Heart failure (HF) is a leading cause of morbidity and mortality. Studies in animal models and individuals with HF revealed a prominent role for CD4+ T cell immune responses in the pathogenesis of HF and highlighted an active cross-talk between cardiac fibroblasts and interferon (IFN)-γ-producing CD4+ T cells that results in profibrotic myofibroblast transformation. Whether cardiac fibroblasts concomitantly modulate pathogenic cardiac CD4+ T cell immune responses is unknown. Here we report that mouse cardiac fibroblasts express major histocompatibility complex type II (MHCII) in two different experimental models of cardiac inflammation. We demonstrate that cardiac fibroblasts take up and process antigens for presentation to CD4+ T cells via MHCII induced by IFN-γ. Conditional deletion of MhcII in cardiac fibroblasts ameliorates cardiac remodeling and dysfunction induced by cardiac pressure overload. Collectively, we demonstrate that cardiac fibroblasts function as antigen-presenting cells and contribute to cardiac fibrosis and dysfunction through MHCII induced by IFN-γ.
In the adult mouse myocardium, cardiac fibroblasts constitute a large fraction of noncardiomyocytes (<20%)1, and are largely responsible for the synthesis and degradation of the extracellular matrix components to maintain tissue homeostasis2. Under pathological conditions, including pressure overload, infection or ischemia, increased fibroblast proliferation and activation is associated with fibrosis2. Activated fibroblasts deposit excessive extracellular matrix components into the cardiac interstitium, resulting in detrimental loss of myocardial compliance and decreased cardiac function. In accordance with this, ablation of activated fibroblasts in mice is associated with decreased fibrosis and improved cardiac function in response to pressure overload and myocardial infarction3,4. In pressure overload-induced HF in mice, the development of cardiac fibrosis coincides with the infiltration of CD4+ T cells into the myocardium and is characterized by the transformation of cardiac fibroblasts to myofibroblasts, which deposit excess collagen5,6. CD4+ T cell-deficient mice are protected from development of cardiac fibrosis5,7, and direct interactions between cardiac fibroblasts and IFN-γ-producing T helper 1 (TH1) cells have been shown to promote cardiac fibroblast transforming growth factor beta production, resulting in myofibroblast transformation6. Here, we aim to uncover the bidirectionality of these cellular interactions and determine if cardiac fibroblasts concomitantly promote CD4+ T cell activation, a new capability that is yet to be established.
CD4+ T cells are classically activated by professional antigen-presenting cells (APCs), which efficiently internalize and process antigens that are displayed by MHCII molecules on the cell membrane8. Additional co-stimulatory signals (CD80 or CD86) expressed by activated APCs are required for full activation of CD4+ T cells. Professional APCs include dendritic cells (DCs), macrophages and B cells, which are all present in the heart at lower frequencies relative to cardiac fibroblasts8. Unlike professional APCs, nonprofessional APCs do not constitutively express MHCII molecules, but the expression of MHCII can be induced in neutrophils, mast cells and endothelial cells by various stimuli9. Additionally, human dermal fibroblasts exposed to IFN-γ in vitro express MHCII, and promote recall antigen-dependent T cell responses10. We hypothesized that the cellular interactions between cardiac fibroblasts and CD4+ T cells in the heart imply a new role for cardiac fibroblasts as nonprofessional APCs central to cardiac CD4+ T cell responses and pathology.
IFN-γ induces MHCII expression in cardiac fibroblasts
Nonprofessional APCs do not typically express MHCII unless induced by specific stimuli9. In conjunction with MHCII, either CD80 or CD86 co-stimulatory signals are required for full activation of CD4+ T cells upon engagement of the T cell receptor (TCR). We isolated the left ventricle (LV) from adult wild-type (WT) mice and cultured purified CD31−CD45−MEFSK4+ cardiac fibroblasts (Fig. 1a). We found that MHCII was not expressed at baseline but was progressively induced over 5 d of IFN-γ stimulation in culture (Fig. 1b,c). Conversely, CD80 was highly expressed at baseline and was refractory to IFN-γ stimulation (Fig. 1d,e). CD86 was neither expressed at baseline, nor induced by IFN-γ (Fig. 1f,g). Expression of MHCII after IFN-γ stimulation in vitro was further demonstrated by immunofluorescence microscopy on cardiac fibroblasts (Fig. 1h). In the heart, the majority of resident cardiac fibroblasts express transcription factor 21 (encoded by Tcf21)11. To delineate cardiac fibroblast expression of MHCII more specifically, we utilized cardiac fibroblast lineage tracing reporter mice (Tcf21iCre/+; R26eGFP) with a tamoxifen-regulated Cre recombinase knocked into one allele of Tcf21 that drives membrane-targeted GFP expression from the Rosa26 locus (R26). GFP+ cells from Tcf21iCre/+; R26eGFP mouse hearts were sorted by fluorescence-activated cell sorting (FACS) and cultured for 72 h in the presence of IFN-γ. Similarly, we found that CD80 was highly expressed at baseline, while MHCII was induced after 72 h of IFN-γ stimulation (Fig. 1i–k). Furthermore, expression of MHCII after IFN-γ stimulation in vitro was demonstrated by immunofluorescence microscopy on GFP+ cardiac fibroblasts derived from Tcf21iCre/+; R26eGFP mice (Fig. 1l). We next evaluated in vivo induction of MHCII and co-stimulatory molecule expression on CD31−CD45−MEFSK4+ cardiac fibroblasts in the myocardial tissue microenvironment by flow cytometry (Fig. 2a). IFN-γ was administered daily to WT mice for 5 d (Fig. 2b). In vehicle-treated mice, 30–50% of cardiac fibroblasts expressed CD80 on the surface but did not express MHCII or CD86 (Fig. 1c–h). However, IFN-γ treatment induced MHCII surface expression on 28–34% of cardiac fibroblasts, while CD80 and CD86 expression was not altered (Fig. 2c–h). These data demonstrate that IFN-γ induces expression of MHCII in cardiac fibroblasts, and, together with the expression of CD80, suggest that cardiac fibroblasts are capable of functioning as nonprofessional APCs that can induce CD4+ T cell activation.
MHCII+ cardiac fibroblasts induce CD4+ T cell activation
We next evaluated the ability of cardiac fibroblasts to activate naïve OTII CD4+ T cells, which express a transgenic TCR specific for chicken ovalbumin (OVA) peptide. CD62LhiCD44lo naïve OTII CD4+ T cells were sorted by FACS (Fig. 3a) and co-cultured with primary LV cardiac fibroblasts stimulated with IFN-γ to induce MHCII expression and loaded with OVA peptide. We identified robust induction of CD4+ T cell proliferation after 72 h of co-culture as determined by CFSE dilution assay (Fig. 3b,d), although not to the same extent as professional antigen-presenting bone marrow-derived dendritic cells (BMDCs; Fig. 3c,d). These responses were restricted by MHCII as MHCII-deficient (MhcII−/−) cardiac fibroblasts did not induce naïve CD4+ T cell proliferation (Fig. 3b). To exclude possible contamination with professional APCs in culture, purified GFP+ CD45− cardiac fibroblasts from Tcf21iCre/+; R26eGFP mice were sorted by FACS (Fig. 3e) and stimulated with IFN-γ to induce MHCII expression and co-cultured with FACS-sorted CD62LhiCD44lo naïve OTII CD4+ T cells in the presence of OVA peptide. Similarly, we identified robust induction of CD4+ T cell proliferation after 72 h of co-culture as determined by CFSE dilution (Fig. 3f,g). TH1 cells are a major cellular source of endogenous IFN-γ and establish intimate contact with cardiac fibroblasts upon infiltration into the heart6. We examined the possibility that antigen presentation to TH1 cells during this contact triggers further IFN-γ production to support sustained antigen presentation. We co-cultured cardiac fibroblasts and differentiated OTII TH1 cells in the presence of OVA peptide (Fig. 4a). After 24 h, TH1 cells showed marked induction of IFN-γ production, as determined by intracellular cytokine staining (Fig. 4b,c). Moreover, 72-h co-cultures of primary adult cardiac fibroblasts with OTII TH1 cells labeled with CFSE, in the presence of OVA peptide, demonstrated increased MHCII surface expression in cardiac fibroblasts as compared to cardiac fibroblasts alone (Fig. 4d,e), and resulted in OTII TH1 cell, but not WT TH1 cell, proliferation (Fig. 4f,g). These data demonstrate the ability of cardiac fibroblasts in the presence of IFN-γ to present peptide antigens to naïve CD4+ T cells. Additionally, cardiac fibroblasts express MHCII during cardiac fibroblast–TH1 cell contact and promote antigen-specific TH1 cell activation.
Cardiac fibroblasts capture antigens for MHCII presentation
Exogenously delivered OVA peptide can directly bind the peptide groove of cell surface MHCII molecules for presentation to CD4+ T cells (Fig. 2). However, defining attributes of APCs include the capacity to internalize soluble and particulate antigens, process them, load peptides onto MHCII in the cytosol and translocate MHCII–peptide complexes to the cell surface to activate CD4+ T cells. Soluble OVA protein is taken up via macropinocytosis or mannose receptor-mediated endocytosis12, whereas the uptake of relatively larger particles involves phagocytosis, with phagosomes containing the ingested material maturing into acidified phagolysosomes13. To track soluble antigen uptake and processing by cardiac fibroblasts, we used DQ OVA, a self-quenched conjugate of OVA protein that fluoresces upon proteolytic degradation. Strikingly, cardiac fibroblasts efficiently took up and processed DQ OVA, as demonstrated by flow cytometry and immunofluorescence, and, unlike MHCII expression, these functions were independent of IFN-γ stimulation (Fig. 5a,b). Moreover, OVA protein internalized by cardiac fibroblasts, combined with IFN-γ stimulation to promote MHCII expression, resulted in naïve OTII CD4 + T cell proliferation (Fig. 5c,d). We next evaluated phagocytic activity of cardiac fibroblasts using pHrodo green Escherichia coli BioParticles, which become fluorescent in acidified phagolysosomes. A strong fluorescent signal emitted from cardiac fibroblasts was detected by both flow cytometry and immunofluorescence, demonstrating efficient phagocytosis independent of IFN-γ stimulation (Fig. 5e,f). Furthermore, E. coli transformed with a plasmid conferring OVA protein expression were engulfed by cardiac fibroblasts and induced antigen-specific naïve OTII CD4+ T cell proliferation, which was remarkably comparable to that induced by BMDCs (Fig. 5g,h). These data demonstrate several functions of cardiac fibroblasts as APCs, including efficient uptake and processing of soluble and particulate antigens, and presentation of peptide fragments via MHCII to generate CD4+ T cell immune responses.
Cardiac fibroblast MHCII expression modulates pathology
Because myocardial IFN-γ is upregulated in response to transverse aortic constriction (TAC), a well-established model of HF6,14, and Chagas disease15,16, we investigated the induction of MHCII on cardiac fibroblasts in vivo, in response to these distinct models of cardiac inflammation. We analyzed the surface expression of MHCII and CD80 on CD31−CD45−MEFSK4+ cardiac fibroblasts by flow cytometry (Fig. 6a). Four weeks of TAC in WT mice resulted in increased expression of both MHCII and CD80 on cardiac fibroblasts relative to Sham control mice (Fig. 6b–e). Similarly, WT mice infected with Trypanosoma cruzi and collected after 19 d, at the peak of parasitemia, demonstrated even greater MHCII induction on cardiac fibroblasts relative to mock infected controls. However, CD80 expression did not change (Extended Data Fig. 1). Expression of MHCII in vivo by cardiac fibroblasts after TAC in LV tissue sections was further demonstrated by immunofluorescence microscopy on alpha smooth muscle actin (α-SMA)-positive cells in WT mice (Fig. 6f), and in GFP+ cells traced from Tcf21iCre/+; R26eGFP reporter mice (Fig. 6g). Additionally, direct contact can be identified between CD4+ T cells and αSMA+ cardiac fibroblasts cells within LV fibrotic lesions in WT TAC mice (Fig. 6h). We next generated Tcf21iCre/+MhcIIfl/fl mice expressing tamoxifen-inducible Cre recombinase driven by Tcf21 expression to conditionally delete the H2Ab1, also known as Ia2, gene (MhcII) in cardiac fibroblasts (Fig. 7a). After tamoxifen treatment of Tcf21iCre/+MhcIIfl/fl mice, MhcII recombination in sorted CD31−CD45−MEFSK4+ cardiac fibroblasts was detected by PCR, and occurred in a cell-specific manner, as CD31−CD45+ cardiac leukocytes did not undergo MhcII recombination (Fig. 7b and Extended Data Fig. 2). Furthermore, CD31−CD45−MEFSK4+ cardiac fibroblasts cultured with 4-hydroxytamoxifen showed 3.5-fold reduction in MHCII protein expression after 3 d of IFN-γ stimulation (Fig. 7h–l), while CD45+CD11b+CD11c+ BMDCs did not show a reduction in MHCII in response to 4-hydroxytamoxifen treatment (Fig. 7e,f). Tcf21iCre/+MhcIIfl/fl mice were treated with tamoxifen by intraperitoneal injection before TAC surgery and maintained on tamoxifen chow for 4 weeks until collection (Fig. 7g). MHCII protein surface expression on myeloid cells in the mediastinal lymph nodes (mLNs) draining the heart (Fig. 7h), as well as in cardiac leukocytes and cardiac endothelial cells, was similar across the treatment groups, with only observed decreased MHCII protein surface expression in cardiac fibroblasts from Tcf21iCre/+MhcIIfl/fl mice treated with tamoxifen (Extended Data Fig. 3). Vehicle-treated and tamoxifen-treated (cardiac fibroblast MHCII-deficient) mice showed similar LV CD4+ T cell infiltration (Fig. 7i,j) and similar CD4+ T cell activation in the mLNs (Extended Data Fig. 4); however, only tamoxifen-treated mice had decreased cardiac fibroblasts compared to vehicle-treated mice in the onset of TAC (Extended Data Fig. 5), and an amelioration of adverse cardiac remodeling, including perivascular fibrosis (Fig. 7k,l) and cardiomyocyte hypertrophy (Extended Data Fig. 4) compared to the vehicle treatment group. Additionally, cardiac systolic function remained preserved in tamoxifen-treated mice in contrast to the vehicle treatment group as demonstrated by preserved fractional shortening (Fig. 7m,n and Table 1). These results demonstrate that IFN-γ-associated cardiac inflammation promotes MHCII expression in cardiac fibroblasts, and that MHCII in cardiac fibroblasts is central to cardiac remodeling and dysfunction in response to TAC.
We report for the first time, to our knowledge, that cardiac fibroblasts function as APCs and contribute to cardiac fibrosis and dysfunction through IFN-γ-induced MHCII. We find that cardiac fibroblasts: (1) express MHCII in vitro and in vivo in response to IFN-γ as well as in two different experimental models of cardiac disease; (2) express the requisite co-stimulatory signal CD80 necessary for CD4+ T cell activation; (3) efficiently capture and process extracellular soluble and particulate antigens for presentation; and (4) induce naïve CD4+ T cell and TH1 cell activation in an MHCII-dependent and antigen-dependent manner. Moreover, we identify a central role for cardiac fibroblast MHCII expression in cardiac remodeling and dysfunction in an experimental model of HF that is highly dependent on IFN-γ+ TH1 cell immune responses.
It has been known for decades that stromal cells, including dermal fibroblasts, can express MHCII in response to IFN-γ in vitro17,18; therefore, this finding in cardiac fibroblasts could be perhaps unsurprising. The key message of our study emerges from the relevance of cardiac fibroblasts as APCs in the context of cardiac inflammation and pathology. Our in vitro results demonstrate that MHCII expressed on cardiac fibroblasts, loaded with a peptide antigen, can induce naïve CD4+ T cell proliferation, a capability considered exclusive to mature DCs8. This serves as decisive validation of the functionality of cardiac fibroblasts as APCs. The quality of TCR interactions with the peptide–MHCII complex is determinant of the strength of CD4+ T cell activation, with higher-affinity interactions generating a more potent response19. In our in vitro studies, we utilize OVA-specific naïve OTII CD4+ T cells against their optimal OVA peptide ligand, thus it is possible that cardiac fibroblasts have a diminished ability to activate CD4+ T cells when presenting suboptimal cognate antigens. However, given the confinement of cardiac fibroblasts to the heart it is unlikely that they establish contact with naïve CD4+ T cells in vivo, but rather with effector CD4+ T cells, which are more sensitive to triggering through the TCR. We have reported strong TCR engagement of CD4+ T cells within the mouse heart20 and induction of T helper responses in the mLNs during HF development6,21. Here, we find that IFN-γ treatment and two cardiac pathologies associated with elevated IFN-γ show elevated expression of MHCII on cardiac fibroblasts. We demonstrate the ability of cardiac fibroblasts to present peptide antigen to TH1 cells in vitro and propose a role in generating recall responses from TH1 cells infiltrated in the heart through cardiac fibroblasts expressing MHCII and the involvement of this axis in cardiac fibrosis and dysfunction.
We identify baseline expression of the co-stimulatory molecule CD80 in >90% of the cells in vitro, and in approximately 20–50% of cardiac fibroblasts in vivo, and detect increased expression in response to cardiac pressure overload. This variability may arise due to heterogeneity of cardiac fibroblasts in the myocardium at varying stages of maturity22, in comparison to synchronized cells in culture. A variety of stimuli have also been shown to increase CD80 expression in APCs, including CD40 ligand, IFN-α, granulocyte–macrophage colony-stimulating factor (GM-CSF), Toll-like receptor ligands and tumor-necrosis factor, which may become expressed in the heart during TAC23,24,25. In addition to direct antigen presentation by cardiac fibroblasts, CD80 expression may have further relevance in global enhancement of CD4+ T cell activation in the heart via trans-co-stimulation, by augmenting co-stimulatory signals delivered to CD4+ T cells already engaged with an APC26. CD80 and CD86 are thought to be interchangeable co-stimulators, and we find that cardiac fibroblasts constitutively express CD80 but not CD86. In contrast to cardiac fibroblasts, CD86 is abundantly expressed on DCs and is thought to play a prominent role in initial T cell activation27. CD80 expression is more slowly induced following DC activation and functional studies suggest that CD80 is a more potent CD28 ligand due its higher relative affinity for CD28 (ref. 24). Our data demonstrate that in the presence of IFN-γ, MHCII and CD80 are available to engage with the TCR and CD28 in CD4+ T cells, respectively, and induce T cell proliferation.
We and others have demonstrated intimate contact between cardiac fibroblasts and immune cells, including TH1 cells and macrophages, in the context of cardiac dysfunction due to pressure overload or age6,28. While these reports focused on the unidirectional signals exerted by immune cells on cardiac fibroblasts and highlighted the requirement for intimate cellular contact to develop cardiac fibrosis, the bidirectionality of this interaction remained unexplored. We now demonstrate that IFN-γ-secreting TH1 cells induce MHCII on cardiac fibroblasts, which subsequently induce further IFN-γ production by TH1 cells, propagating a positive feedback loop that likely exacerbates TH1 cell activation and myofibroblast transformation. Observations on the proximity of MHCII+ cardiac fibroblasts and CD4+ T cells in the TAC hearts, together with our findings in mice lacking MHCII in cardiac fibroblasts having decreased fibrosis, are in support of this loop. We demonstrate the ability of cardiac fibroblasts to capture extracellular antigens and process them into peptide antigens loaded onto MHCII to induce CD4+ T cell activation. Cardiac fibroblasts are nonprofessional phagocytes capable of engulfing apoptotic cells to assist with debris clearance after myocardial ischemia29,30. Our results demonstrate that cardiac fibroblasts internalize both soluble and particulate material, process and load peptides onto MHCII and induce CD4+ T cell proliferation. We additionally report that tamoxifen-treated (cardiac fibroblast MHCII-deficient) Tcf21iCre/+MhcIIfl/fl mice have intact effector CD4+ T cell expansion in the mLNs and CD4+ LV T cell recruitment in response to cardiac pressure overload, as compared to vehicle-treated (MHCII-sufficient) mice. Yet, the lack of cardiac fibroblast MHCII results in decreased cardiac fibroblasts and is sufficient to prevent adverse cardiac remodeling and dysfunction. These results are in line with a central role for DCs in antigen presentation and TH1 cell expansion in the mLNs in the context of heart inflammation as we reported6,20,31, and as demonstrated by DC ablation being effective in preventing cardiac fibrosis and dysfunction32. We recently reported that DCs present cardiac neoantigens to CD4+ T cells, and that interrupting this presentation prevents TCR engagement by CD4+ T cells in the heart and ameliorates cardiac dysfunction20. Because cardiac fibroblast MHCII-deficient mice have intact MHCII expression in myeloid cells, the observed similar number of effector T cells in the lymph nodes, devoid of cardiac fibroblasts, is not surprising. The similar T cell numbers observed in the heart in the presence or absence of MHCII may suggest continuous cardiotropism that is not altered by cardiac fibroblast MHCII expression, as well as a T cell recall response in situ induced by cardiac DCs expressing MHCII that additionally takes place. It is also possible that DCs and cardiac fibroblasts participate in trans-co-stimulation augmenting signals delivered to CD4+ T cells already engaged with an APC and that this is altered in the absence of cardiac fibroblast MHCII, something worth addressing in future studies. While our in vitro results demonstrate cardiac fibroblast induction of T cell proliferation and IFN-γ production through MHCII, we have not directly tested T cell proliferation in the heart in vivo. Nevertheless, our in vivo data demonstrate that cardiac fibroblast expression of MHCII is required for cardiac fibrosis and systolic dysfunction, which are both T cell dependent5,6,7,20. Our new findings endorse the intriguing possibility of treating HF, the leading cause of death in the United States33, with targeted therapies to the cardiac fibroblast that specifically modulate cardiac CD4+ T cell responses without impairing systemic CD4+ T cell activation by DCs, whiich could have undesired immunosuppressive side effects. A detailed investigation of the dynamics of cell-specific MHCII engagement of the TCR in the mLNs and in the heart will identify when such targeted therapies may be more effective.
We acknowledge some limitations in this study. For instance, while we clearly demonstrate that IFN-γ induces MHCII expression of the vast majority of cardiac fibroblasts in vitro, a lower frequency of cardiac fibroblasts express MHCII in vivo in the three experimental models we use (TAC, Chagas or IFN-γ injections) compared to purified cardiac fibroblasts in vitro. It is possible that, in vivo, in the onset of TAC, other factors besides IFN-γ contribute to MHCII expression, and that, given the plasticity of cardiac fibroblasts, specific cardiac fibroblast populations are responsible for antigen presentation. Our data showing that conditional deletion of MHCII in cardiac fibroblasts results in decreased cardiac fibroblast presence in TAC suggest an additional role for cardiac fibroblast MHCII in fibroblast proliferation, which is critical for cardiac fibrosis in pathological conditions that include cardiac pressure overload. Future studies will investigate which fibroblast subsets express MHCII and how MHCII regulates fibroblast function and proliferation. These results are in line with recently published single-cell RNA-sequencing data demonstrating enhanced expression of MHCII in mice 5 weeks after TAC and variable expression in different fibroblast clusters identified in the human cardiac samples obtained from individuals with dilated cardiomyopathy34,35. Noteworthy, recent RNA-sequencing data in individuals with dilated cardiomyopathy before and after left ventricular assist device implantation demonstrate lower expression of cardiac fibroblast HLA-DR in individuals after left ventricular assist device support, in line with our findings that MHCII expression in cardiac fibroblasts contributes to systolic function35. It is also plausible that other APCs sequentially dominate this response during the progression of adverse cardiac remodeling after TAC. These APCs may include lymph node fibroblastic reticular cells, which have been reported to express MHCII in response to IFN-γ36,37. Whether this is the case in mLNs in this context is unknown. For instance, CD4+ T cell engagement of antigen presented by DCs or fibroblastic reticular cells may happen early on in the mLNs, before T cell infiltration in the heart occurs, and continues once T cells infiltrate the heart, coexisting with cardiac fibroblast presentation to intramyocardial CD4+ T cells and cardiac fibroblast proliferation and transformation. The data presented here are limited to one time point after cardiac pressure overload has been induced and future studies will be necessary to determine the dynamics of the cell-specific role of MHCII. This is something worth studying in the immediate future that may have consequences to understand immune tolerance in the heart, which has not been investigated herein. The mechanisms of peptide loading of MHCII in cardiac fibroblasts, and whether these are shared with professional APCs, are also an ongoing research direction we are pursuing. Additionally, we only used male mice in this study, and, given that there are sex-specific differences in HF, it is possible that sex-specific differences also exist in the way cardiac fibroblasts sustain cardiac T cell immune responses. Despite these limitations, our study unveils a new role for cardiac fibroblasts as central contributors to cardiac inflammation and adverse remodeling through MHCII that may have potential implications when targeting inflammation in heart disease (Extended Data Fig. 6).
Taken together, our results contribute to the growing field of cardio-immunology and advance the intriguing possibility that diverse subsets of fibroblasts abundantly dispersed within other organs perform similar functions, serving as sentinel cells that sense local insults and directly boost adaptive immune responses.
Mice were bred and maintained under pathogen-free conditions at Tufts University animal facilities and treated in compliance with the Guide for the Care and Use of Laboratory Animals (National Academy of Science). C57BL/6 WT, OTII, MhcII-floxed mice (all purchased from Jackson Laboratories) and Tcf21-cre mice (provided by J. Davis, University of Washington) were bred and maintained in-house and euthanized at 4–14 weeks of age for tissue collection. Tcf21iCre/+; R26eGFP mice were bred and maintained under pathogen-free conditions at the University of Washington, with all animal experimentation approved by the University of Washington Institutional Animal Care and Use Committee. All animal studies were approved by the Tufts University Institutional Animal Care and Use Committee.
Adult cardiac fibroblasts preparation
Heart ventricles from adult WT and Tcf21iCre/+; R26eGFP mice (4–8 weeks old) were excised, minced and digested at 37 °C for 30 min with agitation in a mixture of 0.25% trypsin and 40 μg ml−1 liberase TL (Roche). Digested tissue was then centrifuged at 300g for 5 min and the pellet resuspended in fibroblast growth medium (Lonza) and plated on 0.1% gelatin-coated plates for 2 h at 37 °C with 5% CO2. Unattached cells were discarded, and adherent cells were further cultured. The digested heart cell suspension was filtered through a 40-µm cell strainer and stained with BV711-conjugated anti-CD45.2 (104), PE-conjugated anti-CD31 (MEC13.3), APC-conjugated anti-feeder cells (mEF-SK4), as outlined below for quantitative flow cytometry and CD31−CD45−MEFSK4+ cells, or GFP+ cells when using Tcf21iCre/+; R26eGFP lineage tracing mice, were sorted on a BD FACSAria and plated on 0.1% gelatin-coated plates at 37 °C with 5% CO2. Once confluent, cardiac fibroblasts were detached with trypsin and either used directly in experiments or passaged one more time to use in experiments. Confirmation of purity was confirmed using these markers, or GFP, in every experiment.
Dendritic cell preparation
Bone marrow was flushed from femurs and tibias of WT mice. Red blood cells were lysed with Tris ammonium chloride buffer (Roche) and cells were cultured in complete RPMI medium with recombinant GM-CSF (15 ng ml−1; PeproTech) for 7 d, with additional medium added on days 3 and 5. CD11c+ DC purity was >85% by FACS analysis.
CD4+ T cell preparation
CD4+ cells were isolated from spleen cell suspensions of OTII mice by positive selection with CD4 microbeads (Miltenyi Biotec) and further FACS sorted based on the expression of CD62L and CD44. Naïve CD4+ T cells were differentiated into TH1 cells by stimulation with plate-bound anti-CD3 (5 µg ml−1) and soluble anti-CD28 (1 µg ml−1) in the presence of interleukin (IL)-12 (0.01 µg ml−1), IL-2 (25 U ml−1) and anti-IL-4 (0.5 µg ml−1). On day 3 of stimulation, TH1 cultures were split at a ratio of 1:1 with fresh medium containing IL-2 (25 U ml−1). All cytokines and blocking antibodies were purchased from PeproTech. Differentiated T cells were collected a day later and immediately used for experiments. TH1 cell generation was confirmed by IFN-γ production upon PMA/ionomycin stimulation. Cells were labeled with 2 µM CFDA-SE tracer (Thermo Fisher) according to the manufacturer’s specifications.
In a 24-well plate, cardiac fibroblasts or BMDCs (100,000 cells per well) were allowed to adhere and were either pulsed for 4 h with 4 µg ml−1 OVA 323-339 (AnaSpec), or incubated overnight with 100 μg ml−1 OVA protein (Sigma), 100 μg ml−1 DQ OVA (Thermo Fisher) or OVA transformed E. coli (MOI 10). Cells were washed twice with PBS and co-cultured for 3 d with 1 million CFDA-SE-labeled OTII CD4 + T cells. For FACS sorting of purified CD44loCD62Lhi naïve CD4+ T cells, bulk splenocytes from OTII mice were stained with FITC-conjugated anti-CD4 (GK1.5), PE-conjugated anti-CD62L (MEL-14), and APC-conjugated anti-CD44 (IM7) as outlined below for FACS sorting on a BD FACSAria (BD Biosciences).
Quantitative flow cytometry
Flow cytometry was performed to analyze cell surface protein expression. The data were acquired on a BD LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software. LV digestion was performed by collagenase type II (0.895 mg ml−1) for 30 min at 37 °C. Cells were then stained with the following antibodies: FITC-conjugated anti-CD4 (GK1.5), APC-conjugated anti-CD4 (RM4-5), BV711-conjugated anti-CD45.2 (104), APC-conjugated anti-CD44 (IM7), PE-conjugated anti-CD62L (MEL-14), Alexa Fluor 488-conjugated anti-I-A/I-E, BV421-conjugated anti-CD80, PerCP/Cy5.5-conjugated anti-CD86 and PE-conjugated anti-CD31 (MEC13.3). All antibodies were purchased from BioLegend. APC-conjugated anti-feeder cells (mEF-SK4) were purchased from Miltenyi Biotec. Cells were surface stained by incubation with the relevant antibodies diluted in PBS + 2% FBS for 20 min at 4 °C, followed by two washes with PBS 2% FBS. When intracellular staining of signature cytokines was performed, cell suspensions were incubated overnight at 37 °C in the presence of 0.1% brefeldin A (BioLegend) and 0.1% monensin (BioLegend). After the incubation, surface staining was performed as indicated above, followed by cell fixation for 20 min at room temperature (RT) with fixation buffer (BioLegend). Upon fixation and washing with PBS 2% FBS, cell suspension was permeabilized with 1× Perm Wash buffer (BioLegend), and stained intracellularly for 30 min at RT in the dark. Absolute cell numbers were quantified using Precision Count Beads (BioLegend).
Cardiac fibroblasts were cultured on 0.1% gelatin-coated glass coverslips in a six-well plate until confluent and treated daily with 100 U ml−1 mouse IFN-γ (PeproTech) for 3 d. At the end of each treatment, cell layers were washed twice with PBS and fixed in 3% paraformaldehyde. Nonspecific binding was prevented by incubation with PBS containing 10% goat serum (Jackson ImmunoResearch) for 1 h. Cells were incubated with primary antibodies against vimentin (Abcam, ab20346) and MHCII (Invitrogen, 14-5321-82) at a 1:250 dilution. The cells were then incubated at 4 °C overnight and washed three times with PBS. As controls, parallel coverslips were incubated with no primary antibody. Alexa Fluor 568-conjugated goat anti-mouse (Invitrogen, A-11004) and Alexa Fluor 488-conjugated goat anti-rat (Invitrogen, A48262) at a 1:500 dilution were used as secondary antibodies. Visualization was performed with a Nikon Ti inverted fluorescence microscope.
Acute T. cruzi Infection
Eight-week-old WT mice were infected with 20,000 T. cruzi trypomastigotes (Colombiana strain, discrete typing unit TcI, the most widely distributed in Colombia). Trypomastigotes were collected from infected Vero cells (American Type Culture Collection), collected from culture supernatant, purified by differential centrifugation, and resuspended in PBS before intraperitoneal injection in mice. Mice were euthanized at the peak of parasitemia, at 19 d after infection.
Mouse model of transverse aortic constriction
Pressure overload was induced by minimally invasive TAC surgery to constrict the transverse aorta (26 G, 0.51 mm outer diameter) of randomized 8- to 10-week-old male mice to induce HF as previously described38. Sham-operated mice underwent the same procedure but without aortic constriction. Four weeks after surgery, mice were euthanized, and tissue was collected for further analysis.
To induce Cre recombinase activity, Tcf21iCre/+MhcIIfl/fl mice were treated with tamoxifen (20 mg ml−1; Millipore Sigma, T5648) dissolved in sunflower oil using 10% ethanol for 5 consecutive daily i.p. injections at 75 mg per kg body weight. After TAC surgery, mice were maintained on a diet containing 400 mg per kg body weight tamoxifen citrate (Envigo, TD.55125) for 4 weeks until collection.
In vivo echocardiography
In vivo transthoracic echocardiography was assessed 1 d before tissue collection. Mice were lightly sedated at 1–2% isoflurane in medical oxygen (0.7 l min−1), on a heated stage in the supine position as previously described38. Heart rates and respiratory rates were continuously monitored via the stage electrodes. Depilatory cream (Nair) was applied to the chest to remove fur and ultrasonic gel was applied to the 22–55-MHz echocardiography transducer (MS550D; Vevo 2100, FUJIFILM VisualSonics) to obtain parasternal short-axis views of the LV in M-mode with a target heart rate of 400–500 b.p.m. LV parameters and heart rate (Table 1) were measured by averaging values obtained from five cardiac cycles. All analyses were performed blindly using Vevo 2100 software (v3.1.1, FUJIFILM VisualSonics).
Immunohistochemistry, and immunofluorescence of cardiac tissue were performed in midpapillary LV tissue, cut into 5-μm sections. Frozen LV sections were incubated with primary antibody against mouse CD4 (BioLegend, clone GK1.5) for 1 h (1:500 dilution) followed by incubation with goat anti-rat biotinylated secondary antibody (1:300 dilution; Jackson ImmunoResearch, 112-065-062). Sections were incubated with Streptavidin-HRP (DAKO, K0675) and visualized using AEC Substrate-Chromogen. CD4+ T cells were quantified by manually scanning the entire LV tissue section and counting stained cells per section. LV sections from tissue fixed in 10% formalin and embedded in paraffin were stained with FITC-conjugated wheat germ agglutinin (Millipore Sigma, L4895) at 5 μg ml−1 to determine cardiomyocyte cross-sectional area by tracing the outline of at least 25 myocytes per section with NIS-Elements software. Antibodies to α-SMA (Sigma, A2547; 1:250 dilution), and to CD4 (BioLegend, 100401; 1:25 dilution), and MHCII (Invitrogen, 14-5321-82; 1:250 dilution), were used in immunofluorescence staining of LV tissue of WT and Tcf21iCre/+; R26eGFP mice subjected to sham and TAC. Fixed sections were stained with Picrosirius red to determine the percentage fibrotic area quantified using the National Institutes of Health (NIH) ImageJ software. All analyses were performed blindly.
Data are presented as the mean ± s.d. Statistical analyses were done by Student’s unpaired t-test (two-tailed) or nonparametric Mann–Whitney test (two-tailed) to adjust for nonequal Gaussian distribution when comparing two groups. Multiple-group comparisons were performed by one-way ANOVA and Tukey’s post hoc test, where indicated, using GraphPad Prism software. Differences were considered statistically significant at P ≤ 0.05.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
All data used to generate figures are included in the accompanying files.
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These studies were supported by NIH grants R01 HL123658 and R01 HL144477 (to P.A.), NIH T32 HL 69770, NIH T32AI007077-34, NIH F31HL140883 and the American Heart Association grant 18PRE34020084 (to N.N.), NIH R01 HL141187 and HL142624 (to J.D.) and NIH T32AG066574 (to D.B.).
The authors declare no competing interests.
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Extended Data Fig. 1 Cardiac fibroblasts express MHCII in vivo in response to acute T. cruzi infection.
(A) Wt mice were inoculated with 20,000 T. cruzi parasites by intraperitoneal injection and the hearts were harvested at 19 days post-infection. (B-E) Ventricular CD31-CD45-MEFSK4+ cardiac fibroblasts were analysed by flow cytometry (≥ 5,000 target cells acquired) to determine surface expression of (B-C) MHCII and (D-E) CD80. n= 4 (mock) and n=3 (Chagas) mice. Error bars represent mean ± SD. (* p≤0.05; *** p<0.001; Mann-Whitney test, two tailed).
Detection of Intact allele (310bp), recombined alleles (475 bp) and GAPDH(156 bp) in sorted CD31-CD45-MEFSK4+ Fibroblasts and leukocytes from Tcf21iCre/+MhcIIfl/fl mice treated with vehicle or Tamoxifen. GAPDH was used as loading control.
Extended Data Fig. 3 Tamoxifen treatment of Tcf21iCre/+/ MhcIIflox/flox mice reduces MHCII surface protein expression specifically in cardiac fibroblasts.
Transverse aortic constriction (TAC) surgery was performed on vehicle and Tamoxifen treated Tcf21iCre/+MhcIIfl/fl mice and the left ventricle was harvested after 4 weeks, digested, stained and analysed by flow cytometry. (A) Representative FACS plot showing gating of CD31+CD45-MESFK4- endothelial cells, CD31-CD45+MESFK4- leukocytes and CD31-CD45-MESFK4+ cardiac fibroblasts. (B-C) Representative FACS plots (B) and quantification of mean fluorescence intensity (MFI) (C) of MHC-II expression in endothelial cells (top), leukocytes (middle) and cardiac fibroblasts (bottom) in the hearts of vehicle and tamoxifen treated mice. n=4 mice/group. Error bars represent mean ± SE. (* p<0.05; Mann-Whitney test, two tailed. C: p value=0.0286).
Extended Data Fig. 4 Cardiac fibroblast MHCII expression does not affect CD4+ T cell activation in the mLNs in response to TAC.
Transverse aortic constriction (TAC) surgery was performed on vehicle and Tamoxifen treated Tcf21iCre/+MhcIIfl/fl mice and the mediastinal lymph nodes were harvested after 4 weeks, stained and analysed by flow cytometry for the indicated T cell activation markers. (A) Representative FACS plot showing CD62L and CD44 staining. (B-C) quantification of CD62LlowCD44hi effector T CD4+ T cell numbers (B) and frequency (C) in vehicle and tamoxifen treated TAC mice. n=5 mice (vehicle) and n=6 mice (tamoxifen). Error bars represent mean ± SD. (Mann-Whitney test, two tailed. C: p value=0.0286).
Extended Data Fig. 5 Cardiac fibroblast MHCII expression contributes to cardiomyocyte hypertrophy and to cardiac fibroblast abundance in response to pressure overload.
Transverse aortic constriction (TAC) was performed on vehicle and Tamoxifen treated Tcf21iCre/+MhcIIfl/fl mice and harvested after 4 weeks. Conditional deletion of MhcII on cardiac fibroblasts was induced by administering Tamoxifen via intraperitoneal injections prior to TAC surgery, followed by Tamoxifen citrate chow for 4 weeks. (A) Wheat germ agglutinin (WGA) staining of frozen LV tissue sections was performed and used to calculate (B) mean cardiomyocyte area. Representative images of n=3 hearts (Sham), n=4 (TAC vehicle) and n=7 (TAC TMX). (C) Gross LV mass was acquired and is shown normalized to tibia length. (D-E) LV tissue was digested, stained for CD31 and MESFK4 and analysed by flow cytometry. Representative FACS plot (D) and quantification (E) of vehicle and tamoxifen treated mice hearts (n=4 mice/group). Error bars represent mean ± SE. (* p<0.01; Mann-Whitney test, two tailed: E: p=0286).
Extended Data Fig. 6 Schematic Diagram showing bidirectionality of cardiac fibroblast and Th1 cell interactions.
Cardiac fibroblasts express the costimulatory molecule CD80 and efficiently capture and process extracellular antigens into small peptides that are uploaded into MHC-II molecules. In response to IFNγ stimulation, cardiac fibroblasts express MHCII and present peptide antigens that induce IFNγ + Th1 cell immune responses and promote cardiac remodelling and dysfunction.
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Ngwenyama, N., Kaur, K., Bugg, D. et al. Antigen presentation by cardiac fibroblasts promotes cardiac dysfunction. Nat Cardiovasc Res 1, 761–774 (2022). https://doi.org/10.1038/s44161-022-00116-7