Arthritis and valvular carditis coexist in several human rheumatic diseases, including systemic lupus erythematosus, rheumatic fever, and rheumatoid arthritis. T cell receptor–transgenic K/BxN mice develop spontaneous autoantibody-associated arthritis and valvular carditis. The common Fc receptor γ (FcRγ) signaling chain is required for carditis to develop in K/BxN mice. FcRγ pairs with numerous receptors in a variety of cells. The aim of this study was to identify the FcRγ-associated receptors and Fcγ receptor (FcγR)–expressing cells that mediate valvular carditis in this model.
We bred K/BxN mice lacking the genes that encode activating Fcγ receptors (FcγRI, FcγRIII, and FcγRIV), and we assessed these mice for valvular carditis. We similarly evaluated complement component C3–deficient K/BxN mice. Immunohistochemistry, bone marrow transplantation, and macrophage depletion were used to define the key FcRγ-expressing cell type.
Genetic deficiency of only one of the activating Fcγ receptors did not prevent carditis, whereas deficiency of all 3 activating Fcγ receptors did. Further analysis demonstrated that FcγRIII and FcγRIV were the key drivers of valve inflammation; FcγRI was dispensable. C3 was not required. FcRγ expression by radioresistant cells was critical for valvular carditis to develop, and further analysis indicated that macrophages were the key candidate FcγR-expressing effectors of carditis.
FcγRIII and FcγRIV act redundantly to promote valvular carditis in K/BxN mice with systemic autoantibody-associated arthritis. Macrophage depletion reduced the severity of valve inflammation. These findings suggest that pathogenic autoantibodies engage Fcγ receptors on macrophages to drive valvular carditis. Our study provides new insight into the pathogenesis of cardiovascular inflammation in the setting of autoantibody-associated chronic inflammatory diseases.
Several systemic autoimmune diseases characterized by autoantibody production, including systemic lupus erythematosus (SLE), antiphospholipid syndrome, acute rheumatic fever, and rheumatoid arthritis (RA), affect both the synovial joints and the heart ([1-3]). Most notable is the increased cardiovascular morbidity and mortality due to atherosclerotic coronary artery disease among patients with SLE and RA. This increased risk is not fully explained by traditional risk factors, strongly suggesting that chronic inflammatory diseases themselves contribute to poor cardiovascular outcomes ([4-6]). Cardiac manifestations of these diseases also include valvular carditis (). The immunologic mechanisms by which these diseases provoke inflammation of the joints and cardiovascular system remain poorly understood.
T cell receptor (TCR)–transgenic K/BxN mice develop spontaneous, fully penetrant, autoantibody-associated arthritis and valvular carditis ([7, 8]). The valve inflammation in these mice shares several pathologic features with the valvular carditis found in patients with rheumatic conditions. Specifically, it affects left-sided valves and is characterized by IgG and complement C3 binding to the valves and a cellular infiltrate comprising predominantly T cells and mononuclear myeloid cells ([2, 7, 9, 10]). In these mice, neutrophils are not found in the inflamed valves (). This mouse model is therefore well-suited for research into how systemic autoimmune inflammatory diseases drive cardiac pathology.
Autoimmunity in K/BxN mice is initiated by a breach of immunologic self-tolerance, when T lymphocytes bearing the KRN TCR transgene recognize peptides derived from the ubiquitously expressed antigen glucose-6-phosphate isomerase (GPI), which is presented by the class II major histocompatibility complex (MHC) molecule I-Ag7 ([8, 11]). This ultimately leads to the sustained production of anti-GPI IgG autoantibodies. Transfer of anti-GPI autoantibodies causes arthritis in recipient mice (). Interruption of any of the events leading to the production of autoantibodies abrogates the development of both arthritis and valvular carditis (). However, the downstream immune effector mechanisms responsible for arthritis and carditis in this model differ. Specifically, arthritis requires complement component C5 but not the common Fc receptor γ signaling chain (FcRγ). Conversely, valvular carditis requires FcRγ but not C5 ().
FcRγ is an immunoreceptor tyrosine–based activation motif (ITAM)–containing signaling molecule that pairs with activating Fcγ receptors and several other types of receptors; it is required for cell surface expression of the receptors and for signal transduction ([13-15]). Fcγ receptors bind the Fc portion of IgG ([13, 15]). There are 2 general categories of Fcγ receptors: activating and inhibitory. In mice, the activating Fcγ receptors are FcγRI, FcγRIII, and FcγRIV. These receptors have unique IgG-binding α-chains, but share the common γ signaling chain, FcRγ. The inhibitory receptor, FcγRII, does not associate with FcRγ. Activating Fcγ receptors have distinct cellular expression patterns, predominantly on myeloid cells. They bind the different IgG subtypes with varying affinities: FcγRI and FcγRIV predominantly bind IgG2a/c and IgG2b, whereas FcγRIII binds IgG1 more strongly than it binds IgG2a/c and IgG2b ([13, 15]). The knowledge that the common γ signaling chain FcRγ is required for valvular carditis in K/BxN mice led to 2 hypotheses: 1) that one or more of the activating Fcγ receptors is required or 2) that a different FcRγ-associated receptor is involved. Herein we used a genetic approach to examine these possibilities, and we also studied the FcRγ-expressing cells that are key drivers of valvular carditis in K/BxN mice.
MATERIALS AND METHODS
KRN TCR–transgenic mice on a C57BL/6 (B6) background and B6 mice congenic for H-2g7 (B6.g7) ([7, 8]) were gifts from Diane Mathis and Christophe Benoist (Harvard Medical School, Boston, MA and Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France). C5-deficient B6 mice congenic for the NOD-derived Hc allele (encoding nonfunctional C5) ([7, 16]) were also a gift from Drs. Mathis and Benoist. C3-deficient mice on a B6 background () were a gift from Michael Carroll (Harvard Medical School, Boston, MA). Mice on a B6 background that were deficient in the gene for FcRγ (Fcer1g) () were purchased from Taconic. Mice with targeted deletion of Fcgr1 have been previously described (). Mice with targeted deletion of Fcgr4 were a gift from Jeffrey Ravetch (The Rockefeller University, New York, NY) and have been previously described ([19, 20]). Generation and characterization of FcγRII/FcγRIII/FcγRIV-deficient mice and FcγRI/FcγRII/FcγRIII/FcγRIV-deficient mice will be described elsewhere (Verbeek JS: unpublished observations). Notably, Fcgr2, Fcgr3, and Fcgr4 are in close proximity on mouse chromosome 1, and this entire region was targeted; Fcgr1 is on mouse chromosome 3. Fcgr3-deficient mice on a B6 background (Fcgr3tm1Sjv; stock no. 003171), B6 mice congenic for CD90.1 (B6.PL-Thy1a/Cy3; stock no. 000406), and Rag1-deficient B6 mice (Rag1tm1Mom; stock no. 002216) ([21, 22]) were purchased from The Jackson Laboratory.
Mice with the described targeted alleles of the genes that encode the various Fcγ receptors and complement components were interbred with KRN mice and B6.g7-congenic mice to generate mice for this study. For ease of nomenclature, we refer to KRN+H-2b/g7 mice as “K/BxN mice” (). Genotype was determined by polymerase chain reaction for all mice and confirmed by flow cytometry for the FcγR-deficient mice. Mice were bred in specific pathogen–free colonies and maintained at the University of Minnesota as per Institutional Animal Care and Use Committee–approved protocols (0909A72086 and 1207A17481).
Antibodies used for flow cytometry included anti-CD3 (145-2C11) and anti-CD4 (RM4-5) (BioLegend), anti-CD4 (RM4-5), anti-CD90.1 (HIS51), anti-CD44 (IM7), anti–interferon-γ (anti-IFNγ) (XMG1.2), anti–interleukin-17 (anti–IL-17) (eBioTc11-18H10.1), anti-CD62L (MEL-14), anti-CD16/32 (clone 93), anti–Gr-1 (RB6-8C5), anti-F4/80 (BM8), and anti-B220 (RA3-6B2) (eBioscience), anti-Vβ6 (RR4-7), anti-CD11b (M1/70), and anti-CD64 (X54-5/7.1) (BD PharMingen), and anti–Armenian hamster IgG (Jackson ImmunoResearch). The anti-FcγRIV–specific antibody (9E9) was generously provided by Jeffrey Ravetch and has been previously described ().
Additional antibodies used for immunohistochemical analysis included anti-CD64 (N-19) (Santa Cruz Biotechnology), anti-CD16/32 (clone 93) and biotinylated anti-F4/80 (BM8) (eBioscience), anti-CD11c (N418) and anti-CD31 (clone 390) (BioLegend), and anti-Langerin (929F3.098) (Imgenex). Biotin-conjugated antibodies recognizing IgG1, IgG2b, and IgG2c were from Jackson ImmunoResearch. Secondary antibodies included bovine anti-goat DyLight 594 and streptavidin Alexa Fluor 488 (Jackson ImmunoResearch), goat anti-rabbit DyLight 594 (Poly4054) and goat anti–Armenian hamster DyLight 594 (Poly4055) (BioLegend), and streptavidin DyLight 550 (Thermo Scientific).
Assessment of arthritis, anti-GPI titers, and histology
Arthritis was assessed as previously described ([24, 25]). Serum anti-GPI total IgG titers and IgG subtype (IgG1, IgG2b, IgG2c, and IgG3) titers were measured as described (). Except where indicated, cardiac valves of 8–10-week-old mice were assessed histopathologically.
Immunohistochemistry and toluidine blue staining
Frozen sections were first blocked for Fcγ receptors and with avidin/biotin (Invitrogen), when necessary. Sections were incubated with the unconjugated primary antibody recognizing the Fcγ receptor of interest (anti-CD64, anti-CD16/32, or FcγRIV-specific antibody) or with appropriate isotype controls, and fluorescence-conjugated secondary antibodies were used for detection of Fcγ receptors. For the detection of CD11c, CD31, and Langerin, frozen sections were stained with the Alexa Fluor 488–conjugated antibodies described above or with appropriate isotype controls. For the detection of F4/80, frozen sections were stained with biotinylated anti-F4/80 followed by fluorescent strepavidin. Nuclei were counterstained with DAPI. Immunohistochemical analysis of IgG1, IgG2b, and IgG2c was performed using biotinylated primary antibodies (Jackson ImmunoResearch) or the appropriate isotype controls. The primary antibodies were detected by application of ImmPACT 3,3′-diaminobenzidine peroxidase substrate with an avidin–biotin–peroxidase kit (Vector). Mast cells were detected in frozen sections by staining with toluidine blue according to the toluidine blue–staining protocol (IHC World). Slides were viewed on an Olympus BX51 microscope equipped with a digital camera and DP-BSW software.
Intracellular cytokine staining for IL-17 was performed according to the instructions of the manufacturer (eBioscience). Flow cytometry was performed using a FACSCalibur and an LSRII (BD Biosciences), and cells were analyzed using FlowJo version 8.8.7 software (Tree Star).
Bone marrow transplantation
Nine–15-week-old Rag1-deficient recipient mice were irradiated with 300 rads. Four hours following irradiation, 3 × 106 donor bone marrow cells were injected intravenously. Hearts were harvested 8 weeks after bone marrow transplantation.
After labeling with carboxyfluorescein succinimidyl ester (CFSE), 5 × 106 donor cells were injected intravenously into H-2g7–expressing recipient mice. Lymphocytes were harvested from recipient mice 40 hours later and analyzed by flow cytometry.
Three-week-old K/BxN mice received weekly intraperitoneal injections (from weeks 3 to 7) of clodronate liposomes or control phosphate buffered saline (PBS)–containing liposomes (100 μl/10 gm of body weight) (ClodronateLiposomes.com). Mice underwent weekly assessment for arthritis. Hearts were harvested at 8 weeks and assessed for carditis. The presence of splenic macrophages was analyzed by flow cytometry.
Pooled serum (150 μl/dose) from K/BxN mice was injected intraperitoneally into recipient mice on days 0 and 2. The mice were monitored for 10 days for the development of arthritis.
Repeated-measures analysis of variance was used to compare arthritis severity scores. Tukey's multiple comparison post hoc test was used when >2 groups were compared. Data on mitral valve thickness were compared by Mann-Whitney 2-tailed U test. All other statistical differences between the mean values were calculated using Student's unpaired 2-tailed t-test. P values less than 0.05 were considered significant.
Antibody production, T cell activation, and antigen presentation occur normally in FcRγ-deficient K/BxN mice
Protection against valvular carditis in K/BxN mice lacking FcRγ (Fcer1g) despite normal production of anti-GPI IgG antibodies has previously been demonstrated (). We found no difference in the production of anti-GPI IgG subtypes (i.e., no difference in titers of IgG1, IgG2b, IgG2c, and IgG3) in FcRγ-deficient K/BxN mice as compared with the production of anti-GPI IgG subtypes in wild-type K/BxN mice (Figure 1A). Because CD4+ T cells are key contributors to the valve pathology in this model (), we questioned whether these cells were activated normally in FcRγ-deficient K/BxN mice. Indeed, we found no difference in T cell expression of CD44 and CD62L or in intracellular cytokine production (IFNγ and IL-17) (Figures 1B and C). KRN T cells that were transferred into FcRγ-deficient or FcRγ-sufficient B6.g7 mice proliferated equivalently (Figure 1D). We therefore concluded that the protection against valvular carditis afforded by FcRγ deficiency in K/BxN mice is not due to impaired T cell or B cell activation or autoantibody synthesis, but rather to later effector events.
Complement is not required for the development of valvular carditis
Because autoantibodies appear critical for the effector phase of valvular carditis, we investigated whether antibody-mediated activation of the complement system was pathogenically important. Previously it was shown that complement component C5 is not required for valvular carditis in this model (). It remained possible, however, that the upstream complement component C3 could play a role, for instance, by binding to receptors for C3 split products, particularly since C3 is bound to the inflamed valves (). We therefore bred K/BxN mice carrying null alleles of the genes encoding FcRγ and/or complement components C3 or C5. We found that C3, like C5, was not necessary for the development of valvular carditis (Figure 2). These findings confirm that the autoantibody-associated valve inflammation in this model depends on FcRγ and not the complement system.
Presence of IgG- and FcγR-expressing cells in the inflamed K/BxN mouse mitral valve
Given the requirement for FcRγ in the development of valvular carditis, we used immunohistochemistry to probe inflamed mitral valves for the presence of individual activating Fcγ receptors and for the various IgG subtypes. Although IgG1 is the predominant autoantibody subtype produced in K/BxN mice, we found that IgG1, IgG2b, and IgG2c were all bound to the inflamed mitral valve () (Figure 3A). We also detected expression of each of the activating Fcγ receptors in the inflamed cardiac valve (Figure 3B). These findings supported the notion that any or all of the activating Fcγ receptors could be the key driver(s) of valvular carditis in K/BxN mice.
No protection against valvular carditis in K/BxN mice lacking single activating Fcγ receptors
We next investigated if the absence of any one of the activating Fcγ receptors protected against valve inflammation. Mice with null alleles of the genes encoding the α-chains of each of the activating Fcγ receptors (Fcgr1−/−, Fcgr3−/−, and Fcgr4−/−) were bred with K/BxN mice and assessed for the development of valvular carditis. We found that K/BxN mice lacking expression of only one of the activating Fcγ receptors developed valvular carditis equivalent in severity to the valvular carditis seen in controls (Figure 3C). As expected, these mice also developed arthritis comparable in severity to the arthritis seen in controls (data not shown), since absence of the gene encoding FcRγ did not reduce the severity of spontaneous arthritis in K/BxN-transgenic mice ().
Because no single activating FcγR was solely responsible for the development of valvular carditis, we considered the possibilities that more than one activating FcγR was contributing (i.e., redundancy) or that an alternative FcRγ-associated receptor, not in the FcγR family, was responsible. To discriminate between these hypotheses, we used recently developed mice that lacked all of the FcγR α-chain genes (Fcgr1, Fcgr2, Fcgr3, and Fcgr4) (Figure 4A). Although the common signaling chain FcRγ is not required for spontaneous arthritis in K/BxN-transgenic mice (), it is required for arthritis that is induced by passive transfer of serum from K/BxN mice into naive recipients ([27, 28]). Similarly, complete protection against serum-transfer arthritis in these new mice lacking all of the FcγR α-chain genes (Figure 4B) confirmed the expected functional defect that the absence of activating Fcγ receptors engenders in the serum transfer model.
FcγRIII and FcγRIV are key mediators of valvular carditis
We bred K/BxN mice lacking FcγRI, FcγRII, FcγRIII, and FcγRIV and found that protection against valvular carditis was evident (Figure 5). Furthermore K/BxN mice lacking only FcγRII/FcγRIII/FcγRIV were also protected, whereas FcγRI deficiency had no effect (Figure 5). Arthritis severity scores were not different between these various groups, with one exception: the Fcgr1+/–,Fcgr2-4–/– group had slightly lower scores than the Fcgr1+/–,Fcgr2-4+/– group (P = 0.044) (data not shown). K/BxN mice deficient in Fcgr2–4 also had slightly lower levels of total anti-GPI IgG than mice sufficient for Fcgr2–4 (data not shown), a somewhat unexpected finding since absence of the inhibitory receptor FcγRII often, but not always, leads to increased autoantibody titers ([29, 30]). These decreases in arthritis severity and antibody titers were slight compared with the dramatic protection against valvular carditis. These findings demonstrate that the FcRγ-associated activating receptors FcγRIII and FcγRIV are the key drivers of cardiac valve inflammation in this model—there is no need to invoke roles for other FcRγ-associated receptors.
FcRγ expression on radioresistant cells promotes valvular carditis
To explore which FcRγ-expressing cells are required for the development of valvular carditis, we performed a reciprocal bone marrow transplantation experiment. We transplanted bone marrow from FcRγ-sufficient or FcRγ-deficient K/BxN mice into sublethally irradiated FcRγ-sufficient or FcRγ-deficient Rag1-deficient host mice. We found that recipient mice lacking FcRγ developed less severe valvular carditis as compared to controls, whereas the FcRγ status of the donor did not influence carditis severity (Figure 6A). Therefore, FcR γ-chain expression of radioresistant host cells, rather than of radiosensitive bone marrow–derived cells, contributed to the development of valvular carditis in K/BxN mice. Candidate cells included mast cells, Langerhans' cells, dendritic cells (DCs), macrophages, and endothelial cells, based on the described radioresistance of subpopulations of these cell types ([31-36]).
FcγR-expressing cells in inflamed mitral valves of K/BxN mice
We next explored which of the candidate cell types were present in the inflamed K/BxN mouse mitral valves and colocalized with one or more of the activating Fcγ receptors. We found that FcγRI (CD64), FcγRII/FcγRIII (CD16/32), and FcγRIV all colocalized with F4/80, a macrophage marker, on the inflamed mitral valve (Figure 6B). We could not detect Langerhans' cells or mast cells in the inflamed mitral valves (Figure 6B). None of the Fcγ receptors colocalized with CD31, an endothelial cell marker, as shown in Figure 3. CD11c, a marker of activated macrophages and DCs, colocalized with FcγRII/FcγRIII and also with F4/80 (Figure 6C), but not with FcγRI or FcγRIV (results not shown). These data indicated that the most likely FcγR-expressing cells mediating valvular carditis are radioresistant macrophages and/or closely related “macrophage-like” inflammatory DCs. Initial activation of KRN T cells depends on conventional DCs and other professional antigen-presenting cells (APCs) (). Our finding that KRN T cells were activated normally in the absence of FcRγ (Figure 1) therefore suggests that FcγR expression by conventional DCs is not required for KRN T cell activation. This indicates that FcγR-expressing macrophages or similar cells have a key role in effecting carditis.
We used clodronate liposomes to deplete macrophages from K/BxN mice, starting at 3 weeks of age, and assessed the mice for the development of valvular carditis at 8 weeks of age. K/BxN mice treated with clodronate liposomes developed less severe valvular carditis compared with mice treated with control PBS-containing liposomes (Figure 6D). We confirmed the efficiency of macrophage depletion by flow cytometric enumeration of CD11b+F4/80+ splenic macrophages, which averaged 4.55 × 105 in mice treated with clodronate liposomes and 2.75 × 106 in mice treated with PBS-containing liposomes (P < 0.0001). Clodronate liposome treatment had no effect on arthritis severity or anti-GPI antibody titers (data not shown). These data suggest that macrophages or similar clodronate-sensitive cells are important cellular effectors of valve inflammation. Collectively, our findings are consistent with a model in which pathogenic autoantibodies engage FcγRIII and FcγRIV on macrophages to drive the development of valvular carditis in K/BxN mice.
Studies in both humans and rodents on the pathogenesis of valvular carditis in rheumatic heart disease, antiphospholipid antibody syndrome, and Libman-Sacks endocarditis have demonstrated the deposition of IgG and complement bound in the subendothelial connective tissue of the valve, as well as the presence of CD4+ T cells and macrophages ([2, 9, 10, 37, 38]). The K/BxN mouse model of inflammatory arthritis and valvular carditis recapitulates these characteristics. We have demonstrated the power of the K/BxN mouse model in the investigation of the immunologic mechanisms that mediate cardiovascular pathology in the setting of systemic autoantibody-associated inflammatory disease. Despite the presence of complement C3 bound to the inflamed valves in K/BxN mice, we found that C3 was not necessary for the development of valvular carditis. Rather, our results suggest that pathogenic antibodies produced by the adaptive immune system engage activating Fcγ receptors on innate immune effector cells to drive the development of valvular carditis.
In this study, we found that no activating FcγR (FcγRI, FcγRIII, or FcγRIV) was solely responsible for the development of valvular carditis in K/BxN mice. This is not surprising given the diversity of FcγR-expressing cells and the presence of multiple IgG subtypes in the inflamed mitral valves. Our findings are consistent with other examples of autoimmune disease that require the contribution of more than one activating FcγR. For example, in a passive mouse model of autoimmune hemolytic anemia induced by IgG2a and IbG2b subclasses of the anti-erythrocyte antibody, FcγRI, FcγRIII, and FcγRIV all contributed to a severe anemia phenotype induced by IgG2a antibodies, while FcγRIII and FcγRIV, but not FcγRI, were required for both the mild and severe anemia phenotypes induced by IgG2b antibodies (). Similarly, in a murine model of acute glomerular inflammation induced by switch variant monoclonal antibodies, FcγRIII and FcγRIV were required, whereas FcγRI was dispensable (). Our results show that FcγRIII and FcγRIV drive the development of valvular carditis in K/BxN mice and that FcγRI is not necessary. Because FcγRIV does not bind to IgG1 (), these findings suggest that although IgG1 is the predominant IgG subtype produced in K/BxN mice, other IgG subtypes (specifically IgG2b and/or IgG2c) are also pathogenically important mediators of carditis.
Our conclusion that macrophages are the critical FcγR-expressing cell type driving the development of valvular carditis is based on our findings that a radioresistant FcγR-expressing cell type is involved, that clodronate liposomes deplete this key cell type, and that macrophage markers colocalize with the activating Fcγ receptors in the inflamed mitral valves. Could DCs also be involved? This question is complicated by the fact that the cell surface markers CD11b, F4/80, and CD11c can be expressed both by activated macrophages and by monocyte-derived DCs; our histologic studies cannot distinguish these cell types () (Figure 6). Furthermore, clodronate liposomes can deplete some DC populations ([41-43]). We consider it unlikely that conventional DCs play a role, based on our finding that professional APC–mediated activation of KRN T cells occurred normally in the absence of FcRγ (Figure 1). The absence of inflammatory cells in the native mitral valve strongly suggests that the CD11b+F4/80+CD11c+ cells present in the inflamed valve are recruited from the circulating blood. Circulating monocytes give rise not only to macrophages but also to inflammatory DCs or tumor necrosis factor/inducible nitric oxide synthase–producing DCs, with phenotypic and functional characteristics that overlap considerably with those of activated macrophages (), leading some investigators to assert that the distinction between these subsets is artificial ().
Our data support the conclusion that the key FcγR-expressing cell types driving the development of valvular carditis in K/BxN mice are macrophages and/or phenotypically and functionally similar monocyte-derived DCs. We therefore postulate that autoantibodies engage FcγRIII and FcγRIV on these cells to drive the development of valvular carditis in K/BxN mice. We are currently investigating how activating FcγR engagement influences macrophage function, with particular attention to defining which of the proinflammatory products are critical mediators of carditis.
We have previously shown that CD4+ T cells are key effectors of valvular carditis in this model (). Our new findings support the notion that CD4+ T cells and macrophages cooperate to provoke cardiac inflammation. Future investigations will focus on evaluating how these cell types are recruited into the valve tissue and on characterizing their molecular interactions.
Our findings provide important insight that can be used to guide the rational choice of agents to treat cardiovascular inflammation in the setting of systemic autoantibody-associated diseases. Interventions that modulate Fc receptor function, such as intravenous immunoglobulin, may be attractive (). Alternatively, the FcγR-expressing effector cells could be eliminated. Additionally, the key Fcγ receptors themselves, or their downstream intracellular signaling molecules, could be targeted. Because the activating Fcγ receptors rely on spleen tyrosine kinase (Syk) for signal transduction, Syk inhibitors, such as fostamatinib (R788) or R406, might be good candidates for the treatment of autoantibody-associated cardiovascular inflammation ([47, 48]). Future investigations to determine which proinflammatory pathways are activated by FcγR engagement on macrophages (or other cell types) to drive the development of valvular carditis in these mice are expected to reveal additional potential therapeutic targets.
In summary, our findings delineate several previously unreported key features in the pathogenesis of autoimmune valvular carditis in K/BxN mice. FcRγ deficiency does not affect the T cell–dependent and B cell–dependent initiation phase in the development of valvular carditis, but rather it affects the downstream effector phase. The activating receptors FcγRIII and FcγRIV drive valvular carditis, while FcγRI is dispensable. Moreover, macrophages contribute critically to the development of carditis in this model. Our findings provide new insight into the pathogenesis of cardiovascular inflammation in the setting of autoantibody-associated chronic inflammatory diseases and offer new directions from which to pursue research on basic pathogenic mechanisms and rational therapeutic approaches.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Binstadt had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Hobday, Auger, Verbeek, Binstadt.
Acquisition of data. Hobday, Auger, Schuneman, Haasken, Binstadt.
Analysis and interpretation of data. Hobday, Haasken, Verbeek, Binstadt.
We thank Drs. Diane Mathis, Christophe Benoist, and Michael Carroll for the mice used in this study. We also thank Jeffrey Ravetch for the mice used in this study and for the anti-FcγRIV specific antibody (9E9).