The healing myocardium mobilizes a distinct B-cell subset through a CXCL13-CXCR5-dependent mechanism

Abstract

Aims

Recent studies have revealed that B cells and antibodies can influence inflammation and remodelling following a myocardial infarction (MI) and culminating in heart failure—but the mechanisms underlying these observations remain elusive. We therefore conducted in mice a deep phenotyping of the post-MI B-cell responses in infarcted hearts and mediastinal lymph nodes, which drain the myocardium. Thereby, we sought to dissect the mechanisms controlling B-cell mobilization and activity in situ.

Methods and results

Histological, flow cytometry, and single-cell RNA-sequencing (scRNA-seq) analyses revealed a rapid accumulation of diverse B-cell subsets in infarcted murine hearts, paralleled by mild clonal expansion of germinal centre B cells in the mediastinal lymph nodes. The repertoire of cardiac B cells was largely polyclonal and showed no sign of antigen-driven clonal expansion. Instead, it included a distinct subset exclusively found in the heart, herein termed ‘heart-associated B cells’ (hB) that expressed high levels of Cd69 as an activation marker, C-C-chemokine receptor type 7 (Ccr7), CXC-chemokine receptor type 5 (Cxcr5), and transforming growth factor beta 1 (Tgfb1). This distinct signature was not shared with any other cell population in the healing myocardium. Moreover, we detected a myocardial gradient of CXC-motif chemokine ligand 13 (CXCL13, the ligand of CXCR5) on Days 1 and 5 post-MI. When compared with wild-type controls, mice treated with a neutralizing CXCL13-specific antibody as well as CXCR5-deficient mice showed reduced post-MI infiltration of B cells and reduced local Tgfb1 expression but no differences in contractile function nor myocardial morphology were observed between groups.

Conclusion

Our study reveals that polyclonal B cells showing no sign of antigen-specificity readily infiltrate the heart after MI via the CXCL13-CXCR5 axis and contribute to local TGF-ß1 production. The local B-cell responses are paralleled by mild antigen-driven germinal centre reactions in the mediastinal lymph nodes that might ultimately lead to the production of specific antibodies.

Myocardial infarction, B cells, Cardiac remodelling, Heart failure and immune cells

1. Introduction

Myocardial infarction (MI) is still the leading cause of death worldwide. In Europe, it accounts for ∼20% of all-cause mortality.¹ Due to timely and widely available acute treatment, such as catheter interventions performed according to standardized algorithms in medical centres, the mortality rates related to ischaemic heart disease and acute heart failure (HF) have steadily decreased over the past three decades.² In sharp contrast, patients surviving acute MI often develop chronic HF with significant rates of morbidity and mortality due to poor myocardial healing capacity³ and because less is known about optimal, long-term treatment of post-MI patients. Therefore, it is crucial to better understand and control post-MI tissue repair processes in order to eventually improve therapies and long-term survival.

Immune-inflammatory mechanisms critically impact post-MI and HF outcomes.⁴ Myeloid cells account for the largest fraction of leucocytes infiltrating the infarcted myocardium, and their activity in situ has been scrutinized by many groups over the past decade.^5,6 In addition to acute myeloid cell responses, MI also elicits lymphocyte responses that can impact healing outcomes.^7–10

Previous studies performed using animal models of MI have revealed that Blymphocytes infiltrate the infarcted myocardium as early as Day 1 and persist until the healing phase (Day 7).⁵ Zouggari et al.⁸ reported that depleting B cells using a CD20-specific antibody attenuated post-MI inflammation and improved cardiac functional outcome in mice. More recently, Adamo et al.¹¹ further showed that targeting B cells also had beneficial effects in experimental models of ischaemia–reperfusion and toxin-induced myocardial injury. The mechanisms by which B cells influence myocardial function are not fully elucidated though. It has been reported that B cells secrete chemokines recruiting monocytes⁸ and produce pathogenic antibodies that aggravate adverse remodelling. In a recent study of experimental MI, we observed that transgenic mice, harbouring B cells that are unable to secrete immunoglobulins, show attenuated cardiac remodelling and better preservation of cardiac function in chronic HF.¹²

The B-cell compartment comprises a heterogeneous population of cells exhibiting considerable phenotypic plasticity. In the context of MI, it was shown that mature follicular B cells activated after MI secrete chemokine C-C motif ligand 7 (CCL7), thereby promote the recruitment of pro-inflammatory monocytes, hence contributing to excessive inflammation.⁸ However, protective CD5⁺B1 B cells that secrete high levels of IL-10 were also found enriched in murine pericardial adipose tissues.¹³

In light of this remarkable phenotypic plasticity, a more refined characterization of post-MI B-cell responses is necessary. It remains unclear whether B cells infiltrating the injured myocardium target specific antigens (e.g. myosin), as observed for cardiac T cells,^14,15 or if they exert more innate-immune functions. Moreover, the chemokine ligand-receptor axis mediating the mobilization of B cells into the injured heart has not yet been revealed, in marked contrast to other leucocyte subsets.^6,16 Thus, in the present study, we sought to characterize post-MI B-cell responses by dissecting the mechanisms underlying their mobilization into the myocardium and to assess their antigen specificity profile.

2. Methods

2.1 Animals and study approval

Male C57BL/6 mice aged 7–8 weeks were purchased from Charles River (Sulzfeld, Deutschland) and housed in specific pathogen-free conditions with a controlled light-dark cycle for at least 1 week prior to experimentation. CXCR5^−/− mice were purchased from the Jackson Laboratory and housed under the same conditions. All animal procedures were approved by the local authorities (Regierung von Unterfranken) and conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.

2.2 Experimental MI

Experimental MI was induced by permanent ligation of the left coronary descending artery (LAD) using the guidelines for experimental models of MI.¹⁷ In preparation for surgery, animals were anaesthetized by isoflurane inhalation (initially 4%, then maintenance dosing at 2.0–2.5% v/v O₂) and intubated for mechanical ventilation. Next, mice were submitted to a left thoracotomy and the LAD was permanently ligated (MI group). In sham-operated animals, a similar procedure was conducted with the exception of LAD ligation. At the end of the surgical procedure, the isoflurane concentration was reduced to 0.5% (v/v O₂) and the animals were extubated as soon as spontaneous respiration reoccurred. Body temperature was controlled during the operation, and mice received pre- and post-operative pain management for 3 days [buprenorphine (0.1 mg/kg body weight subcutaneously, every 12 h)]. An experienced experimenter performed all surgeries and the health status of the animals was monitored daily until the endpoint analyses.

2.3 Organ harvesting and storage

Infarcted and sham-operated animals were sacrificed by cervical dislocation at the time points indicated, ranging from Day 1 to 14 after MI. Upon euthanasia, mice were perfused with PBS-heparin (50 IU/mL) to flush the coronary circulation and remove blood–borne leucocytes, as previously described.¹⁸ Afterwards, hearts and mediastinal lymph nodes were rapidly excised and processed according to the intended downstream application. Heart samples were first dissected to separate the injured left ventricle from the remote, i.e. healthy, myocardium by visual examination. Samples designated for quantitative PCR were subsequently stored in RNAlater^® (Invitrogen, Carlsbad, USA) at 4°C overnight and finally kept at −80°C for further processing (detailed below). Heart samples for protein analyses were similarly dissected, snap-frozen in liquid nitrogen, and then kept at −80° C for further processing. In experiments designed for single-cell sequencing, mice were also injected intravenously with an anti-CD45.2 antibody (4 µg per mouse, clone 104) 3 min prior to euthanasia, to further eliminate potentially contaminating circulating leucocytes, as previously established.¹⁹ The rationale of this approach is that cells in contact with the vasculature stain positive for antibodies injected i.v., in contrast to the bona fide parenchymal cells that remain unlabelled until post-digestion staining. For flow cytometry experiments, hearts and lymph nodes as well as spleen and liver samples were harvested in cold Hanks’ balanced salt solution supplemented with 0.5% BSA and processed immediately as detailed below.

2.4 Flow cytometry and cell sorting

Heart samples were enzymatically digested in type II collagenase (1000 IU/ml, Worthington Biochemical Corporation, Lakewood, NJ, USA) for 30 min at 37°C. Digested hearts and mediastinal lymph nodes were gently filtered through a 30 µm mesh (Miltenyi Biotec, Bergisch Gladbach, Germany). Following a wash step, all samples were resuspended in flow cytometry buffer (PBS containing 1% BSA, 0.1% sodium azide, and 1 mM EDTA), and surface staining was performed in the presence of an Fc-blocking antibody (anti-CD16/CD32, clone 2.4G2, BD Pharmingen). The following fluorophore-conjugated antibodies [obtained from Biolegend (San Diego, CA, USA) or eBioscience/Thermo Fisher Scientific (Waltham, MA, USA), unless otherwise indicated] were used for surface staining: anti-CD45 (clone 30-F11), anti-CD45.2 (clone 104), anti-CD19 (clone 6D5), anti-CD45/B220 (clone RA3-6B2), anti-CXCR5 (clone L138D7), anti-CCR7 (clone 4B12), anti-CD69 (clone H1.2F3), anti-Ly6G (clone 1A8), anti-CD11b (clone M1/70), anti-CD95 (clone SA367H8), and anti-GL7 (clone GL7). Additionally, AmCyan Zombie Aqua fixable viability dye (Biolegend, San Diego, CA, USA) was used to exclude dead cells. Flow cytometry measurements were performed using either an Attune-NxT (Thermo Fisher Scientific, Waltham, MA, USA) or a FACS Canto (BD, Heidelberg, Germany) instrument. Cell sorting for single-cell RNA-sequencing was performed using a FACS Aria III instrument (BD, Heidelberg, Germany). Sodium azide was omitted in the buffers used for sorting purposes. After sorting, the cells were washed once in PBS containing 1% fetal calf serum, and each sample was labelled separately with an anti-CD45/H2-K hashtag antibody (Biolegend TotalSeq-C antibodies, San Diego, CA, USA, 1:150 dilution). Following a washing step, all samples were pooled and suspended in 100 µl of PBS/0.04% BSA, filtered through a 40 µm cell strainer, and loaded in the 10x Genomics Chromium system as one multiplexed sample.

2.5 Single-cell RNA-sequencing

Chromium™ Controller was used for partitioning single cells into nanolitre-scale Gel Bead-In-EMulsions (GEMs) and Chromium Next GEM Single Cell 5’ v1.1 kits for reverse transcription, cDNA amplification, and library construction (10x Genomics, Pleasanton, CA, USA), following manufacturer’s specifications. A SimpliAmp Thermal Cycler was used for amplification and incubation steps (Applied Biosystems, Foster City, CA, USA). Libraries were quantified by a Qubit^TM 3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and quality was checked using a 2100 Bioanalyser with High Sensitivity DNA kit (Agilent, Santa Clara, CA, USA). Libraries were pooled and sequenced using the NovaSeq 6000 platform (S2 Cartridge, Illumina, San Diego, CA, USA) in paired-end mode to reach at least 70 000 reads per single-cell for gene expression and 7000 reads for the B-cell receptor repertoire and hashtags. The Cell Ranger Software Suite (10x Genomics, Pleasanton, CA, USA) was used for sequence alignment, barcode processing, and sample demultiplexing. The Seurat R package v3.1.4²⁰ was used for filtering out low-quality cell data, normalizing gene expression, and for clustering. For B-cell receptor repertoire analysis, the R package scRepertoire (https://f1000research.com/articles/9-47/v1) was used. A more detailed explanation is found in the Supplementary material online.

2.6 Quantitative PCR

Myocardial RNA was extracted using the Qiagen RNeasy Mini kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The final RNA content was eluted in nuclease-free water, and its concentration and quality were determined based on the absorption curves at 260/280 nm and 260/230 nm (NanoDrop 2000c spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA was synthesized from 1 µg RNA using an iScript kit (Bio-Rad Laboratories, Munich, Germany), and the relative expression of chemokine genes of interest was monitored using TaqMan probes. The following TaqMan probes were used in this study: Cxcl13 (Mm00444534_m1), Ccl19 (Mm00839967_g1), Ccl21 (Mm03646971_gH), Il6 (Mm00446190_m1), Il1b (Mm0043228_m1), Tnf (Mm99999068_m1), and Tgfb1 (Mm01178820_m1) and the gene expression levels were normalized to those of the housekeeping gene Gapdh (Mm99999915_g1) using the delta-delta ΔΔC_t method (where C_t is the cycle threshold).

2.7 Protein extraction and ELISA

To extract proteins from heart tissue, samples were homogenized in ice-cold 0.9% NaCl (B. Braun Melsungen AG, Melsungen, Germany) supplemented with 10 µL protease inhibitor and 10 µL phosphatase inhibitor (Halt Protease Inhibitor Cocktail and Halt Phosphatase Inhibitor Cocktail, ThermoFisher Scientific, Waltham, MA, USA). Then, samples were centrifuged at 16 110 g, and the protein content in the cleared supernatant was monitored based on its absorbance at 280 nm (NanoDrop 2000c spectrophotometer, Thermo Scientific, Waltham, USA). CXCL13 content was subsequently assessed in the scar tissue and remote myocardium extracts using a commercial ELISA kit (Quantikine ELISA Kit by R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions. The ELISA reaction was monitored with a Dynex MRX^e tc microplate reader (Magellan Biosciences, North Billerica, USA).

2.8 Histology, immunofluorescence, and light-sheet fluorescence microscopy

B-cell numbers in the myocardium were monitored by immunofluorescence microscopy to simultaneously assess B-cell infiltration into the infarct zone and the remote myocardium. Seven micrometre thick heart slices were prepared with a cryotome (Leica CM 1850, Leica Microsystems, Wetzlar, Germany), dried and then stored at −20°C. To prepare the sections for staining, they were fixed in ice-cold acetone (−20°C). Afterwards, the staining of specific antigens was performed according to standard protocols established in our lab.¹⁴ B cells were detected using a B220-specific antibody (RA3-6B2) conjugated with Alexa647. Moreover, we stained F-actin with phalloidin-Alexa488 to distinguish in the left ventricle the vital myocardium (remote) from the infarct area (scar).²¹ Images were acquired using a fluorescence microscope (Axio Imager.Z1m., Carl Zeiss, Jena, Germany) at 20× magnification. Subsequently, picture files were processed using Zen lite software (Zeiss, Jena, Germany).

Light-sheet fluorescence microscopy of cardiac samples was performed as previously described.¹⁴ In brief, the perfused hearts were fixed in 4% (w/v) formaldehyde diluted in PBS and then bleached in 15% (w/v) hydrogen peroxide diluted in methanol. The hearts were then incubated with anti-CXCR5-Alexa647 (clone L138D7, Biolegend, San Diego, CA, USA) for 7 days. After a washing step, the samples were dehydrated in increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 99%) and then cleared in 1:3 benzyl alcohol/benzyl acetate. Sequential multicolour stacks (5 µm interval between images, up to 1 mm in depth) were acquired using a 5× objective lens, and the data were analysed using Fiji ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA).

2.9 CXCL13 neutralization

To functionally assess CXCL13-mediated chemoattraction, mice were administered intraperitoneally with 200 µg of a neutralising CXCL13-specific antibody (monoclonal Rat IgG2A, clone: 143614, R&D Systems, Minneapolis, USA) 3 h prior to LAD ligation, as previously described.²² As a control, animals received a nonspecific, monoclonal rat isotype control IgG2A (clone 54447, R&D Systems, Minneapolis, USA).

2.10 Echocardiography

Mouse echocardiography was performed using a Vevo 1100 (VisualSonics, Amsterdam, Netherlands) system equipped with a 30 MHz probe on Day 28 post-operation. Mice were kept under slight isoflurane inhalation anaesthesia (0.5–1.5% volume/volume O₂), and short-axis images were acquired at the mid-papillary and apical levels of the left ventricle (B- and M-mode) according to the guidelines for measuring cardiac physiology in mice.²³ Only animals with a basal heart rate >450 b.p.m. were included in the analysis.

2.11 Statistical analyses

Unless otherwise stated, the results are given as the mean ± the standard error of the mean (SEM) along with the distribution of all individual values in each group. The sample size for each group is described in each graph legend. The statistical analyses were performed with GraphPad Prism (version 7.0 or 9.0.0, GraphPad Software, San Diego, CA, USA). Unless otherwise stated, the following tests were used in this study: for comparisons between two groups of data following a normal distribution, we used an unpaired two-tailed t-test. For multiple comparisons between more than two groups, one- or two-way analyses of variance (ANOVA) were conducted followed by a post hoc test always specified in each graph legend. Differences were considered significant when P < 0.05 (*). The B-cell receptor sequencing data were statistically analysed using R.

3. Results

3.1 MI triggers complementary B-cell responses in the heart and in the mediastinal lymph nodes

Previous studies have already reported the recruitment of B-lymphocytes into the infarcted myocardium but did not shed light on the spatial distribution of the cells within the injured myocardium.^5,8 To further assess the localization of infiltrating B cells, we histologically analyzed heart sections obtained from MI- and sham-operated mice that were co-stained to detect B cells (CD45⁺B220⁺) and F-actin (phalloidin). The F-actin⁺ regions correspond to the intact remote myocardium, whereas the F-actin⁻ regions identify the site of MI. B-cell numbers were counted in relation to area (in mm²). As shown in Figure 1A, the planimetric evaluation revealed that B cells infiltrated the infarcted myocardium already on Day 1 post-MI (inflammatory phase) and preferentially localized within the F-actin⁻ scar area. Flow cytometry analyses provided further details about the temporal dynamics of myocardial B-cell (CD19/B220⁺) infiltration and revealed a peak response at Day 7 post-MI (Figure 1B).

Figure 1

MI triggers complementary B-cell responses in the heart and in the mediastinal lymph nodes. (A) Representative micrograph (20× magnification) showing B cells (B220⁺DAPI⁺) infiltrating the infarcted myocardium (Day 7 post-MI). Viable myocardium was defined based on phalloidin staining: F-actin⁺ areas correspond to remote tissue, whereas infarcted zones are defined as F-actin⁻ (scale bar: 100 µm). The kinetics of B-cell infiltration in the remote and infarcted myocardium and in sham-operated controls were assessed by planimetry during the post-MI inflammatory (Day 1–3) and healing (Day 7–14) phases. Statistical test: Two-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05 and ****P < 0.0001 when comparing scar tissue to the remote myocardium, whereas §§§§P < 0.0001 when comparing scar tissue to the sham group. (B) The number of B cells in scar tissue was also investigated by flow cytometry at different time points post-MI. The bar graphs indicate the group mean values ± SEM per group (n = 4–5), and the distribution of each individual value. Statistical test: one-way ANOVA followed by Dunnett’s multiple comparison test. *P < 0.05. (C) Flow cytometry gating strategy and absolute numbers of germinal centre (GC) B cells (defined as CD19/B220⁺GL7⁺CD95⁺) found in the mediastinal lymph nodes (med-LN) 5-day post-MI. (D) Flow cytometry gating strategy and absolute numbers of class-switched IgG⁺ B cells (defined as CD19/B220⁺IgG⁺IgM⁻) found in the mediastinal lymph nodes (med-LN) 5-day post-MI. The bar graphs indicate the group mean values ± SEM per group (n = 4), and the distribution of each individual value. Statistical test: unpaired t-test. *P < 0.05.

In parallel to the rapid mobilization of B cells into the infarcted myocardium, we also observed a mild increase in the numbers of germinal centre B cells in the mediastinal (heart-draining) lymph nodes (defined as CD19/B220⁺CD95⁺GL7⁺), and in the IgG⁺-switched B cells (defined as CD19/B220⁺IgG⁺IgM⁻) on Day 5 post-MI (Figure 1C and D). Taken together, these findings indicate that MI triggers dual and simultaneous B-cell responses in the scar tissue and in the draining lymph nodes.

3.2 Single-cell RNA-sequencing reveals a broad phenotypic diversity of B cells infiltrating the infarcted heart

To assess in greater depth how MI impacts the phenotypes of B cells in both sites, we performed single-cell RNA and B-cell receptor sequencing of B cells purified from the heart and the mediastinal lymph nodes at Day 5 after MI (defined as live, singlet, CD45⁺CD19/B220⁺). In addition, we also purified germinal centre B cells sorted from the mediastinal lymph nodes of infarcted animals to enrich for this rare population in the downstream analyses. Circulating cells were also excluded by intravascular anti-CD45 staining performed shortly before euthanasia. All sorted cell subsets and groups were multiplexed using barcoded hashtag antibodies (anti-CD45/-MHC-I Total-seq-C) and then pooled and sequenced in a single library preparation to avoid batch effects²⁴ (Figure 2A). We opted to focus our analyses on Day 5 after MI based on the time-course of myocardial B-cell infiltration described in previous studies^5,8 and because it marks the transition between the inflammatory and healing phases of post-MI responses, in which important regulatory mechanisms take place. As shown in Figure 2B and C, our analysis yielded nine clusters of cardiac and mediastinal lymph node B cells, including the canonical subsets of B1 B cells (expressing Fcrl5, Cd9, Itgb1, Cd5^variable) and B2 B cells comprising follicular B cells (Fo1 and Fo2 expressing Ighm, Ighd, Sell, and Fcer2), marginal zone B cells (expressing Cr2, Ebf1, Pax5), amongst others (Figure 2, Supplementary material online, Figure S1 and Table SI).²⁵ All these clusters were found expanded in infarcted hearts in comparison to sham-operated controls. Most strikingly, our data revealed a distinct B-cell subset exclusively found in heart samples but not in the mediastinal lymph nodes, herein termed as heart-associated B cells (hB cells, light green subset shown in Figure 2B and C and detailed below). We also subtracted out the immediate-early genes previously described to be induced by tissue digestion with collagenase,²⁶ and yet, the distribution of all B-cell clusters remained unaltered (Supplementary material online, Figure S1A).

Figure 2

The phenotypic diversity of B cells in the heart and the mediastinal lymph nodes in the context of MI. (A) Experimental pipeline used for single-cell RNA-sequencing and demultiplexing. (B) Gene expression according to the single-cell RNA-sequencing data from 6588 B cells plotted by UMAP dimensionality reduction, revealing nine different B-cell clusters present in the heart and in the mediastinal lymph nodes (med-LNs), including heart-associated B cells (hB) and germinal-centre B cells (GC). Each point represents a single cell and is coloured according to the cluster assignment made by the Seurat package. (C) Jitter plot representing the distribution of clusters among the different samples in heart and lymph nodes (LN)—Heart MI, Heart Sham, LN MI, and LN Sham—after demultiplexing based on the hashtag antibody signal. Fo1 (follicular B-cell cluster 1), Fo2 (follicular B-cell cluster 2), hB (heart-associated B-cell cluster), MZ (marginal zone B cells), B1 (B1 B cells), CD74⁺ (CD74-expressing B cells), IFNR (B cells enriched in interferon-responsive transcripts), CYC (Cycling B cells), GC (germinal-centre B cells). The single-cell RNA-sequencing experiment was performed after pooling samples obtained from n = 5 animals per group at Day 5 after MI. (D) UMAP plots of B-cell receptor sequencing analyses depicting the number of clones of the same B-cell receptor clonotype in both MI and sham hearts compared to mediastinal lymph nodes. Each cell plotted in the UMAP is coloured according to their clonal size, i.e. number of cells expressing the same B-cell receptor. The colour legend is shown within the panel. The sequencing experiment was performed after pooling samples obtained from n = 5 animals per group at Day 5 after MI.

As shown in Figure 2B and C, besides the follicular, marginal zone, B1 and hB-cell subsets, we detected 3 additional minor B-cell clusters comprising B-cell subsets expressing high levels of genes involved in the antigen presentation process (e.g. Cd74 and H2-Aa, termed CD74⁺ cluster), B cells enriched in interferon-responsive transcripts (e.g.: Ifit3, Ifit3b, Ifit1, Ifit2, Irf7, Isg15, Stat1, termed IFNR subset), and a cluster enriched in transcripts related to cell cycle (e.g. Trp53, Cdc27) and ribosome biogenesis (Mrto4, Nhp2, Ranbp1, and Ncl Gnl3), which might represent cycling B cells (Cyc). The top genes defining each B-cell cluster are presented in Supplementary material online, Figure S1B and Table SI.

3.3 hB cells are not clonally expanded and show no sign of antigen specificity

In parallel to the heterogeneous myocardial B-cell responses, in our single-cell sequencing analyses, we also found a small but consistent population of proliferating germinal centre B cells in the mediastinal lymph nodes of infarcted animals (Figure 2B and C, Supplementary material online, Table SI) that was identified by the expression of key genes related to antibody class-switching, affinity maturation and antibody production (e.g. Aicda, Ki67, Mef2b, Bcl6).²⁷

B-cell specificity is conferred by the expression of somatically rearranged antigen receptors on the cell surface. Therefore, we also conducted sequencing of the B-cell receptors at a single-cell level as an approach to uncover the clonality of post-MI B-cell responses. The rationale for these experiments is the fact that the natural (unstimulated) B-cell repertoire is largely polyclonal but antigen recognition leads to the proliferation of specific clones expressing unique B-cell receptors, causing a bias in their repertoire distribution.

B-cell receptor sequencing analyses revealed that the vast majority of B cells found in the heart express unique receptors, meaning that their largely polyclonal repertoire shows no sign of antigen-driven clonal expansion (Figure 2D). Mild clonal expansion was though observed among germinal centres in the mediastinal lymph nodes of infarcted mice. Taken together, our B-cell receptor repertoire analyses indicate that acute MI rapidly mobilizes non-specific B cells into the injured myocardium and triggers, in parallel, antigen-specific germinal centre responses in the mediastinal lymph nodes that might ultimately lead to the production of specific antibodies at later stages.

3.4 hB cells express markers of cell activation and show a distinct chemokine receptor signature

Next, we sought to characterize the transcriptome profile of the hB-cell cluster in further detail. Interestingly, hB cells showed a distinct chemokine receptor signature that was enriched in Cxcr5 and Ccr7 transcripts (Figure 3). In silico analysis using publicly available single-cell sequencing data generated in a similar experimental setting²⁸ revealed that among all major cell populations found in infarcted hearts, B cells were the only subset that expressed Cxcr5, whereas Ccr7 was only expressed in cardiac B cells and T cells (Supplementary material online, Figure S2). While certain B cells in other clusters expressed either Cxcr5 or Ccr7, we found that only hB cells exhibited a double-positive signature (Figure 3A and B). These cells additionally showed up-regulated expression of Cd69, a B-cell activation marker (Supplementary material online, Figure S3A). The surface expression levels of the proteins encoded by these genes were validated and confirmed by flow cytometry (Figure 3C). Of note, cardiac CCR7⁺CXCR5⁺ hB cells also expressed higher levels of surface CD69 when compared with double negative non-hB cells (Supplementary material online, Figure S3B).

Figure 3

hB cells exhibit a distinct chemokine receptor signature. (A, B) Feature plots showing the expression levels in single-positive Cxcr5 (green dots) and Ccr7 (red dots) and double-positive (yellow dots) cells in the different B-cell clusters found in the heart (A) and in mediastinal lymph node (med-LN) samples (B). Heart-associated B cells (hB cells) were purified from sham and infarcted animals (Day 5 post-MI), each dot represents a single cell and the relative gene expression levels are indicated by the colour threshold. The single-cell sequencing experiments were performed after pooling samples obtained from n = 5 animals per group. (C) Flow cytometry gating strategy according to the surface expression levels of CXCR5 and CCR7 amongst B cells (defined as live, singlet CD19/B220⁺ events) purified from the heart to identify hB cells. (D) The frequency of hB cells in the heart was investigated over time after MI. The bar graphs indicate the group mean values ± SEM per group (n = 4 or 5), and the distribution of each individual value. Statistical test: ordinary one-way ANOVA followed by Dunnett’s multiple comparison test. **P < 0.01 and ****P < 0.0001. (E) Mediastinal lymph nodes (med-LN), subiliac lymph nodes (si-LN), spleen, and liver were monitored as control tissues in parallel by flow cytometry (Day 5 post-MI). The bar graphs indicate the group mean values ± SEM per group (n = 3–8), and the distribution of each individual value. Statistical test: two-way ANOVA followed by Tukey’s multiple comparisons test. **P < 0.01 and ****P < 0.0001.

Heart-associated B cells after MI were further investigated by flow cytometry over time (Figure 3D). Double-positive CCR7⁺CXCR5⁺ B cells were found to be enriched in the scar tissue with increasing numbers peaking at Day 7 after MI. In sharp contrast, double-positive CCR7⁺CXCR5⁺ B cells were rarely observed in control organs (subiliac lymph nodes, spleen, and liver samples) and their numbers did not change after MI (Figure 3E). The flow cytometry gating strategies are shown in Supplementary material online, Figure S4.

The single-cell sequencing analysis of differentially expressed genes in MI- vs. sham-operated animals revealed up-regulation of Tgfb1 in hB cells sorted at Day 5 post-MI, when compared with non-hB cells or hB cells purified from sham-operated animals. On the other hand, cytokines, such as Tnf, Il1b, Il6, Ifng, and Il10, were not expressed by hB cells or up-regulated after MI (Figure 4A). Interestingly, hB cells purified from infarcted hearts expressed higher levels of transcripts involved in pro-inflammatory signalling via the NF-κB pathway (Rela, Irf2, Irf5, Irf8, and Stat3) while the inhibitory molecule Lyn was down-regulated (Figure 4B).

Figure 4

hB cells are characterized by increased Tgfb1 expression. (A) Single-cell RNA-sequencing of hB cells sorted at Day 5 after MI was performed and followed by a post hoc analysis on differential expression of various cytokines between MI and sham as well as between hB- and non-hB cells. (B) In the same experimental set-up, we investigated differential expression of genes involved in inflammatory processes (Rela, Irf2, Irf5, Irf8, and Stat3). The term ‘identity’ refers to the experimental condition (MI vs. sham) of hB-cell purification, whereas the term ‘features’ refers to the transcripts analysed.

These data suggest that the CD19/B220⁺CCR7⁺CXCR5⁺ cells found in the infarcted myocardium represent a bona fide novel population of heart-associated B cells (hB cells) distinct from peripheral B cells. These hB cells are largely polyclonal and do not seem to be enriched for cardiac-specificity but they up-regulate Tgfb1 and the activation marker Cd69.

3.5 hB cells infiltrate the myocardium guided by a local CXCL13 gradient

Next, we quantified the relative gene expression levels of Cxcl13 (ligand of CXCR5), Ccl19 and Ccl21 (ligands of CCR7) mRNA in heart extracts of scar tissue (5-day post-MI) vs. myocardial tissue obtained from sham-operated controls. As shown in Figure 5A, Cxcl13 and Ccl19 but not Ccl21 gene expression were upregulated in myocardial scar tissue. Notably, Cxcl13 expression in infarcted hearts was approximately seven-fold higher than Ccl19 expression. A sandwich ELISA approach confirmed that CXCL13 protein levels were indeed increased in the scar tissue (Figure 5B) leading to a concentration gradient between the infarcted and remote area. Myocardial CXCL13 expression peaked on Day 1 post-MI, suggesting an important role of this chemokine for the rapid B-cell influx observed. As shown in Figure 5C, immunofluorescence microscopy analyses of heart sections from Day 1 post-MI revealed that B220⁺ B cells (blue) found in the injured myocardium colocalized with CXCL13-expressing cells (red). In addition, three-dimensional rendering of MI hearts achieved through light-sheet fluorescence microscopy revealed that the infiltrating CXCR5⁺ cells were distributed in discrete patches within the scar tissue (Figure 5D). Taken together, these observations suggest that the CXCL13-CXCR5 axis could lead to selective B-cell recruitment into the injured heart.

Figure 5

The infarcted myocardium shows increased CXCL13 expression. (A) The relative myocardial expression levels of Cxcl13 (ligand of CXCR5), Ccl19 and Ccl21 (ligands of CCR7) were assessed by quantitative PCR (qPCR) in sham-operated and infarcted mice (Day 5 post-MI). The bar graphs indicate the group mean values ± SEM per group (n = 3–5), and the distribution of each individual value. Statistical test: Mann–Whitney test. *P < 0.05. (B) Myocardial CXCL13 concentration was detected by ELISA at Day 1 and Day 5 post-MI. The bar graphs indicate the group mean values ± SEM per group (n = 5–6), and the distribution of each individual value. Statistical test: one-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01. (C) Representative micrograph depicting the distribution of B cells in the infarcted myocardium (B220⁺) in close association with CXCL13-expressing cells at Day 3 after MI. White arrowheads indicate the CXCL13-B220 interaction. Scale bar: 100 µm. (D) Light-sheet microscopy imaging showing the distribution of CXCR5⁺ cells (yellow) in the injured myocardium 5-day post-MI. White arrowheads indicate areas enriched in CXCR5⁺ cells. Scale bar: 150 µm, Z-stack: 500 µm (5× magnification).

To test this hypothesis, we treated mice with a neutralizing anti-CXCL13 antibody 3 h before induction of MI according to protocols established in previous studies in other disease models.²² In our study, CXCL13 neutralization efficiently blocked B-cell influx into the infarcted myocardium (Figure 6A), confirming that this chemokine confers B-cell cardiotropism in the context of MI.

Figure 6

B cells infiltrate the infarcted heart through a CXCL13-CXCR5-dependent mechanism. (A) Numbers of B cells in the heart (scar vs. remote tissue) were assessed planimetrically at Day 3 after MI after intraperitoneal treatment with 200 µg of a neutralizing CXCL13-specific antibody (α-CXCL13) prior to MI induction. As a control, animals were injected with 200 µg of an isotype control antibody (Ctrl). The bar graphs represent the mean ± the standard error of the mean per group (n = 5). (B) Numbers of B cells in the heart (scar vs. remote tissue) were investigated (by flow cytometry) in CXCR5^−/− vs. wild-type mice at Day 7 after MI. The bar graphs represent the mean ± the standard error of the mean per group (n = 4–5). Statistical tests: Two-way ANOVA followed by Tukey’s post hoc test (A and B). *P < 0.05. (C) Frequencies of B cells were investigated in the spleens of CXCR5^−/− vs. wild-type mice at Day 7 after MI. The bar graphs represent the mean ± the standard error of the mean per group (n = 3–4). Statistical test: Unpaired t-test. (D) Echocardiographic analyses were performed in α-CXCL13-treated vs. control animals on Day 28 post-MI and included assessment of fractional shortening and end-diastolic area. In CXCR5^−/− vs. wild-type mice, echocardiographic analyses were performed on Day 7 post-MI and also included assessment of fractional shortening and end-diastolic area. The bar graphs represent the mean ± the standard error of the mean per group (n = 5–14). (E) In CXCR5^−/− vs. wild-type mice the expression levels of cytokines (Il6, Il1b, Tnf, and Tgfb1) were assessed at Day 7 after MI. The bar graphs represent the mean ± the standard error of the mean per group (n = 8–9). Statistical test: two-way ANOVA followed by Sidak’s multiple comparisons test. *P < 0.05.

To further scrutinize these findings, we performed additional experiments using CXCR5-deficient mice (CXCR5^−/−) and observed that these mice subjected to MI showed significantly reduced B-cell infiltration in the myocardial scar tissue (Figure 6B). The numbers of B cells present in the intact remote myocardium or in the spleen were not impacted by CXCR5-deficiency (Figure 6C). The numbers of cardiac granulocytes were not impacted by CXCR5-deficiency either (Supplementary material online, Figure S5).

After identifying a specific chemokine-receptor signature responsible for mobilizing B cells into the infarcted myocardium, we were able to functionally dissect the impact of local B cells on myocardial inflammation and repair processes, advancing therefore from previous studies that have targeted B cells systemically.⁸ Despite of the reduced myocardial B-cell infiltration, no differences in echocardiographic assessment, including fractional shortening and end-diastolic area, were observed between placebo and anti-CXCL13 treated groups. Functional analyses of post-infarction CXCR5^−/− vs. wild-type mice further confirmed these findings (Figure 6D). Moreover, we also monitored the expression levels of Tnf, Il6, Il1b, and Tgfb1 by quantitative PCR in myocardial samples obtained from CXCR5⁻/⁻ vs. wild-type infarcted mice. Consistently with the single-cell RNA-sequencing data presented in Figure 4A, we observed that myocardial extracts obtained from infarcted CXCR5-deficient animals exhibited significantly lower expression level of Tgfb1 when compared to wild-type controls (Figure 6E).

These findings reveal that the B cells infiltrating the infarcted myocardium are an important source of myocardial TGF-ß1, although they do not critically influence cardiac morphometry and function after MI. In contrast, the B cells’ effects on myocardial biology reported in previous findings, based on global depletion approaches^8,29 might be primarily related to systemic mechanisms, such as the production of antibodies or other soluble factors.¹²

4. Discussion

Taken together, our study shows that MI triggers a rapid influx of polyclonal B cells into the myocardial scar tissue which is accompanied by a mild expansion of oligoclonal germinal centre B cells in the heart-draining lymph nodes. Interestingly, we could identify a B-cell subset specifically associated with the heart and with a distinct phenotype (CXCR5⁺CCR7⁺) that we herein coined as hB cells. By identifying CXCL13:CXCR5 as the chemokine axis that selectively confers cardiotropism to Bcells and by characterizing the transcriptome of post-MI hB cells, we could shed more light on the complex immunological phenomena induced by MI.

Previous studies have also reported the presence of B cells associated with healthy myocardial tissue in steady-state conditions.^11,29,30 In a recent study, Adamo et al. reported the presence of B cells in the healthy myocardium which recirculate between the myocardial parenchyma and blood and show a distinct transcriptomic profile that overlap with the hB cells we report here. In our study, we provide the first description of the B-cell repertoire of these cells suggesting that they are unlikely to be enriched for myocardial specificity. Moreover, we report, for the first time, how these hB cells shift their transcriptomic profile in response to permanent LAD ligation and significantly contribute to local TGF-ß1 production in the infarcted myocardium.

Based on gene and protein expression data as well as functional evidence through CXCL13 neutralization experiments and exploiting CXCR5-deficient mice, we put forward that B-cell mobilization in the injured myocardium is selectively mediated by the CXCL13:CXCR5 axis. Under physiological conditions, CXCL13 is a key mediator that organizes lymphocyte trafficking in secondary lymphoid organs such as lymph nodes and spleen. Outside of lymphoid organs, CXCL13 has already been shown to play a decisive role in mediating B-cell recruitment to sites of inflammation, such as atherosclerotic plaques,³¹ pericardial adipose tissue,¹³ cerebrospinal fluid,³² rheumatoid synovium,³³ and the central nervous system.³⁴

In the context of MI, B cells were the only cardiac cell subset found to express the receptor for CXCL13, CXCR5. Moreover, Martini et al.³⁵ recently reported that cardiac B cells also showed increased Cxcr5 gene expression in a model of pressure overload-induced HF.

Based on the selective distribution of CXCR5 among B cells and on the detected myocardial CXCL13 expression, we hypothesized that both CXCL13 and CXCR5 could serve as selective targets to modulate cardiac B-cell responses without affecting other neither leucocyte populations nor B-cell distribution systemically.

Our experiments using antibody-mediated CXCL13 neutralization and CXCR5-deficient animal models confirmed this hypothesis and revealed that hB cells are an important source of myocardial TGF-ß1 at Day 7 post-MI which marks the peak of the healing phase.³ The cellular source of myocardial CXCL13 was not identified in the present study but it has been widely reported that fibroblasts are the main source of CXCL13 within the lymph node follicles.^36–38 Moreover, it has been reported that monocytes can also express CXCL13 in sites of inflammation.³⁹ Monocytes and fibroblasts are both abundant in the infarcted myocardium and might account for the local CXCL13 production.

TGF-ß1 is an important soluble mediator that can impact myocardial survival, immune cell phenotype, and fibroblast activity.⁴⁰ It has been implicated in myocardial tissue repair and fibrosis, but its complex effects are largely dependent on timing and context.⁴¹ Post-MI fibrosis can be a beneficial response, which helps to preserve the structure of the heart but exaggerated fibrosis and remodelling can lead to functional impairment. The cardiac reparative fibrosis process is tuned by immune cells and the long-term effects of hB-derived TGF-ß1 on post-MI fibrosis and remodelling require further investigation.

Despite of being a relevant source of local TGF-ß1, hB cells do not seem to impact the contractile function of the heart in vivo, as their selective ablation did not critically affect outcomes, such as fractional shortening and end-diastolic area. Previous studies focused on post-MI immune responses have mostly employed tools to systemically deplete a specific cell type, thereby interrogating its distinct pathophysiological roles.⁸ Such global depletion approaches (e.g. with antibodies, toxins, genetic ablation) have led to important discoveries but they do not help to resolve whether the cells of interest modulate the myocardial biology through their activity in situ or though other systemic mechanisms. After identifying a defined chemokine signature underlying B-cell cardiotropism, we were able to selectively target the myocardial B-cell influx while leaving their systemic distribution and activity unaltered. Based on our observations, we argue that the cardiac phenotypes previously reported in models of systemic B-cell ablation⁸ might be, at least in part, related to the systemic effects mediated by B cells, including the production of specific antibodies and other soluble mediators. This is in agreement with previous findings showing that antibody-deficient mice also exhibit a better preserved cardiac function in experimental models of MI.¹²

In addition to in situ polyclonal B-cell responses, our study also revealed the presence of germinal centre B cells in the heart-draining mediastinal lymph nodes of infarcted mice. The observed post-MI response was mild when compared to classical immunization conditions, yet the B-cell receptor repertoire analyses also indicated that the germinal centre cells were clonally expanded, a hallmark of antigen-driven immune response. Germinal centre B cells expressed Mef2b, Bcl6, and Aicda, which are essential for immunoglobulin class switching and the generation of high-affinity antibodies.^27,42,43 In future studies, we will further investigate whether the germinal centre B cells expanding in the heart-draining lymph nodes impact chronic adverse remodelling via the production of heart-specific antibodies.¹²

Taken together, our study reveals that MI elicits complementary B-cell responses in the affected myocardium as well as in the heart-draining lymph nodes. Polyclonal B cells readily infiltrate the infarcted heart via a distinct CXCL13-CXCR5 axis and contribute to local TGF-ß1 production, whereas mild antigen-driven germinal centre B cells proliferate in the mediastinal lymph nodes and might ultimately lead to the production of specific antibodies.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Author contribution

M.H., D.A., G.W., U.H., S.F., K.H., C.C., and G.C.R. made substantial contributions to the conception and design of the present work. M.H., D.A., J.S., L.B., G.W., C.C., A.E.M., P.A., and G.C.R. acquired and analysed data. M.H., D.A., U.H., S.F., and G.C.R. drafted the manuscript. All co-authors critically revised it, made significant intellectual contributions, and approved the manuscript in its final version to be published.

Acknowledgements

The authors greatly appreciate the skillful technical assistance of Elena Vogel, Lisa Popiolkowski, and Andrea Leupold. They thank Prof Richard Schulz for the insightful comments and suggestions. The graphical abstract figure was prepared using resources freely available by the Servier Medical Art.

Conflict of interest: none declared.

Funding

This work was supported by the Interdisciplinary Centre for Clinical Research Würzburg [E-354 to GCR, E-353 to CC, Z-6 to P.A., and the clinician scientist programme to M.H.], the European Research Area Network—Cardiovascular Diseases [ERANET-CVD JCT2018, AIR-MI Consortium grant 01KL1902 to G.C.R.], the German Research Foundation [DFG grant 411619907 to G.C.R.], the German Ministry of Research and Education [BMBF 01EO1504 to C.C. and A.E.S.] and by the Else-Kröner-Fresenius-Stiftung (EKFS) to G.W. L.B. received a scholarship from the German Cardiac Society (DGK).

Data availability

The single-cell RNA-sequencing data presented in this study has been deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession number GSE150140.

Time for primary review: 26 days

Translational perspective

We performed a deep phenotyping of B cells engaged in immune responses following a myocardial infarction and shed some light on their roles in myocardial inflammation and repair in mice. The novel description of a distinct subset of heart-associated B cells (CCR7⁺, CXCR5⁺) and the identification of the mechanism underlying their mobilization into the injured myocardium might help to dissect their local vs. systemic implications to myocardial biology and heart failure. In the future, these results might pave the way to a better understanding of B-cell responses also in humans.

Author notes

Margarete Heinrichs and DiyaaElDin Ashour authors contributed equally to the study.