and result in subsequent expansion of autoreactive CD8+ T cells [72]. Thus, the adaptive immune response may drive fibrosis through cytokine-mediated direct mechanisms, as well as indirect mechanisms promoting the innate arm of the immune response (Figure 3C).

5.3. Myocarditis

In nonischemic HF, myocarditis, defined broadly as inflammation of the myocardium, is a common trigger of inflammation, and encompasses a range of clinical presentations from acute inflammation to dilated cardiomyopathy [1]. Infectious myocarditis elicits a robust innate immune response initiated to clear infection and debris resulting from dead infected cardiac cells, followed by fibrotic repair to replace the myocardium lost. Murine models of immunization with cardiac protein (to model autoimmune myocarditis, EAM), infection with Coxsackie virus of group B (CVB), or infection with Trypanosoma cruzi (to model Chagas disease) are commonly used to investigate the active interplay between cardiac fibrosis and inflammation.

Autoimmune myocarditis has recently gained attention as a severe consequence of checkpoint inhibitor therapy for cancer, prompting renewed investigation into the mechanisms of aberrant inflammation in the myocardium [73]. Furthermore, cardiomyopathies are common in several autoimmune disorders, such as systemic lupus erythematosus (SLE). Using cardiac magnetic resonance imaging, fibrosis can be detected in a significant proportion of patients not presenting with cardiovascular disease symptoms, suggesting the presence of subclinical cardiac remodeling in the context of systemic autoimmunity that must be closely monitored [74], [75]. Additionally, diabetes mellitus, a common comorbidity in heart disease, presents with increased myocardial fibrosis in patients with aortic stenosis [76]. This action is likely T cell-mediated, as fingolimod administration (restricting T cell activity) in a streptozotocin-induced diabetic mouse model reduced fibrosis [77]. Lastly, cardiovascular disease in response to SARS-COV2 infection has gained widespread attention. While the virus does have tropism for cardiac tissue, and myocardial abnormalities have been studied in patients, available autopsy data to date does not identify fibrotic remodeling as a key driver of pathology [78], [79], [80].

The immune and fibrotic responses in EAM have been extensively reviewed, and we have summarized them in Figure 3D. In this setting, fibroblasts again function as semi-professional pro-inflammatory immune cells by expression of chemokines (e.g. CCL2, CXCL1) and cytokines (IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in response to IL-17 induced by immunization predominantly released by neutrophils and Th17 cells [81], [82]. However, while this model makes use of specific heart-reactive T cells to study fibrosis and inflammation, it has also been demonstrated that heart non-specific T cells do not contribute to severity and may be protective against fibrosis in chronic stages, suggesting a dynamic and complex role for T cell immunity and the interplay between T cells and fibroblasts in fibrotic disease [83].

In the CVB model of infectious myocarditis, the immune and fibroblast response is similar to ischemic disease, in which infiltrated monocytes and neutrophils promote inflammation through secretion of alarmins such as S100A8 and S100A9 [84]. Additionally, IL-1β secretion perpetuates the inflammatory response, and its neutralization significantly attenuates cardiac fibrosis and myocardial damage [85]. Furthermore, as discussed, T cells are major drivers of pathology, and suppression of T cell responses with Treg adoptive transfer quells inflammation and fibrotic remodeling, suggesting interplay between fibroblasts and T cells [86]. Somewhat surprisingly, while T cell-derived IFNγ drives fibrosis in other disease contexts, IFNγ-deficient mice have more severe disease, fibrosis, and immune cell infiltration (specifically, a large amount of degranulating mast cells) [87]. This reiterates the intricacy of fibroblast and immune cell crosstalk in driving fibrosis, and how strongly these interactions are shaped by specific components of the cardiac microenvironment.

Lastly, chronic myocarditis is a common manifestation in Chagas disease, a parasitic infection caused by Trypanosoma cruzi. Acute infections are often treatable, but long-standing infection results in chronic Chagas cardiomyopathy, wherein sustained inflammation contributes to ongoing myocardial damage and fibrotic remodeling. Acute infection elicits rapid inflammation that damages the myocardium and is harmful to the parasite, resulting in counter efforts by the parasite itself to modulate the immune response through secretion of resolvins [88]. While a robust immune response is necessary to contain infection, its improper resolution facilitates entry into the chronic phase of disease, governed by different inflammatory processes that promote fibrosis. Immunity and fibrosis are also intricately linked in this phase, as anti-inflammatory treatment strategies aiming to polarize the immune cell compartment are able to limit fibrotic remodeling [89]. Myocardial infection with T. cruzi triggers a rapid inflammatory response recruiting macrophages and CD8+ T cells to the heart in a CCL5-dependent manner [90]. Furthermore, disruption of the CCL5/CCR5 axis in mice ameliorates myocardial damage [91]. The importance of this axis is corroborated by patient data demonstrating chronically-ill patients with more severe disease also had increased CCR5+ CD8+ T cells [92].

Taken together, fibrotic remodeling and inflammation in nonischemic disease is established over a longer period of time, oftentimes as a result of long-standing cardiac stress. Indeed, there are exceptions, as in infectious myocarditis, in which a rapid inflammatory response necessitates rapid fibroblast activity to replace lost myocardium, similar to what is seen in ischemic disease. While there are similarities in immune regulation of fibrotic remodeling in nonischemic disease, the specific mechanisms of immune cell and fibroblast crosstalk also differ, as the specific components of the inflammatory cardiac milieu differ depending on the nature of the insult. These differences, as well as how they affect fibroblast/inflammatory cross-talk, must be kept in mind when exploring therapeutic opportunities.

6. Immune-mediated cardiac fibrosis in “inflammaging’

One hallmark comorbidity of cardiovascular disease and fibrotic cardiac remodeling is age, which has gained attention as advances in science and medicine have increased the global life expectancy. As pro-inflammatory cells drive cardiac fibrosis in heart disease, it may seem expected that increased systemic inflammation would naturally result in cardiac fibrosis in aged individuals. Indeed, cardiac macrophages are expanded in aged mice (in part due to replenishment by infiltrating monocytes), and activate cardiac fibroblasts in an IL-10-dependent manner, resulting in diastolic dysfunction [93]. Furthermore, while T cell numbers in the myocardium are not significantly increased in the hearts of aged mice, there is a significant increase in IFNγ-producing effector CD4+ T cells in the mediastinal lymph nodes, suggesting the presence of active T cell-mediated local inflammation in older age [94]. This is supported by recent findings demonstrating blockade of T cell co-stimulation with the FDA-approved drug abatacept (which blocks cytotoxic T lymphocyte associated antigen 4, CTLA4) in aged mice significantly reduces cardiac inflammation, fibrosis, and systolic and diastolic dysfunction [95]. In addition to experiencing heightened systemic inflammation, the myocardium itself undergoes functional changes in advanced age, notably due to accumulation of ECM proteins (including collagen) and changes in MMP secretion that affect ECM turnover and the processing of cytokines and growth factors [96]. Such proteins, such as osteopontin, a matricellular protein elevated during myocardial stress, mediate age-related fibrosis and cross-talk with immune cell compartments, including those migrating from local visceral adipose depots [97]. Furthermore, infiltrating granulocytes in aged mice express pro-fibrotic Extracellular Matrix protein 1 (ECM1), which stimulates collagen production in cardiac fibroblasts, revealing another ECM protein serving as an intermediate between pro-inflammatory immune cells and fibrotic remodeling [98]. Despite the seemingly clear association between inflammation, age, and heart failure, our understanding of the mechanisms by which immunity contributes to age-associated cardiac fibrosis remain incomplete. Although the role of the fibroblast in inflammation initiation has not been studied, it is likely this cell contributes to inflammaging directly, in coordination with the immune senescence phenotype. Studies investigating age-associated cardiac phenotypes in animals with other cardiac pathologies, a clinically-relevant objective given many patients present with several comorbidities, reveal a complex role for age and immunity. For instance, transgenic aged mice overexpressing macrophage MMP-9 had decreased angiogenesis and collagen cross-linking one week post-MI compared to age-matched wild-type controls, suggesting improved wound healing [99]. Indeed, meticulous study and sophisticated technology are necessary to relate the inflammatory changes perpetuating fibrosis in the heart to systemic inflammaging in older age (Figure 3E).

7. New and insightful areas of investigation

Numerous methodologies used to understand the initiation and progression of fibrosis have proved insufficient in developing effective therapeutics, suggesting the need for an innovative paradigm. While many drugs targeting exclusively pro-fibrotic signaling in fibroblasts have been proposed, few have been successful at preventing ongoing fibrosis, and none have been successful at reversing existing fibrosis. Similarly, strictly anti-inflammatory drugs have shown little promise in reversing cardiac dysfunction and adverse cardiac remodeling in patients. Understandably so—cardiac fibrosis involves a complex system of molecular interactions that combine the adaptive and innate immune signaling axis with fibroblast activity and the cardiovascular system. Furthermore, the mechanisms by which immune cells and fibroblasts coordinate to drive cardiac pathology change across the course of time in disease and depending on the presence of other comorbidities. In recent years, the development of innovative tools, and a better comprehension of these complex systems, have led to exciting novel approaches exploring the pathophysiology of this disorder. Two such promising strategies highlighted below include targeting fibrosis through immunomodulation and the exploration of the interaction between the microbiome and the immune system in the onset of cardiac fibrosis. These domains represent often overlooked areas of opportunity for novel discoveries and will serve to unify previously isolated regions of cardio-immunology by focusing on the details of their interplay, rather than targeting them in isolation.

7.1. Targeting fibrosis through immunomodulation

As cardiac fibrosis is driven by fibroblasts and immune cells working in unison, inflammation has emerged as a novel target for anti-fibrotic therapy. Immunomodulation is an attractive strategy in part because there are several therapeutic approaches, such as monoclonal antibodies, cell therapies, and small molecule inhibitors, in use for other diseases, such as cancer and autoimmunity. As such, inflammation is a targetable and can be used to limit ongoing pro-fibrotic processes.

Nanoparticles are emerging as a viable drug delivery system in part due to their ability to be mass produced, as well as their ability to be conjugated to factors that hone them to particular tissues. One application of nanoparticle technology involves the scavenging of reactive oxygen species (ROS) and polarization of macrophages towards anti-inflammatory phenotypes in an MI murine model, resulting in decreased fibrosis. This was achieved through administration of graphene oxide, which serves as an antioxidant and effective gene carrier, diminishing ROS production and delivering polarizing genes, such as IL-4, to macrophages [100]. Other nanoparticle approaches target macrophage CSF-1 signaling, which is necessary for monocyte production and expansion, in an EAM myocarditis murine model. Encapsulated siRNA targeting CSF-1 effectively attenuates chronic cardiac fibrosis and preserves systolic function 30 days post-induction [101]. In addition to nanoparticles, small molecules targeting the activity of the immune system have shown promise in murine models of cardiac fibrosis and HF. Macrophage targeting by an anti-inflammatory semi-synthetic of betulinic acid reduces fibrosis in a murine T. cruzi myocarditis model to a greater extent than benznidazole (an antiparasitic drug commonly used to treat Chagas disease), and increases the expression of macrophage-associated anti-inflammatory markers, such as arginase-1 and IL-10, in the heart [89]. Furthermore, small molecule inhibition of CD40, which prevents monocyte activation of T cells expressing CD40L, limits cardiac fibrosis in a TAC model of pressure overload [59]. Lastly, inhibition of transient receptor potential ankyrin 1 (TRPA1), which is expressed in the endothelium and is involved in the pathologies of several cardiovascular diseases, decreases cardiac fibrosis and macrophage infiltration in a TAC murine model, representing an indirect immunomodulatory strategy that may exert anti-fibrotic benefits through changing macrophage activity [102].

A very novel therapeutic strategy emerging in the last few decades is the use of cell therapy for the treatment of disease. The use of mesenchymal stem cells overexpressing growth factors, such as insulin-like growth factor-1 (IGF-1), has been successful in limiting cardiac fibrosis and pro-inflammatory cytokine production in a murine T. cruzi myocarditis model, uniting the anti-inflammatory properties of mesenchymal stem cells with those of IGF-1 [103]. In addition to regenerative medicine, adoptive transfer of anti-inflammatory cell types, such as Tregs, reduces the expression and deposition of left ventricular collagen in a murine CVB3 myocarditis model [86]. Lastly, given the recent success of chimeric antigen receptor (CAR) T cells in treating cancer, these cells emerged as a possible method by which specific cell types can be targeted for inhibition. Engineered CD8+ T cells targeting fibroblast activation protein (FAP), a protein upregulated in dilated cardiomyopathy patients, prevents systolic dysfunction in an Ang-II hypertensive murine model. While these cells do not demonstrate the ability to reverse existing fibrosis, their administration shortly after angiotensin-II induction prevents excessive fibrosis compared to untreated controls [104]. Though cellular therapies present as exciting alternatives to traditional drugs, this strategy has several limitations for the development of viable therapeutics in humans – several cellular therapies are successful in murine models, and fail in humans, likely due to distinct and critical differences in the inflammatory pathways and physiology of mice and humans.

Success in murine models has prompted several attempts at developing anti-inflammatory and/or anti-fibrotic therapeutics for the treatment of patients. Disappointingly, several of these approaches, including anti-cytokine, anti-inflammatory, and immunomodulatory therapies, have failed in clinical trials for reasons that have been reviewed previously [105]. Current trials, several using repurposed drugs approved for other conditions, continue to monitor or target inflammation and fibrosis to improve adverse cardiac remodeling and function (Table 1). For example, the combination sacubitril and valsartan, which are neprilysin inhibitors and angiotensin receptor blockers respectively, is being tested in patients with hypertension, HFpEF, or HIV. Additionally, the effects of SGLT-2 inhibition, a strategy already used to treat diabetes, is being tested for its effects on myocardial fibrosis and inflammation. Other trials are investigating cardiac fibrosis and function in response to broad scale immunomodulation, or direct targeting of inflammatory factors, such as IL-1 receptor antagonism. These indirect ways to modulate inflammation and fibrosis may hold promise in the treatment of cardiac fibrosis.

Table 1: Ongoing clinical trials that modulate inflammation and fibrosis and heart disease.

Current clinical trials using receptor antagonists/agonists, inhibitors, broad and/or specific immunomodulation aiming to evaluate safety of therapy or improve cardiac function/limit adverse cardiac remodeling. These trials are ongoing and target several patient populations, including patients with heart failure, diabetes, Chagas Disease, or HIV.

Study	Therapeutic	Disease/Target/Objective	Clinical Trials Identifier
REDHART2	Anakinra (IL-1 receptor antagonist)	Aerobic exercise capacity in recently decompensated systolic heart failure	NCT03797001
SGLT-2 Inhibition	Dapagliflozin (SGLT-2 inhibitor)	Myocardial fibrosis and inflammation in patients with Type 2 Diabetes Mellitus	NCT03782259
iMP Cell Injection	Immunomodulatory progenitor cells (iMP cells)	Myocardial fibrosis following injection during coronary artery bypass graft (CABG) surgery	NCT03515291
REVERSE-LVH	Combination sacubitril/valsartan	Myocardial fibrosis in hypertensive heart disease	NCT03553810
ENCHANTMENT HIV	Combination sacubitril/valsartan	Myocardial fibrosis and inflammation in HIV patients	NCT04153136
PRISTINE-HF	Combination sacubitril/valsartan	Microvascular function and fibrosis in HFpEF patients	NCT04128891
COACH	Colchicine (anti-inflammatory drug)	Myocardial fibrosis and inflammation in Chagas Disease	NCT03704181
Beta3_LVH	Mirabegron (beta3-adrenergic receptor agonist)	Left ventricular mass index and diastolic function in patients with structural cardiac pathologies	NCT02599480
GIPS-IV	Sodium thiosulfate (H2S-releasing agent)	Myocardial infarct size following injection during primary percutaneous coronary intervention post-MI	NCT02899364
MIRACLE HIV	Eplerenone (mineralcorticoid receptor antagonist)	Myocardial inflammation in HIV patients	NCT02740179

7.2. The microbiome – immune system axis in cardiac fibrosis

Exciting advancements in microbiology and next-generation sequencing have linked the microbiome in regulating the functions of the adaptive and innate immune signaling axes [106]. In addition to other chronic inflammatory diseases, alterations in microbial populations have been implicated in HF. Depleting the gut microbiota in various mouse models has been shown to protect from autoimmune myocarditis, Ang-II induced hypertension, and TAC-induced HF, while simultaneously being harmful during myocardial infarction [107], [108], [109], [110]. These distinct responses of the microbiota highly dependent on the cardiac insult have been directly associated with both immune cell activation and cardiac fibrosis associated with these conditions, and suggest that specific microbes and derived metabolites modulate immune responses in heart disease, a less explored but promising area of research.

Gut microbiota can transmit signals across the intestinal epithelium by interacting with PRRs on the surface of mucosal cells. These interactions are initiated via structural components of gut microbes, specifically microbe-associated molecular patterns (MAMPs), such as lipopolysaccharide (LPS) and peptidoglycans. LPS can successfully traffic to lymphatic circulation by chylomicron co-transport and paracellular diffusion to promote disease [111], [112]. It is possible that microbial-derived inflammatory components may bind TLRs expressed in the myocardium, possibly in the cardiac fibroblasts themselves, particularly in the context of HF driven by systemic inflammation. However, not always gut dysbiosis in heart disease is associated with increased LPS serum levels, and often diet metabolites converted by the microbiota function as modulators of immune responses. Dietary fiber, cholesterol, animal-derived lipids, and antibiotics are metabolized and converted into bile acids, Short Chain Fatty Acids (SCFAs), tryptophan derivatives, and other endotoxins. Many of the metabolites produced by the gut microbiota are absorbed into systemic circulation, where they can traffic to numerous sites and function as biologically active agents. This metabolite-dependent signaling pathway mimics the endocrine system to modulate normal physiological function at proximal and distal loci [113], [114]. SCFA from dietary fiber intake can reduce hypertensive fibrosis through the transcription factor Egr1 and prevent HF in mice [115]. SCFA activate many G-protein coupled receptors (GPRs) such as GPR41 and GPR43 [116]. As these receptors are expressed in the heart, SCFA may also exert local anti-fibrotic actions. We recently reported that mice undergoing TAC experience microbiota alterations that include a significant decrease in the Lactobacillus bacterial genus—a highly active generator of tryptophan metabolites. TAC resulted in a significant decrease in tryptophan related metabolic pathways as well as decreased expression of their receptor, the aryl hydrocarbon receptor, in the gut epithelial cells and in the heart [110]. These data support that microbiota processed metabolites may exert actions in immunity and fibrosis acting on receptors at distal sites beyond the gut, and a promising area of investigation in the context of cardiac inflammation and fibrosis.

Lastly, trimethylamine N-oxide (TMAO), a metabolite derived from dietary choline and other trimethylamine-containing elements, induces cardiac fibrosis indirectly by accelerating fibroblast-myofibroblast differentiation via activation of the TGF-βRI/Smad2 pathway, and directly via the TGF-βRI/Smad3 pathway, a pathways that when inhibited prevented the pathological effects of TMAO [117], [118].

Understanding the role of the gut microbiome in modulating cardiac fibrosis presents an excellent opportunity for developing potential therapeutics. The metabolites generated by commensal bacteria have wide-ranging effects, including regulation of the adaptive and innate immune signaling axis, through their ability to traffic to distal sites as well as modulate local inflammatory processes (Figure 4). More emphasis must be placed on understanding the ligand-specific and cell-specific mechanisms of fibrosis perpetuated by microbiota processed metabolites to design potential dietary and pro-biotic therapies in cardiac fibrosis and HF.

Figure 4: Multi-faceted regulation of cardiac fibrosis and inflammation.

The stress response interconnects inflammation, and fibrosis, which can be further influenced by the gut microbiome though derived metabolites. Fibrosis is heavily influenced by inflammation, and cardiac fibroblasts and immune cells are plastic and have effects on each other’s actions. Stress induces fibroblast responses to damage-associated molecular patterns (DAMPs) and advanced glycation end products (AGEs), and activates TGFβ signaling pathways, αSMA expression, and fibrosis. Additionally, stress induces pro-inflammatory mediators that invoke innate and adaptive immune cell activity and result in inflammation. The gut microbiome, known to modulate immunity locally, also regulates immunity in distal sites possibly through derived metabolites with actions in immune cells, and potentially on cardiac cells, something that remains largely unexplored. At the interface of fibrosis and inflammation, modulation of the microbiome or derived metabolites may be considered for therapeutic purposes to treat cardiac inflammation and fibrosis in heart failure.

8. Concluding remarks and the future of immune-regulated fibrosis research

Cardiac fibrosis is a major contributor to HF, a syndrome with extensive global burden. While fibrosis in cardiovascular disease has been extensively investigated for many decades, it was often studied in isolation or in parallel with other processes during cardiac disease. Emerging research into the integral roles inflammation play in HF pathophysiology reveal a tight interplay between the activity of resident and infiltrating immune cells and cardiac fibroblasts. The actions of immune cells and fibroblasts are coordinated, interdependent, and integral to cardiac homeostasis and fibrotic cardiac remodeling. Comprehensive study of fibrosis in heart conditions, especially with therapeutic goals, necessitates thorough appreciation of this relationship.

There have been several instances in the field in which anti-fibrotic therapies demonstrated significant efficacy in preclinical models, then proceeded to fail in human trials. As such, a key limitation in much of the research in immune-driven cardiac fibrosis, including work reviewed herein, is that it is done largely in rodents. Surely, we can expect significant differences in key immunological processes governing cardiac function, homeostasis, and repair between humans and rodents, and many of these differences are already well-recognized. While this discordance must be appreciated, they should not detract from the many benefits of preclinical models, which allow for thorough characterizations of mechanism and the continued development of biological tools for further studies.

Recent advancements in the field of cardio-immunology show promise in generating effective therapeutic strategies against fibrosis. This pathology is a result of a dynamic interplay between the adaptive and innate immune system, the gut microbiome, and the cardiovascular system. With the advent of novel mechanistic discoveries and breakthrough technologies, we are beginning to understand the significant interactions between multiple biological systems and their role in the development and progression of heart failure. This work has the potential for novel approaches wherein the immune system itself is modulated to attenuate, or possibly reverse, fibrosis. Indeed, vaccination as an approach for the treatment or prevention of other cardiovascular diseases, such as atherosclerosis has recently begun to be explored [119]. Nonetheless, these potentials can only be realized if fibrosis is studied in relation to immunity and systemic factors that impact inflammation. Study of either inflammation or fibroblast activity in isolation of one another is limited, incomplete, and unlikely to yield successful therapeutic strategies. To this end, a comprehensive understanding of the modulatory role of the immune system in fibrosis and elucidating the immunomodulatory function of microbiome metabolites will help in the development of functional therapeutics against fibrosis and ultimately heart failure.

Highlights.

• Cardiac fibroblast activation and immune cell activation are two mechanistic domains necessary for fibrotic remodeling in the heart. We discuss their interdependence in modulating each other’s actions in response to different cardiac insults.

• We highlight the pro-fibrotic role of immune cells as well as the less explored pro-inflammatory role of cardiac fibroblasts, which adopt “immune cell like” functions that have an impact on cardiac and immune cells, and discuss therapeutic opportunities that modulate these responses.

• We review the gut-heart axis as an example of a novel perspective that may contribute to our understanding of how immune and fibrotic modulation may be indirectly modulated as a potential area for therapeutic research.

Abbreviations

HF

Heart failure

PRRs

Pattern recognition receptors

ECM

Extracellular matrix

CCR2

CC-chemokine receptor 2

TGFβ

Transforming growth factor β

CCR2

CC-chemokine receptor 2

DAMPs

Damage-associated molecular patterns

RAGE

Receptor for advanced glycation end products

αSMA

α-smooth muscle actin

MI

Myocardial infarction

MMP

Matrix metalloproteinase

NETs

Neutrophil extracellular traps

IL

Interleukin

TLR4

Toll-like receptor 4

Tregs

T regulatory cells

IFNγ

Interferon-γ

IL-1β

Interleukin-1β

TAC

Transverse aortic constriction

Ang-II

Angiotensin-II

CVB3

Coxsackie virus of group b

EAM

Experimental autoimmune myocarditis

SCFA

Short chain fatty acids