Introduction

Numerous biological mechanisms converge to remodel the heart after myocardial infarction (MI). Adverse postischemic cardiac remodeling and progression to heart failure may be promoted by macroscopic alterations in left ventricular geometry and function, as well as microscopic alterations in the cellular and molecular landscapes of the ischemic heart. Recent evidence implicates both infiltrated and resident immune cells in post-MI remodeling.^1,2 Notably, B lymphocytes have been shown to produce the chemokine CCL7 and promote cardiac inflammatory monocyte recruitment after acute MI, leading to enhanced tissue damage and myocardial dysfunction.³ Likewise, rapid elimination of cardiac B cells along with reduced CCL7 expression prevented pathologic remodeling and heart failure.⁴ Modulation of different subsets of B lymphocytes has also been shown to control tissue remodeling in various experimental models of cardiac disease.^5-7 Large B-cell clusters were identified in epicardial adipose tissue of patients with coronary artery disease and in infarcted mice. B-cell depletion reduced cardiac neutrophil infiltration as well as fibrosis and preserved left ventricular ejection fraction after acute MI.⁸ Conversely, interleukin-10–producing CD5⁺ B cells were expanded in pericardial adipose tissues and activated the resolution of MI-induced inflammation.⁹ Together, these results indicate that B cells control left ventricular remodeling, but the cellular mechanisms governing the effects of distinct B lymphocyte subtypes from diverse tissue origin remain to be defined. This is highly important given the ongoing and planned clinical trials aiming at targeting B cells in patients with acute MI.¹⁰

microRNAs (miRs) are fundamental to the development of inflammatory cells and also capable of regulating almost every leukocyte-related activity. Notably, they play prominent roles in B-cell maturation, and different stages of normal B-cell differentiation are characterized by distinct miR expression profiles.^11,12 Among these miRs, several reports have shown a key role for microRNA-21 (miR21) in inflammatory cells, and increased miR21 expression is coupled with conditions involving altered immune response, including cardiovascular diseases.¹³ In B cells, overexpression of miR21 leads to a pre–B cell malignant lymphoid-like phenotype, suggesting that miR21 may act as an oncogene in B cells.¹⁴

In the current study, we show, for the first time, the involvement of splenic marginal zone B (MZB) cells in the regulation of cardiac function and remodeling after acute MI. We also show that endogenous activation of the miR21/hypoxia-inducible factor-1α (HIF-1α)–related pathway is an integral component of the splenic MZB cell–dependent damaging effect on post-ischemic cardiac remodeling.

Methods

Experimental procedure

All experiments were conducted according to the ethical committee for animal experimentation (University of Paris, CEEA 34) and the National Charter on the ethics of animal experimentation from the French Minister of Higher Education and Research under the reference MESR no. 01373.01. Acute MI was induced in mice by permanent ligation of the left anterior descending coronary artery. Cardiac function and remodeling were analyzed by using echocardiography and immunohistochemistry.

For evaluation of the role of HIF-1α and HIF-2α in B cells, Cd79a^cre/+ mice were crossed with Hif1a^flox/flox and/or Hif2a^flox/flox animals to generate Cd79a^cre/+/Hif1a ^flox/flox (referred to as the Hif1a^–/– B-cell group); Cd79a^cre/+/Hif2a^flox/flox (referred to as the Hif2a^–/– B-cell group); Cd79a^cre/+/Hif1a-Hif2a ^flox/flox (referred to as the Hif1a-2a^–/– B-cell group) mice and their littermates Cd79a^+/+/Hif1a ^flox/flox, Cd79a^+/+/Hif2a ^flox/flox or Cd79a^+/+/Hif1a- Hif2a ^flox/flox (referred to as the wild-type [WT] B-cell groups). For evaluation of the role of MZB cells, Cd79a^cre/+ animals were crossed with Rbpjk ^flox/flox mice to generate Cd79a^cre/+/Rbpjk ^flox/flox (referred to as the MZB^Δ group lacking MZB cells) and Cd79a^+/+/Rbpjk^flox/flox (referred to as the MZB^WT group) animals.

Statistical analysis

Kruskal-Wallis one-way analysis of variance was used to compare 3 or more independent experimental groups. Comparisons between groups were then performed by using Dunn’s multiple comparisons test when the analysis of variance test was statistically significant. The Mann-Whitney U test was used to compare 2 groups. A P value <0.05 was considered significant. Results are expressed as means with SDs or as boxes and whiskers with maximum and minimum values. P values presented in this report have not been adjusted for multiplicity, and therefore inferences drawn from these statistics may not be reproducible.

A detailed description of all experimental procedures is provided in the Supplemental Methods.

Results

Role of B cell–specific miR21 in the response to acute MI

Because mature B cells mainly reside in the spleen, we first assessed miR21 expression in the spleen of WT mice, which underwent sham operation or were subjected to a permanent left anterior descending coronary artery ligation to induce acute MI, at different time points after the onset of ischemia. We found that miR21 was up-regulated at day 1 after the ischemic insult in the spleen, compared with sham-operated animals (Figure 1A). Using fluorescence-activated cell sorting analysis, we then determined miR21 levels in distinct splenic inflammatory cells at day 1 after MI and showed that miR21 was strongly expressed in B cells compared with other types of splenic inflammatory cells, including neutrophils, T lymphocytes, monocytes, and macrophages.

Splenic B cells are known to emerge from B-cell progenitors within the bone marrow (BM) and subsequently undergo final maturation in the spleen.¹⁵ To assess the role of miR21 in mediating B cell–dependent effects on post-MI cardiac remodeling, we generated WT chimeras through lethal irradiation and reconstitution with BM cells isolated from WT or miR21-deficient animals. Transplantation of miR21-deficient BM did not affect the number of splenic B cells (Figure 1B) but reduced miR21 levels in splenic B cells compared with mice transplanted with WT BM (Figure 1C). Left ventricular ejection fraction was similar in both un-operated and sham-operated chimeric animals (Supplemental Figure 1A). After induction of acute MI, cardiac function was higher in WT mice reconstituted with miR21^–/– BM cells compared with WT chimeras (Figure 1D). Interstitial fibrosis was 1.7-fold lower in WT mice reconstituted with miR21^–/– BM compared with those receiving WT BM (Figure 1E). Infarct size and capillary density were unaffected.

We also substantiated the role of B cell–specific miR21 in re-supplementation experiments using B-cell transplantation in immunodeficient animals (Supplemental Results, Supplemental Figures 1B to 1D). Altogether, these results indicate that miR21 controls the B cell–dependent impact on post-ischemic cardiac remodeling.

B cell–specific miR21 affects the inflammatory response post-MI

Toll-like receptor (TLR) activation has been shown to trigger CCL7 secretion by B cells and governs BM-derived monocyte mobilization and infiltration in the ischemic milieu.^3,4 Increased miR21 expression is also associated with TLR activation.¹⁶ Treatment with a TLR agonist, cytosine-phosphate-guanine (CpG), for 24 hours led to a 1.8-fold increase of miR21 expression in cultured splenic B cells (Figure 1F). CpG stimulation was also associated with activation of CCL7 production by cultured WT B cells, and this effect was blunted in cultured B cells isolated from miR21^–/– mice (Figure 1G). CCL7 circulating levels were reduced in chimeric miR21^–/– animals at day 3 after MI (Figure 1H).

We then evaluated the impact of CCL7 alteration on the number of monocytes.^3,17 Within the population of CD45⁺CD11b⁺Ly6G^– cells, classical inflammatory monocytes were characterized as Ly6C^High cells (Supplemental Figure 2). miR21 deficiency was associated with a reduction in both circulating and cardiac Ly6C^High monocytes (Figure 1I). Re-supplementation experiments using WT and miR21-deficient B cells also revealed variation of CCL7 and inflammatory monocyte levels (Supplemental Results, Supplemental Figures 1E and 1F). Thus, miR21 expression fosters CCL7 production by activated B cells, which promotes mobilization and infiltration of Ly6C^High monocytes leading to adverse ventricular remodeling and cardiac dysfunction.

miR21 controls HIF-1α levels in B cells

miR21 has been shown to enhance HIF-1α signaling.^18-20 HIF-1α is a master transcription factor that controls B-cell function.^21-23 Interestingly, aberrant regulation of HIF-1α by miRNAs is a hallmark of chronic lymphocytic leukemia B cells.²⁴ HIF-1α also drives B cell–related effects in exacerbated collagen-induced arthritis and experimental autoimmune encephalomyelitis.²⁵ We then speculated that miR21-induced regulation of HIF-1α controlled B cell–related effects on post ischemic cardiac homeostasis. For this purpose, HIF-1α levels were examined in splenic B cells isolated from WT and miR21^–/– mice and stimulated with CpG or anti–immunoglobulin M (IgM). Up-regulation of HIF-1α protein content was blunted in activated miR21^–/– B cells compared with activated WT B cells (Figure 2A).

HIF-1α mediates B cell–related effects in mice with acute MI

To evaluate the role of HIF-1α in B cells, we generated mice with a B-cell lineage–specific conditional deletion of HIF-1α (Hif1a^–/– B cells). HIF-1α deficiency did not affect cardiac function in un-operated and sham-operated animals (Supplemental Figure 3A). After MI, mice with Hif1a^–/– B cells exhibited improved cardiac function compared with WT animals (Figure 2B). Cardiac function recovery was associated with a reduction in both infarct size and interstitial fibrosis, whereas capillary density was unaffected. HIF-1α invalidation did not reduce the total B-cell number in the spleen after MI (Figure 3A). CCL7 levels were decreased in the blood of mice with Hif1a^–/– B cells (Figure 3B). CpG treatment increased HIF-1α transcriptional activity (Figure 3C) as well as CCL7 messenger ribonucleic acid (mRNA) and protein levels (Figure 3D) in WT cultured B cells. Of note, CCL7 up-regulation was abrogated in cultured Hif1a^–/– B cells treated with CpG. As a consequence, the number of Ly6C^High monocytes was lowered in the blood and the infarcted heart (Figure 3E). Similarly, cardiac macrophage accumulation (gating strategy is shown in Supplemental Figure 2), as well as the expression of classical pro-inflammatory cytokines such as interleukin-1ß and interleukin-6, were reduced in mice with Hif1a^–/– B cells compared with WT animals, at day 7 after MI (Figure 3F).

HIF-2α is not involved in B cell–related effects post-MI

In mammalian cells, HIF-α activity is mainly regulated by 2 α subunits, HIF-1α and HIF-2α. Although HIF-1α and HIF-2α are paralogs and share extensive sequence homology, these two α subunits also have nonoverlapping and sometimes even opposing roles.²⁶ Furthermore, HIF-1α deficiency has been shown to increase levels of HIF-2α, and mutual antagonism between HIF-α isoforms can shape HIF-α activity.²⁷ We found that Hif2a mRNA levels were enhanced in miR21^–/– and Hif1a^–/– splenic B cells, 1 day after the onset of ischemia (Supplemental Figure 3B). Cardiac function and post-MI remodeling were similar in mice with Hif2a^–/– B cells and their WT littermates as well as in un-operated and sham-operated animals (Supplemental Figures 3C and 3D). On the same note, Hif2a deletion did not affect B-cell numbers in the spleen, did not modulate blood CCL7 levels, and did not alter circulating and cardiac Ly6C^High monocyte number (Supplemental Figures 3E to 3G). In contrast, whereas cardiac function was similar in un-operated and sham-operated animals (Supplemental Figure 3H), Hif1a and Hif2a deletion improved cardiac function after MI (Figure 4A). Cardiac function recovery was associated with a reduction in infarct size, whereas interstitial fibrosis and capillary densities were unaffected. Hif1a and Hif2a deficiency also reduced Ly6C^High monocytes and CCL7 levels in the blood along with a reduction of cardiac macrophage number and Il1ß and Il6 mRNA levels (Figures 4B-4E). Altogether, these results indicate that HIF-2α does not control B cell–related functions and that the HIF-1α isoform plays a prominent role in the orchestration of B cell–induced deleterious effects on post-MI cardiac remodeling.

MZB cells are instrumental in the regulation of cardiac function and remodeling post-MI

Several subsets of B cells exist. They are broadly divided into innate-like B1 cells and B2 cells, which comprise MZB and follicular B (FOB) cells with specific developmental pathways and functional properties.¹⁵ Within the population of IgM⁺CD19⁺ B cells, MZB cells were identified as CD23^low/−CD21^high, and FOB cells as CD23^highCD21^low/− (Supplemental Figure 2). MZB cells displayed higher expression of miR21 compared with FOB cells, and miR21 expression was markedly up-regulated in the MZB cells of infarcted animals compared with sham-operated mice (Figure 5A). To determine the specific role of MZB cells, we then used mice with MZB cell deficiency (MZB^Δ).²⁸ As expected, MZB cell numbers were markedly decreased in the spleen of MZB^Δ mice (Figure 5B). Interestingly, MZB cell deficiency curbed MI-induced miR21 up-regulation in splenic B cells (Figure 5C). Cardiac function was similar in un-operated and sham-operated MZB^Δ and MZB^WT animals (Supplemental Figure 3I). The selective depletion of MZB cells improved post-MI cardiac function and the remodeling process (Figures 5D to 5G), suggesting that MZB cells expressing miR21 played a significant role in the regulation of cardiac function and repair.

On the same note, CpG stimulation enhanced CCL7 release in cultured MZB^WT cells but not in MZB^Δ cells (Figure 6A). MZB cell–deficient mice also displayed altered circulating levels of CCL7 (Figure 6B) and reduced circulating and cardiac Ly6C^High monocytes compared with their WT littermates (Figure 6C). As a consequence, at day 7 after MI, the number of cardiac macrophages, along with Il1b and Il6 mRNA levels, were decreased in the infarcted hearts of MZB^Δ mice compared with MZB^WT animals (Figure 6D). In an additional set of experiments, WT and MZB cell–deficient animals were challenged with a sequence of ischemia-reperfusion for 40 minutes. MZB cell deficiency also improved cardiac function and reduced interstitial fibrosis in this experimental setting (Supplemental Figure 4).

miR21/HIF-1α signaling controls MZB cell–related effects in mice with acute MI

We then assessed the role of miR21/HIF-1α signaling selectively in MZB cells. CpG- and IgM- induced HIF-1α up-regulation was abrogated in B cells isolated from MZB^Δ mice compared with those isolated from MZB^WT animals (Figure 7A). To substantiate the role of miR21 in HIF-1α regulation within MZB cells, we cultured FOB and MZB cells from WT and miR21-deficient mice and analyzed HIF-1α protein levels (Figure 7B). We showed that miR21 deficiency hampered CpG- and IgM-induced up-regulation of HIF-1α protein levels and CCL7 release in MZB but not in FOB cultured cells.

Finally, we generated chimeric mice in which MZB cell–deficient mice have been partially irradiated and reconstituted with BM cells isolated from MZB^Δ, MZB^WT, miR21^–/–, or Hif1a^–/– animals. Reconstitution restored the number of MZB cells in chimeric animals (Figure 7C) without affecting cardiac function in un-operated and sham-operated animals (Supplemental Figure 5A). Chimeric mice transplanted with MZB miR21^–/– cells displayed reduction of miR21 content in MZB cells (Supplemental Figure 5B), whereas chimeric mice transplanted with MZB Hif1a^–/– cells exhibited decreased Hif1a mRNA levels in MZB cells (Supplemental Figures 5B and 5C). miR21 and Hif1a mRNA levels were unaffected in FOB cells in chimeric mice. Of great interest, transplantation with WT, miR21-deficient MZB, or Hif1a-deficient MZB cells reduced circulating CCL7 levels and improved cardiac function compared with chimeric MZB^Δ animals (Figures 7C and 7D). These effects were associated with a reduction in infarct size and (Figure 7D), interstitial fibrosis, and cardiac macrophage as well as Il1ß and Il6 mRNA contents (Supplemental Figures 5D to 5F).

Thus, miR21 expression controls HIF-1α content in activated MZB cells, and MZB cells govern inflammation-dependent ventricular remodeling and cardiac function (Central Illustration).

Discussion

In the current work, we found that deletion of MZB cells or selective miR21 deficiency in MZB cells abrogated B cell–induced adverse ventricular remodeling after MI, indicating a determinant role for miR21-expressing MZB cells in this context. Although previously unsuspected, this new role of MZB cells in response to acute ischemic injury is consistent with their innate-like properties and their strategic positioning at the blood–lymphoid interface, where they can sense changes in the systemic environment and mount immediate innate immune responses. miR21 up-regulation was correlated with activation of HIF-1α–related signaling, and conditional deletion of HIF-1α counteracted MZB cell–induced adverse cardiac remodeling and dysfunction. In mature B cells, persistent (re)-induction of HIF transcription factors limits proliferation, isotype switching, and levels of high-affinity antibodies in splenic germinal centers after immunization.²⁹

It is unlikely that those adaptive B-cell functions were involved in our experimental conditions very shortly after ischemic injury. However, one can speculate that dying cells and matrix fragments within the ischemic infarcted milieu may produce specific signals, including cytokines or damage-associated molecular patterns, and activate innate-like MZB cells. In this line of reasoning, TLR activation is sufficient to activate the miR21/HIF-1α pathway in cultured MZB cells. Nevertheless, further investigations are needed to elucidate how B cells are able to recognize these specific biological entities originating from the injured myocardium and how the ischemic milieu commands B-cell effector functions.

Our work also identifies a determinant role for miR21 and HIF-1α in the regulation of CCL7 production by MZB cells. CCL7 directs Ly6C^High monocyte mobilization from the BM and recruitment to inflammatory cardiac tissue.^3,30 Ly6C^High pro-inflammatory monocytes are responsible for the scavenging of debris and secretion of pro-inflammatory cytokines and matrix-degrading proteases, which contribute to the myocardial injury.^3,17 Of note, bioinformatics analysis and chromatin immunoprecipitation assays revealed 3 functional hypoxia-response elements in the Ccl7 promoter, indicating that Ccl7 is a direct HIF-1α target gene.³¹ However, additional work will be required to thoroughly address the mechanisms through which miR21 and HIF-1α regulate CCL7 production.

As B cells have emerged as important players in atherogenesis and post-MI cardiac remodeling, our novel results add to a number of experimental and clinical evidence suggesting that B cells represent a promising therapeutic target for cardiovascular diseases. Several B cell–directed therapies are already available such as rituximab, an anti-CD20 B cell–depleting monoclonal antibody. Small-scale human studies have previously reported some beneficial effects on the cardiovascular system, such as variable degrees of reduction in carotid intima-media thickness, improved flow-mediated dilation, or decreased arterial thickness in rituximab-treated patients.³² We recently conducted a Phase I/II clinical trial investigating the impact of rituximab in patients with acute MI and found that infusion of rituximab within 24 hours of MI was safe and efficiently depleted circulating B cells.¹⁰

The detrimental role of MZB cells at the acute phase of MI shown in the current study contrasts with their previously reported atheroprotective properties through modulation of T follicular helper cell functions.²⁸ What, therefore, are the clinical implications of these opposing properties? We propose that a therapeutic strategy that depletes MZB cells needs to be both selective and limited to the acute phase after MI, allowing for MZB cell recovery after the acute phase and reducing any potential harm, which can result from a loss of their atheroprotective properties. The B cell–depleting strategy currently being tested at the acute phase of MI in humans uses one single infusion of rituximab.¹⁰ Rituximab depletes both FOB and MZB cells. It is interesting to note, however, that transitional B cells with innate-like immune properties seem to recover after a few months of a single low dose of rituximab,¹⁰ suggesting that a low dose may limit the loss of potentially atheroprotective B cells. In addition, selective targeting of miR21 or HIF-1α signaling in MZB cells, which would probably not affect the atheroprotective properties of MZB cells, would be another potential therapeutic strategy in patients with acute MI.

Study limitations

This study did not attempt to identify the specific cardiac-derived signals that could activate miR21/HIF-1α signaling in MZB cells. It is also likely that miR21 and HIF-1α control additional targets that could modulate splenic, circulating, or cardiac B cell–related effects.

Conclusions

This work reveals a prominent role for the miR21/HIF-1α axis in guiding MZB cell function in response to MI through activation of CCL7-dependent mobilization and infiltration of pro-inflammatory monocytes into the ischemic myocardium.

Footnotes