Aging influences the cardiac macrophage phenotype and function during steady state and during inflammation

Aging‐mediated immune dysregulation affects the normal cardiac immune cell phenotypes and functions, resulting in cardiac distress. During cardiac inflammation, immune activation is critical for mounting the regenerative responses to maintain normal heart function. We investigated the impact of aging on myeloid cell phenotype and function during cardiac inflammation induced by a sub‐lethal dose of LPS. Our data show that hearts of old mice contain more myeloid cells than the hearts of young mice. However, while the number of monocytic‐derived suppressor cells did not differ between young and old mice, monocytic‐derived suppressor cells from old mice were less able to suppress T‐cell proliferation. Since cardiac resident macrophages (CRMs) are important for immune surveillance, clearance of dead cells, and tissue repair, we focused our studies on CRMs phenotype and function during steady state and LPS treatment. In the steady state, we observed significantly more MHC‐II^low and MHC‐II^high CRMs in the hearts of old mice; however, these populations were decreased in both young and aged mice upon LPS treatment and the decrease in CRM populations correlated with defects in cardiac electrical activity. Notably, mice treated with a liver X receptor (LXR) agonist showed an increase in MerTK expression in CRMs of both young and old mice, which resulted in the reversal of cardiac electrical dysfunction caused by lipopolysaccharide (LPS). We conclude that aging alters the phenotype of CRMs, which contributes to the dysregulation of cardiac electrical dysfunction during infection in aged mice.     

Adding insult to injury - Inflammation at the heart of cardiac fibrosis

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.     

Senescent cardiac fibroblasts: A key role in cardiac fibrosis

Cardiac fibroblasts are a cell population that controls the homeostasis of the extracellular matrix and orchestrates a damage response to maintain cardiac architecture and performance. Due to these functions, fibroblasts play a central role in cardiac fibrosis development, and there are large differences in matrix protein secretion profiles between fibroblasts from aged versus young animals.Senescence is a multifactorial and complex process that has been associated with inflammatory and fibrotic responses. After damage, transient cellular senescence is usually beneficial, as these cells promote tissue repair. However, the persistent presence of senescent cells within a tissue is linked with fibrosis development and organ dysfunction, leading to aging-related diseases such as cardiovascular pathologies. In the heart, early cardiac fibroblast senescence after myocardial infarction seems to be protective to avoid excessive fibrosis; however, in non-infarcted models of cardiac fibrosis, cardiac fibroblast senescence has been shown to be deleterious. Today, two new classes of drugs, termed senolytics and senostatics, which eliminate senescent cells or modify senescence-associated secretory phenotype, respectively, arise as novel therapeutical strategies to treat aging-related pathologies. However, further studies will be needed to evaluate the extent of the utility of senotherapeutic drugs in cardiac diseases, in which pathological context and temporality of the intervention must be considered.     

Nontuberculous mycobacterium M. avium infection predisposes aged mice to cardiac abnormalities and inflammation

Biological aging dynamically alters normal immune and cardiac function, favoring the production of pro‐inflammatory cytokines (IL‐1β, IL‐6, and TNF‐α) and increased instances of cardiac distress. Cardiac failure is the primary reason for hospitalization of the elderly (65+ years). The elderly are also increasingly susceptible to developing chronic bacterial infections due to aging associated immune abnormalities. Since bacterial infections compound the rates of cardiac failure in the elderly, and this phenomenon is not entirely understood, the interplay between the immune system and cardiovascular function in the elderly is of great interest. Using Mycobacterium avium, an opportunistic pathogen, we investigated the effect of mycobacteria on cardiac function in aged mice. Young (2–3 months) and old (18–20 months) C57BL/6 mice were intranasally infected with M. avium strain 104, and we compared the bacterial burden, immune status, cardiac electrical activity, pathology, and function of infected mice against uninfected age‐matched controls. Herein, we show that biological aging may predispose old mice infected with M. avium to mycobacterial dissemination into the heart tissue and this leads to cardiac dysfunction. M. avium infected old mice had significant dysrhythmia, cardiac hypertrophy, increased recruitment of CD45⁺ leukocytes, cardiac fibrosis, and increased expression of inflammatory genes in isolated heart tissue. This is the first study to report the effect of mycobacteria on cardiac function in an aged model. Our findings are critical to understanding how nontuberculous mycobacterium (NTM) and other mycobacterial infections contribute to cardiac dysfunction in the elderly population.     

Deficiency of CD73 activity promotes protective cardiac immunity against Trypanosoma cruzi infection but permissive environment in visceral adipose tissue

Damaged cells release the pro-inflammatory signal ATP, which is degraded by the ectonucleotidases CD39 and CD73 to the anti-inflammatory mediator adenosine (ADO). The balance between ATP/ADO is known to determine the outcome of inflammation/infection. However, modulation of the local immune response in different tissues due to changes in the balance of purinergic metabolites has yet to be investigated. Here, we explored the contribution of CD73-derived ADO on the acute immune response against Trypanosoma cruzi parasite, which invades and proliferates within different target tissues. Deficiency of CD73 activity led to an enhanced cardiac microbicidal immune response with an augmented frequency of macrophages with inflammatory phenotype and increased CD8+ T cell effector functions. The increment of local inducible nitric oxide (NO) synthase (iNOS)⁺ macrophages and the consequent rise of myocardial NO production in association with reduced ADO levels induced protection against T. cruzi infection as observed by the diminished cardiac parasite burden compared to their wild-type (WT) counterpart. Unexpectedly, parasitemia was substantially raised in CD73KO mice in comparison with WT mice, suggesting the existence of tissue reservoir/s outside myocardium. Indeed, CD73KO liver and visceral adipose tissue (VAT) showed increased parasite burden associated with a reduced ATP/ADO ratio and the lack of substantial microbicidal immune response. These data reveal that the purinergic system has a tissue-dependent impact on the host immune response against T. cruzi infection.     

MHC class II antigen-expressing cells in cardiac ganglia of the rat

Cardiac ganglia develop destructive ganglionitis in chronic Chagas disease and rheumatic heart disease. This ganglionitis is associated with periganglionic infiltrations and is suspected of developing secondary to epicardial inflammation. If so, it would be expected that cardiac ganglia (1) are equipped with an inventory of immune competent cells allowing the initiation of inflammatory processes, and (2) are not effectively protected from the milieu of the surrounding tissue by metabolically active diffusion barriers. These problems were addressed in specified pathogen-free rats by electron microscopy and immunohistochemistry with markers for dendritic cells, monocytes/macrophages, and perineurial barriers. In contrast to nerve fascicles, cardiac ganglia are only partially enveloped by perineurial cells. Inside the ganglia, ramified cells with major histocompatibility complex class II antigen (reacting with monoclonal antibody OX6) on their surface and exhibiting an ultrastructure typical of dendritic cells are numerous, comprising nearly 5% of all cells within ganglia. The ratio of the number of these cells to that of neurons is 1:2. Cells reacting with monoclonal antibodies ED1 and ED2, markers for monocytes/macrophages, constitute 1.8% and 1.6% of the ganglionic cell population, respectively. Such cells are less frequent in the cervical trunk of the vagus nerve. Thus, the inventory of immune competent cells in rat cardiac ganglia is consistent with the view that the abundance of antigen-presenting cells correlates with the permeability of the barriers providing protection from blood-borne and tissue-borne factors.     

H2S protects from oxidative stress-driven ACE2 expression and cardiac aging

Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H₂S) plays an essential role in preserving cardiac functions. Angiotensin-converting enzyme 2 (ACE2) acts as the negative regulator of the renin-angiotensin system, exerting anti-oxidative stress and anti-inflammatory properties within the body. The interplays of CSE/H₂S signaling and ACE2 in cardiac aging are unclear. In this study, the regulatory roles of H₂S on ACE2 expression in mouse heart tissue and rat cardiomyocytes under different stress conditions were investigated. It was found that ACE2 protein level was lower in heart tissues from old mice (56-week-old) than young mice (8-week-old), and the knockout of CSE (CSE KO) induced moderate oxidative stress and further inhibited ACE2 protein level in mouse hearts at both young and old age. Incubation of rat cardiac cells (H9C2) with a low dose of H₂O₂ (50 µM) suppressed ACE2 protein level and induced cellular senescence, which was completely reversed by co-incubation with 30 µM NaHS (a H₂S donor). Prolonged nutrient excess is an increased risk of heart disorders by causing metabolic dysfunction and cardiac remodeling. We further found high-fat diet feeding stimulated ACE2 expression and induced severe oxidative stress in CSE KO heart in comparison with wild-type heart. Lipid overload in H9C2 cells to mimic a status of nutrient excess also enhanced the expression of ACE2 protein and induced severe oxidative stress and cell senescence, which were significantly attenuated by the supplementation of exogenous H₂S. Furthermore, the manipulation of ACE2 expression partially abolished the protective role of H₂S against cellular senescence. These results demonstrate the dynamic roles of H₂S in the maintenance of ACE2 levels under different levels of oxidative stress, pointing to the potential implications in targeting the CSE/H₂S system for the interruption of aging and diabetes-related heart disorders.

     

Immunological aspects of age-related diseases

The proportion of elderly people rises in the developed countries. The increased susceptibility of the elderly to infectious diseases is caused by immune dysfunction, especially T cell functional decline. Age-related hematopoietic stem cells deviate from lymphoid lineage to myeloid lineage. Thymus shrinks early in life, which is followed by the decline of na?ve T cells. T-cell receptor repertoire diversity declines by aging, which is caused by cytomegalovirus-driven T cell clonal expansion. Functional decline of B cell induces antibody affinity declines by aging. Many effector functions including phagocytosis of myeloid cells are down regulated by aging. The studies of aging of myeloid cells have some controversial results. Although M1 macrophages have been shown to be replaced by antiinflammatory(M2) macrophages by advanced age, many human studies showed that pro-inflammatory cytokines are elevated in older human. To solve this discrepancy here we divide age-related pathological changes into two categories. One is an aging of immune cell itself. Second is involvement of immune cells to age-related pathological changes. Cellular senescence and damaged cells in aged tissue recruit pro-inflammatory M1 macrophages, which produce pro-inflammatory cytokines and proceed to agerelated diseases. Underlying biochemical and metabolic studies will open nutritional treatment.     

Acute heart inflammation: ultrastructural and functional aspects of macrophages elicited by Trypanosoma cruzi infection

The heart is the main target organ of the parasite Trypanosoma cruzi , the causal agent of Chagas' disease, a significant public health issue and still a major cause of morbidity and mortality in Latin America. During the acute disease, tissue damage in the heart is related to the intense myocardium parasitism. To control parasite multiplication, cells of the monocytic lineage are highly mobilized. In response to inflammatory and immune stimulation, an intense migration and extravasation of monocytes occurs from the bloodstream into heart. Monocyte differentiation leads to the formation of tissue phagocytosing macrophages, which are strongly activated and direct host defence. Newly elicited monocyte-derived macrophages both undergo profound physiological changes and display morphological heterogeneity that greatly differs from originally non-inflammatory macrophages, and underlie their functional activities as potent inflammatory cells. Thus, activated macrophages play a critical role in the outcome of parasite infection. This review covers functional and ultrastructural aspects of heart inflammatory macrophages triggered by the acute Chagas' disease, including recent discoveries on morphologically distinct, inflammation-related organelles, termed lipid bodies, which are actively formed in vivo within macrophages in response to T. cruzi infection. These findings are defining a broader role for lipid bodies as key markers of macrophage activation during innate immune responses to infectious diseases and attractive targets for novel anti-inflammatory therapies. Modulation of macrophage activation may be central in providing therapeutic benefits for Chagas' disease control.     

Visceral leishmaniasis with cardiac involvement in a dog: a case report

A dog presented with cutaneous nodules, enlarged lymph nodes and oedema in limbs, face and abdomen. The diagnosis of visceral leishmaniasis was established by identification of Leishmania amastigotes within macrophages from skin and popliteal lymph node biopsies. At necropsy, lesions were found in different organs, but it was particularly striking to observe large areas of pallor in the myocardium. Histological examination revealed an intense chronic inflammatory reaction in many organs, and numerous macrophages were found to contain amastigote forms of Leishmania. The inflammatory reaction was especially severe in the heart, where large areas of the myocardium appeared infiltrated with huge numbers of mononuclear immune cells, causing cardiac muscle atrophy and degeneration. Despite the severe inflammation, the number of parasitized macrophages was low in the myocardium, as revealed by immunohistochemical staining of Leishmania amastigotes. Because cardiac involvement is not usually described in this condition, this dog represents a very rare case of canine visceral leishmaniasis with affection of the myocardium.     

SDF-1 induces TNF-mediated apoptosis in cardiac myocytes

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation. One such chemokine, Stromal cell-derived factor-1 (SDF-1) also known as CXCL12, and its receptor, CXCR4, are expressed and functional in cardiac myocytes. SDF-1 both stimulates and enhances the cellular signal which attracts potentially beneficial stem cells for tissue repair within the ischemic heart. Paradoxically however, this chemokine is known to act in concert with the inflammatory cytokines of the innate immune response which contributes to cellular injury through the recruitment of inflammatory cells during ischemia. In the present study, we have demonstrated that SDF-1 has dose dependent effects on freshly isolated cardiomyocytes. Using Tunnel and caspase 3-activation assays, we have demonstrated that the treatment of isolated adult rat cardiac myocyte with SDF-1 at higher concentrations (pathological concentrations) induced apoptosis. Furthermore, ELISA data demonstrated that the treatment of isolated adult rat cardiac myocyte with SDF-1 at higher concentrations upregulated TNF-α protein expression which directly correlated with subsequent apoptosis. There was a significant reduction in SDF-1 mediated apoptosis when TNF-α expression was neutralized which suggests that SDF-1 mediated apoptosis is TNF-α-dependent. The fact that certain stimuli are capable of driving cardiomyocytes into apoptosis indicates that these cells are susceptible to clinically relevant apoptotic triggers. Our findings suggest that the elevated SDF-1 levels seen in a variety of clinical conditions, including ischemic myocardial infarction, may either directly or indirectly contribute to cardiac cell death via a TNF-α mediated pathway. This highlights the importance of this receptor/ligand in regulating the cardiomyocyte response to stress conditions.     

Immune regulation of cardiac fibrosis post myocardial infarction

Pathological changes resulting from myocardial infarction (MI) include extracellular matrix alterations of the left ventricle, which can lead to cardiac stiffness and impair systolic and diastolic function. The signals released from necrotic tissue initiate the immune cascade, triggering an extensive inflammatory response followed by reparative fibrosis of the infarct area. Immune cells such as neutrophils, monocytes, macrophages, mast cells, T-cells, and dendritic cells play distinct roles in orchestrating this complex pathological condition, and regulate the balance between pro-fibrotic and anti-fibrotic responses. This review discusses how molecular signals between fibroblasts and immune cells mutually regulate fibrosis post-MI, and outlines the emerging pharmacological targets and therapies for modulating inflammation and cardiac fibrosis associated with MI.     

An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile

Cardiac tissue macrophages (cTMs) are a previously uncharacterised cell type that we have identified and characterise here as an abundant GFP(+) population within the adult Cx(3)cr1(GFP/+) knock-in mouse heart. They comprise the predominant myeloid cell population in the myocardium, and are found throughout myocardial interstitial spaces interacting directly with capillary endothelial cells and cardiomyocytes. Flow cytometry-based immunophenotyping shows that cTMs exhibit canonical macrophage markers. Gene expression analysis shows that cTMs (CD45(+)CD11b(+)GFP(+)) are distinct from mononuclear CD45(+)CD11b(+)GFP(+) cells sorted from the spleen and brain of adult Cx(3)cr1(GFP/+) mice. Gene expression profiling reveals that cTMs closely resemble alternatively-activated anti-inflammatory M2 macrophages, expressing a number of M2 markers, including Mrc1, CD163, and Lyve-1. While cTMs perform normal tissue macrophage homeostatic functions, they also exhibit a distinct phenotype, involving secretion of salutary factors (including IGF-1) and immune modulation. In summary, the characterisation of cTMs at the cellular and molecular level defines a potentially important role for these cells in cardiac homeostasis.     

Stem cells as therapy for cardiac disease - a review

Acute myocardial infarction (AMI) is one of the most significant causes of morbidity and mortality worldwide. Stem cells represent an enormous chance to rebuild damaged heart tissue. Correct definition of the cardiac progenitors is necessary to understand heart development, and would pave the way for the use of cardiac progenitors in the treatment of heart disease. Identifying, purifying and differentiating native cardiac progenitor cells are indispensable if we are to overcome congenital and adult cardiac diseases. To understand their functions, physiology and action, cells are tested in animal models, and then in clinical trials. But because clinical trials yield variable results, questions about proper cardiac stem cells remain unanswered. Transplanted stem cells release soluble factors, acting in a paracrine fashion, which contributes to cardiac regeneration. Cytokines and growth factors have cytoprotective and neovascularizing functions, and may activate resident cardiac stem cells. Understanding all these mechanisms is crucial to overcoming heart diseases.     

The effect of immune cell-derived exosomes in the cardiac tissue repair after myocardial infarction: Molecular mechanisms and pre-clinical evidence

After a myocardial infarction (MI), the inflammatory responses are induced and assist to repair ischaemic injury and restore tissue integrity, but excessive inflammatory processes promote abnormal cardiac remodelling and progress towards heart failure. Thus, a timely resolution of inflammation and a firmly regulated balance between regulatory and inflammatory mechanisms can be helpful. Molecular- and cellular-based approaches modulating immune response post-MI have emerged as a promising therapeutic strategy. Exosomes are essential mediators of cell-to-cell communications, which are effective in modulating immune responses and immune cells following MI, improving the repair process of infarcted myocardium and maintaining ventricular function via the crosstalk among immune cells or between immune cells and myocardial cells. The present review aimed to seek the role of immune cell-secreted exosomes in infarcted myocardium post-MI, together with mechanisms behind their repairing impact on the damaged myocardium. The exosomes we focus on are secreted by classic immune cells including macrophages, dendritic cells, regulatory T cells and CD4⁺ T cells; however, further research is demanded to determine the role of exosomes secreted by other immune cells, such as B cells, neutrophils and mast cells, in infarcted myocardium after MI. This knowledge can assist in the development of future therapeutic strategies, which may benefit MI patients.     

G-MDSCs promote aging-related cardiac fibrosis by activating myofibroblasts and preventing senescence

Aging is one of the most prominent risk factors for heart failure. Myeloid-derived suppressor cells (MDSCs) accumulate in aged tissue and have been confirmed to be associated with various aging-related diseases. However, the role of MDSCs in the aging heart remains unknown. Through RNA-seq and biochemical approaches, we found that granulocytic MDSCs (G-MDSCs) accumulated significantly in the aging heart compared with monocytic MDSCs (M-MDSCs). Therefore, we explored the effects of G-MDSCs on the aging heart. We found that the adoptive transfer of G-MDSCs of aging mice to young hearts resulted in cardiac diastolic dysfunction by inducing cardiac fibrosis, similar to that in aging hearts. S100A8/A9 derived from G-MDSCs induced inflammatory phenotypes and increased the osteopontin (OPN) level in fibroblasts. The upregulation of fibroblast growth factor 2 (FGF2) expression in fibroblasts mediated by G-MDSCs promoted antisenescence and antiapoptotic phenotypes of fibroblasts. SOX9 is the downstream gene of FGF2 and is required for FGF2-mediated and G-MDSC-mediated profibrotic effects. Interestingly, both FGF2 levels and SOX9 levels were upregulated in fibroblasts but not in G-MDSCs and were independent of S100A8/9. Therefore, a novel FGF2-SOX9 signaling axis that regulates fibroblast self-renewal and antiapoptotic phenotypes was identified. Our study revealed the mechanism by which G-MDSCs promote cardiac fibrosis via the secretion of S100A8/A9 and the regulation of FGF2-SOX9 signaling in fibroblasts during aging.Subject terms: Senescence, Cardiovascular diseases