FcγRIIb on CD11c⁺ cells modulates serum cholesterol and triglyceride levels and differentially affects atherosclerosis in male and female Ldlr^−/− mice

Abstract

Background and aims:

Circulating levels of oxidized lipoprotein (oxLDL) correlate with myocardial infarction risk and atherosclerosis severity. Our previous study demonstrates that oxLDL immune complexes (oxLDL-ICs) can signal through FcγRs on bone marrow-derived dendritic cells (BMDCs) and enhance their activation and inflammatory cytokine secretion. While global FcγR^−/− studies have shown that activating FcγRs are proatherogenic, the role of the inhibitory FcγRIIb is unclear. We sought to determine the role of DC-specific FcγRIIb in atherosclerosis.

Methods:

Bone marrow chimeras were generated by rescuing lethally irradiated Ldlr^−/− mice with hematopoietic cells from littermate CD11c-Cre⁺ or CD11c-Cre^- Fcgr2b^fl/fl donors. Four weeks following transplant, recipients were placed on a Western diet for eight weeks. Various tissues and organs were analyzed for differences in inflammation.

Results:

Quantitation of atherosclerosis in the proximal aorta demonstrated a 58% increase in female CD11c-Cre⁺ Fcgr2b^fl/fl recipients, but a surprising 44% decrease in male recipients. Hepatic cholesterol and triglycerides were increased in female CD11c-Cre⁺ Fcgr2b^fl/fl recipients. This was associated with an increase in CD36 and MHC Class II expression on hepatic CD11c⁺CD11b⁺ DCs in female livers. In contrast, male CD11c-Cre⁺ Fcgr2b^fl/fl recipients had decreased hepatic lipids with a corresponding decrease in CD36 and MHC Class II expression on CD11c⁺ cells. Interestingly, both sexes of CD11c-Cre⁺ Fcgr2b^fl/fl recipients had significant decreases in serum cholesterol and TGs with corresponding decreases in liver Fasn transcripts.

Conclusions:

The absence of FcγRIIb expression on CD11c⁺ cells results in sex-dependent alteration in liver inflammation influencing atherogenesis and sex-independent modulation of serum cholesterol and TGs.

Graphical Abstract

Introduction

Cardiovascular disease (CVD) leads all-cause mortality in both men and women in the U.S, claiming more lives than cancer and chronic respiratory lung disease combined. The American Heart Association predicts that by 2035, over 130 million American adults (45.1%) will have some form of CVD.¹ Atherosclerosis is a chronic inflammatory disease of the vessel wall that underlies the vast majority of CVD disorders. Cholesterol, and in particular, oxidized lipoproteins (oxLDL) are well established mediators of atherosclerotic plaque formation. Interestingly, up to 90% of circulating oxLDL can complex with specific antibodies to form oxLDL immune complexes (oxLDL-ICs).² These ICs have long been known to positively correlate with myocardial infarction risk, carotid atherosclerosis severity, and poor CVD outcomes.^{2, 3} Despite this, oxLDL-ICs have only recently been implicated in disease pathogenesis. OxLDL-ICs were found to be more potent primers of the NLRP3 inflammasome and elicited greater IL-1β production than free oxLDL in bone marrow-derived dendritic cells (DCs).⁴ However, the mechanism behind oxLDL-ICs enhanced inflammatory effects is unknown.

Our data suggest that oxLDL-ICs’ effects are partially mediated via Fc gamma receptors (FcγRs). FcγRs are found on antigen presenting cells (APCs) such as macrophages, DCs, and B-cells and have high affinity for the Fc region of IgG antibodies. In vitro studies demonstrate that oxLDL-ICs can signal through Fc receptors in both human macrophage cells lines and murine bone marrow-derived DCs, likely in concert with toll-like receptor 4 and the scavenger receptor CD36.^{4, 5} FcγRs can be either activating or inhibitory, containing an immunoreceptor tyrosine-based activation motif or an immunoreceptor tyrosine inhibitory motif, respectively.⁶ The activating murine FcγRs, FcγRI, FcγRIII, and FcγRIV, have been shown to be proatherogenic.⁷

The lone inhibitory FcγR, FcγRIIb, is well conserved across species, and is present on most immune cells.⁶ It is the only FcγR expressed on B-cells and has been studied most extensively in relation to autoimmune diseases. Global FcγRIIb^−/− mice develop a spontaneous lupus-like phenotype with elevated anti-nuclear antibody titers and glomerulonephritis.⁸ Additionally, polymorphisms in Fcgr2b are associated with susceptibility to systemic lupus erythematosus (SLE) and rheumatoid arthritis in humans.^9–11 Although the inhibitory FcγRIIb is expressed at lower levels compared to the activating FcγRs in bone marrow-derived DCs, several studies support a role for FcγRIIb in regulating DC function.⁴ In a bovine type II collagen-induced model, CD11c-conditional FcγRIIb knockout (KO) mice were more susceptible to developing arthritis.¹² Furthermore, FcγRIIb knockout mice demonstrated increased dermal DC migration to lymph nodes.¹³ DC FcγRIIb is also reported to strongly regulate the other activating FcγRs in vitro.¹⁴ These data highlight a role for FcγRIIb in regulating DCs.

The impact of FcγRIIb on atherogenesis, however, remains unclear. Male Apoe^−/−Fcgr2b^−/− mice placed on a Western diet experience increased atherosclerosis independent of lipid levels, characterized by increased antibody titers and pro-inflammatory cytokines in the aorta.¹⁵ Conversely, a separate group reported that Apoe^−/−Fcgr2b^−/− mice demonstrated decreased carotid and aortic root atherosclerosis, with higher numbers of circulating T_REGS and higher serum levels of IL-10 and TGF-β.^{16, 17} The authors suggested that these conflicting results are likely due to strain differences, as the former may have increased expression of lupus-associated Slam genes located near Fcgr2b while the latter did not.¹⁷

Given that oxLDL-IC stimulation of FcγRs on DCs affected inflammasome activation, cytokine production, and downstream T-cell responses in vitro, we sought to determine how FcγRIIb expression on DCs impacts atherosclerosis in vivo. We hypothesized that a conditional knockout of this inhibitory receptor in DCs would result in increased atherosclerosis. Surprisingly, we found that female Ldlr^−/− recipients of CD11c-Cre⁺ Fcgr2b^fl/fl bone marrow experienced increased atherosclerosis, while male Ldlr^−/− recipients of CD11c-Cre⁺ Fcgr2b^fl/fl bone marrow had an unexpected decrease in atherosclerosis compared to littermate controls. Changes in atherosclerosis occurred despite both sexes having improved serum cholesterol and triglyceride profiles with CD11c⁺ specific FcγRIIb knockdown. Instead, atherosclerosis seemed to be more related to sex-specific differences in APC functional markers within the liver. Collectively, these findings support a novel role for FcγRIIb in CD11c⁺ cells for maintaining liver cholesterol homeostasis and reveal unexpected sex-dependent differences in the inflammatory response during atherosclerosis.

Materials and methods

Mice

C57BL/6J-Tg Itgax-cre,-EGFP 4097Ach/J (CD11c-Cre-GFP; Stock # 007567) and B6.129S7-Ldlr^tm1Her/J (Ldlr^−/−; Stock # 002207) mice were originally obtained from the Jackson Laboratory (Bar Harbor, ME). Mice homozygous for the Fcgr2b^fl alleles were generously gifted from Jeffery V. Ravetch (The Rockefeller University, New York City, NY) and bred with CD11c-Cre⁺ mice to obtain CD11c-Cre⁺ Fcgr2b^fl/fl mice and CD11c-Cre^- mice as determined by PCR. (Please see Major Resources Table in the Supplemental Material). All mice were subsequently housed and maintained at Vanderbilt University. Procedures were approved by the Vanderbilt University Institutional Animal Care and Use Committee.

Bone marrow transplants and atherosclerosis quantification

Eight to twelve-week-old Ldlr^−/− mice were lethally irradiated (900 rad) by a ¹³⁷Cs source. After four hours, up to 5 × 10⁶ bone marrow cells harvested from the femurs of CD11c-Cre⁺ Fcgr2b^fl/fl mice or CD11c-Cre^- littermates were transferred into irradiated Ldlr^−/− recipients via retro-orbital injection. Transplanted mice were cohoused and maintained on a normal chow diet for 4–5 weeks during reconstitution. Following reconstitution, mice were placed on a Western diet (21% saturated fat, 0.15% cholesterol, TD.88137, Envigo, Indianapolis, IN). Mice were sacrificed after 8 weeks of Western diet and atherosclerotic lesion burden was quantified in the proximal aorta by oil-red-O staining as previously described.¹⁸

Serum amyloid A and anti-oxLDL antibody ELISAs

Serum Amyloid A was measured using a commercially available kit (Abcam, Cambridge, MA) and following the manufacturer’s instructions. Samples were assessed at a 1:1000 dilution. For anti-oxLDL antibody ELISAs, Nunc MaxiSorp plates were coated with 10μM oxLDL at 4°C overnight. OxLDL was generated as previously described.⁴ Plates were washed three times in PBS containing 0.05% Tween and blocked in 1% BSA/PBS (Fisher Scientific, Waltham, MA) at room temperature (RT) for 2 hours. Plates were washed in PBS-Tween x3 and serum samples diluted in 1% BSA/PBS were added and incubated for 2 hours at RT. Plates were washed five times with PBS-Tween. For Total IgG quantification, anti-mouse IgG-HRP (Promega, Madison, WI) diluted 1:5000 was added to the wells and incubated overnight at 4°C. Plates were washed with PBS-Tween x5 and OptEIA TMB substrate (BD Biosciences, San Diego, CA) was added. Plates developed for 10 minutes prior to quenching with 2M HCl and immediately read at 450nM.

For IgM and IgA quantification, goat anti-mouse IgA-biotin (Southern Biotechnology, Birmingham, AL) and goat anti-mouse IgM biotin (Southern Biotechnology) were added at a 1:5000 dilution to the wells overnight at 4°C. Plates were washed with PBS-Tween ×5 and incubated with streptavidin-peroxidase (MilliporeSigma, St Louis, MO) at 2.5μg/mL for 30 minutes at RT. Plates were developed for 25 minutes in TMB substrate and quenched as described above.

Macrophage immunohistochemistry

5 μM sections of the proximal aorta were fixed in cold acetone and incubated at room temperature in 2% BSA/PBS. Slides were treated with avidin block (Vector, Olean, NY), biotin block (Vector), and peroxidase-activity block (9:1 ratio of methanol:30% H₂O₂). Macrophages were stained using a 1:25 dilution of rat-anti mouse macrophage/monocyte, clone MOMA-2 (MilliporeSigma) in 2% BSA/PBS for 1 hour at RT. Next, slides were stained with a 1:200 dilution of biotin goat-anti rat (BD Biosciences) in 2% BSA/PBS for 30 minutes at 37°C.

Strepavadin-HRP (Biogenex, Fremont, CA) was applied for 20 minutes followed by AEC substrate (Abcam) for 2 minutes. Hematoxylin counterstain was applied for 2 minutes and slides were imaged using a Q-Color5™ imaging system (Olympus, Center Valley, PA). Quantification of macrophage/monocyte area was performed using ImageJ and averaged for four sections.

Serum, liver, and fecal cholesterol and triglyceride assays

Mice were fasted on paper bedding for four hours and blood was collected via the retroorbital sinus. Lipids were extracted from stool samples and liver segments using a modified version of Folch-Lees.¹⁹ Briefly, 10 mg aliquots of feces dried at 70°C for 1h were crushed into a powder, resuspended in 2:1 chloroform-methanol, and incubated at 60°C for 30 minutes under constant agitation. The suspension was centrifuged at 1000 × g for 10 minutes and water was added to the supernatant. Phase separation was induced by low-speed centrifugation and the chloroform phase was removed and evaporated until dry. Samples were re-suspended in chloroform-2% Triton X-100, evaporated until dry, and then re-suspended in diH₂O with 2% Triton X-100. For the liver segments, 50–100 mg aliquots of tissue were homogenized in 2:1 chloroform-methanol and agitated overnight on an orbital shaker at 4°C and then treated as described above. Cholesterol and triglycerides were measured using a commercially available kit (Raichem, San Marcos, CA).

Isolation of serum lipoproteins by FPLC

Size-exclusion chromatology was carried out on a Superose 6 10/300 GL column (GE Healthcare, Uppsala, Sweden) and elutions monitored at an absorbance of 280 nm. Samples were prepared for injection by thawing on ice, centrifuging at 10,000 × g for 5 minutes, and pooling two samples to reach a total volume of 100μL. Collected fractions were analyzed for cholesterol and triglyceride content using the commercially available kits as described above.

Isolation of immune cells from livers and adipose tissue

Livers were perfused through the heart with PBS, minced, and digested in 1 mg/mL of Collagenase, type II (Worthington Biochemical, Lakewood, NJ) in HBSS with calcium and magnesium for 30 minutes at 37°C. Liver tissue was passed through a 40 μM strainer and debris was separated by decanting. Cells were collected by centrifugation at 1500 rpm for 10 minutes and subsequently resuspended in 40% Percoll. After underlaying a layer of 60% Percoll, the gradient was spun at 2000 rpm for 20 minutes. Leukocytes were collected from the interface of the 40% and 60% layers.

Gonadal fat pads were harvested and processed as previously described.²⁰ Briefly, fat pads were minced in 1% FBS in PBS and then digested in 2mg/mL of Collagenase, type II (Worthington Biochemical) for 40 minutes at 37°C in a rotational shaker at 200rpm. The homogenate was triturated several times before passing through a 40μM strainer. RBCs and adipocytes were lysed by resuspension in ACK buffer (Gibco, Langley, OK) for 3 minutes on ice. Cells were then passed through a FACS filter cap prior to staining.

Flow cytometry

Flow cytometry was performed on leukocytes isolated from organs as described above. Cells were washed in FACS buffer containing HBSS, 1% BSA, 4.17mM sodium bicarbonate, and 3.08 mM sodium azide and incubated for 15 minutes at room temperature with Fc Block (anti-CD16/32; Tonbo, San Diego, CA) except for stains for FcγRIIb. Cells were then incubated for 30 minutes at 4°C with the following antibodies: anti-CD11c-FITC (Tonbo), anti-CD11b-V450 (Tonbo), anti-CD45.2-APCCy7 (Tonbo), anti-CD64-PE (BD, San Jose, CA), anti-CD36-APC (BD), anti-MHC Class II-PerCP-Cyanine5.5 (Tonbo), anti-CD32b-PE (Thermofisher, Waltham, MA), anti-TCRβ-V450 (Tonbo), and anti-CD4-PeCy7 (Tonbo). All samples were washed, and either fixed in 2% paraformaldehyde (PFA) or permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA) for intracellular staining overnight at 4°C. Cells were then stained an additional 30 minutes with anti-FoxP3-FITC (eBioscience) followed by washing and fixation in 2% PFA. Samples were run using a MACSQuant Analyzer (Miltenyi, Auburn, CA) and analyzed using FlowJo software.

Liver qPCR

10 mg liver segments were stored at −20°C in RNAlater Solution (Thermofisher) until RNA was purified using the tissue protocol from the Norgen Biotek Total RNA Purification Kit (Ontario, Canada). RNA concentrations were normalized, and RNA was reversed transcribed with a High-Capacity RNA-to-cDNA kit (Applied Biosystems, Grand Island, NY). The reverse transcription product was used for detecting mRNA expression by quantitative real-time PCR with TaqMan™ probes (Thermofisher) for Fasn, Msr1, Abca1, Abcg1, Cd36, Hmgcr, and Srebp1 on the QuantStudio 6 Flex Real-Time PCR System (Life Technologies). The cycling-threshold (C_T) value for each gene was normalized to the housekeeping gene Ppia, and the relative expression was calculated by the change in cycling threshold method (ΔΔCT). Probe catalog numbers can be found in the supplemental methods.

Statistical analyses

Statistical significance between experimental and control groups was determined using a Student’s t test normally distributed data and A Mann-Whitney test for non-normally distributed data in Graph Pad Prism (San Diego, CA).

Results

FcγRIIb expression on CD11c⁺ cells modulates atherosclerosis in a sex-dependent manner.

To study the effects of FcγRIIb expression in CD11c⁺ cells on atherosclerosis, we generated bone marrow chimeras with Ldlr^−/− recipients and CD11c-Cre⁺ Fcgr2b^fl/fl or littermate control CD11c-Cre^- donors (Figure 1A). Transplanted mice were cohoused and fed a Western diet for 8 weeks. Flow cytometry on splenic and hepatic leukocytes confirmed reduced FcγRIIb expression on CD11c^hi CD11b^hi expressing cells in both female and male recipients (Figure 1B–C). Quantification of atherosclerosis by Oil-red-O (ORO) area and total lesion area in the aortic root resulted in a near 60% increase in total atherosclerotic lesion size in female recipients of CD11c-Cre⁺ Fcgr2b^fl/fl bone marrow (Figure 1D–E). However, male recipients of CD11c-Cre⁺ Fcgr2b^fl/fl bone marrow had an unexpected 44% decrease in total lesion area.

Figure 1.

Sex influences the impact of a CD11c conditional FcγRIIb KO on atherosclerosis. (A) Study design. Bone marrow from 5-week old CD11c-Cre⁺ and CD11c-Cre^- Fcgr2b^fl/fl mice was transplanted into lethally irradiated 8–12-week-old male and female Ldlr^−/− mice. After 4–5 weeks of reconstitution on a normal chow diet, mice were fed a Western diet for 8 weeks prior to euthanasia. (n=8–12 mice per group, 3 independent experiments) (B) Spleen FcγRIIb expression in CD11c^hi CD11b^hi cells from CD11c-Cre⁺ Fcgr2b^fl/fl and littermate control bone marrow recipients quantified by mean fluorescence intensity (MFI). (C) Liver FcγRIIb expression on CD11c^hi CD11b^hi cells from CD11c-Cre⁺ Fcgr2b^fl/fl recipients quantified by MFI. (D) Representative Oil-Red-O stained atherosclerotic lesions from the aortic root, quantified in (E) by Oil-Red-O (ORO) area and total lesion area. Error bars shown above represented standard error. *, **, and *** indicate significance at p <0.05, p <0.01, and p <0.001 respectively by Student’s t test.

CD11c-Cre⁺ Fcgr2b^fl/fl recipients have sex-dependent differences in serum markers of inflammation and macrophage composition in the proximal aorta.

To determine if the differences in atherosclerosis could be attributed to differences in inflammation, we measured serum cytokine and amyloid A levels. While no differences were observed in serum IFNγ, IL-1β, IL-6, IL-10, IL-17A, IL-23, or TNFα between CD11c-Cre⁺ Fcgr2b^fl/fl recipients and controls (Supplemental Figure 1A), female CD11c-Cre⁺ Fcgr2b^fl/fl recipients had significantly elevated serum amyloid A levels (Figure 2A). There were no differences in serum amyloid A levels in male recipients.

Figure 2. Sex differences in serum markers of inflammation and macrophage infiltrate in atherosclerotic lesions.

(A) Serum amyloid A was measured using a commercially available kit. (B) Serum anti-oxLDL IgM, IgG, and IgA was determined by ELISA at a 1:50, 1:250, and 1:50 dilution respectively. (C) Macrophages in the proximal aorta were quantified by immunohistochemistry. 5μM sections were stained with 2% BSA/PBS (left panels) or anti-mouse Macrophage/Monocyte (MOMA) (right panels). Slides were developed using AEC substrate and counterstained with hematoxylin. Quantification was performed based on total red area and % of lesion area. Error bars shown above represented standard error. * and ** indicate significance at p <0.05 and p <0.01 respectively, by Student’s t test.

Given our previous work with oxLDL-ICs in vitro⁴, we hypothesized that there may be differences in anti-oxLDL antibody titers. Serum studies revealed elevated anti-oxLDL IgM in male CD11c-Cre⁺ Fcgr2b^fl/fl recipients with a trend towards decreased anti-oxLDL IgM in female recipients (Figure 2B). There was no difference in anti-oxLDL IgG, IgA or any of the IgG subtypes (Figure 2B, Supplemental Figure 1B). No differences were seen in immune cell composition or DC activation markers in the spleens of CD11c-Cre⁺ Fcgr2b^fl/fl recipients compared to controls (Supplemental Figure 2).

Immunohistochemistry of the proximal aortic plaques showed that macrophages composed less of the total lesion in female CD11c-Cre⁺ Fcgr2b^fl/fl recipients (Figure 2C). However, as the female lesions were greater in size, the total macrophage area was the same. Conversely, macrophages made up the same percentage of the plaque area in males, but because there was less atherosclerosis in the male CD11c-Cre⁺ recipients, there was a decreased total macrophage area in male mice (Figure 2C).

CD11c-Cre⁺ Fcgr2b^fl/fl recipients gain less weight, have improved serum lipid profiles, and excrete less cholesterol irrespective of sex.

Although we observed sex differences in atherosclerosis, both sexes trended towards less weight gain with a CD11c-conditional FcγRIIb KO (Figure 3A). Despite this, CD11c-Cre⁺ Fcgr2b^fl/fl recipients did not have any differences in gonadal fat pad weight (Supplemental Figure 3A). Male CD11c-Cre⁺ Fcgr2b^fl/fl recipients did have smaller spleen and liver masses compared to littermate controls, however, no differences were seen between female cohorts (Supplemental Figure 3B–C).

Figure 3. CD11c-Cre⁺ Fcgr2b^fl/fl recipients gain less weight, have improved serum lipid profiles, and excrete less cholesterol.

(A) Percent weight gain of female and male mice after 8 weeks of Western diet. (B) Serum triglyceride (TG) and cholesterol (CHOL) after 8 weeks of Western diet for female mice (upper panels) and male mice (lower panels) as measured by commercially available kits. (C) Triglycerides (TG) and cholesterol (CHOL) excreted in the stool of CD11c-Cre⁺ Fcgr2b^fl/fl recipients and littermate controls after 8 weeks of Western diet. Lipids were extracted from 10mg aliquots of dried stool collected after fasting using a modified version of the Folch method in 2:1 chloroform-methanol. Values are normalized to littermate controls. Error bars shown above represented standard error. * and **indicate significance at p <0.05 and p <0.01 by Student’s t test.

We also examined the serum lipid profiles of the transplanted mice. In both sexes, CD11c-Cre⁺ Fcgr2b^fl/fl recipients had a substantial decrease in fasting serum triglycerides and a slight, but significant, decrease in serum cholesterol (Figure 3B). FPLC analysis demonstrated that this reduction was present in both VLDL-TG and LDL-CHOL fractions, but not in HDL (Supplemental Figure 4C–D). This improved serum lipid profile was surprising given that female CD11c-Cre⁺ Fcgr2b^fl/fl recipients had increased atherosclerosis. Because decreased circulating lipid may result from increased excretion in feces, stool samples were collected and analyzed for cholesterol content. Even prior to starting a Western diet, CD11c-Cre⁺ Fcgr2b^fl/fl recipients excreted less cholesterol, despite having similar serum cholesterol levels at baseline (Supplemental Figure 4A–B). After 8 weeks of Western diet, both male and female CD11c-Cre⁺ Fcgr2b^fl/fl recipients continued to trend towards decreased stool lipid excretion (Figure 3C). These results indicate that improved serum lipids in CD11c-Cre⁺ Fcgr2b^fl/fl recipients is not due to increased excretion through feces, but is rather likely related to lipid production, handling, or storage.

CD11c-Cre⁺ Fcgr2b^fl/fl recipients have increased pro-inflammatory dendritic cells in their adipose tissue.

Given the effect of a CD11c-conditional FcγRIIb KO on weight gain and serum lipids, APC populations were analyzed in white adipose tissue (WAT), as WAT is an important site of triglyceride and free fatty acid storage.²¹ Studies were focused on the gonadal fat pads as those are often the first to develop signs of inflammation and insulin resistance among fat tissues.²²There was a substantial increase in the percent of CD11c^hi CD11b^hi expressing cells in CD11c-Cre⁺ Fcgr2b^fl/fl recipients regardless of sex (Figure 4A and Supplemental Figure 5). As both AT macrophages (ATMs) and AT dendritic cells (ATDCs) can express CD11c and CD11b, we used CD64 to distinguish between ATMs (CD11c^hi CD11b^hi CD64⁺ cells) and ATDCs (CD11c^hi CD11b^hi CD64⁻ cells).²³ We observed a significant decrease in the percent of ATMs with a concurrent increase in ATDCs in male WAT (Figure 4B–C). WAT from female CD11c-Cre⁺ Fcgr2b^fl/fl recipients also demonstrated a two-fold increase in percent of ATDCs but showed no change ATMs. ATDCs are known to accumulate in WAT in obesity and are the dominant CD11c⁺ population with moderate high fat diet exposure, which is consistent with our findings of increased atherosclerosis in female mice.^{23, 24}

Figure 4. CD11c-Cre⁺ Fcgr2b^fl/fl recipients have increased pro-inflammatory dendritic cells in their white adipose tissue (WAT).

Gonadal fat pads from transplanted mice were digested with collagenase, 40μM cell strainer passage, and RBC/adipocyte lysis prior to staining with CD45.2, CD11c, CD11b, CD64, MHC Class II, and CD36 by flow cytometry. (A) Percent of CD45⁺ cells that are CD11c^hi CD11b^hi in male WAT (left) and female WAT (right). (B) Percent of CD11c^hi CD11b^hi CD64⁺ macrophages and CD11c^hi CD11b^hi CD64⁺ dendritic cells of CD45⁺ cells in male and female WAT. (C) Representative contour plots of CD11c versus CD64 (gated on CD11c^hi CD11b^hi) to examine ATM and ATDC populations. (D) CD36 expression on CD11c^hi CD11b^hi CD64⁺ WAT macrophages in male mice (upper panels) and female mice (lower panels) quantified by MFI. (E) MHC Class II expression on CD11c^hi CD11b^hi CD64⁻ WAT dendritic cells in male (left) and female mice (right) quantified by MFI. Data are representative of N=4–5 mice per group. Error bars shown above represent standard error. * indicates p <0.05 and ** p <0.01 by Student’s t test.

We next hypothesized that CD36 expression in the WAT may be contributing to the metabolic differences we observed as previous reports found that Cd36^−/− mice had lower body weights despite increased triglyceride levels.²⁵ While there was no difference in CD36 expression between male CD11c-Cre⁺ Fcgr2b^fl/fl recipients and littermate controls, there was a near 50% reduction in CD36 on female ATMs (Figure 4D). As a general marker of activation, we also examined MHC Class II expression on ATDCs and found that it was increased on CD11c-Cre⁺ Fcgr2b^fl/fl recipients in both males and females (Figure 4E). Collectively these data demonstrate that there is an increase in pro-inflammatory ATDCs in CD11c-Cre⁺ Fcgr2b^fl/fl recipients with increased maturation and activation as measured by expression of MHC Class II.

A CD11c-conditional FcγRIIb KO alters hepatic liver storage and synthesis in a sex-dependent manner.

Given the liver’s role in cholesterol and TG storage and synthesis, we hypothesized that hepatic DCs may be involved in modulating serum lipid levels. The lipid fraction from liver segments was purified and cholesterol and TG content was quantified (Figure 5A). In males, liver cholesterol and TG was lower in the CD11c-Cre⁺ Fcgr2b^fl/fl recipients compared to littermate controls. In contrast, female liver cholesterol and TG was increased in CD11c-Cre⁺ Fcgr2b^fl/fl mice. Therefore, liver lipid content was reflective of each sex’s underlying atherosclerosis burden, but not their serum lipid profile.

Figure 5. Male and female CD11c-Cre⁺ Fcgr2b^fl/fl recipients produce less Fasn transcripts, but only female recipients store more cholesterol and triglycerides in their liver.

(A) Lipids were purified from male and female liver segments by the Folch method methods in 2:1 chloroform-methanol and triglycerides (TG) and cholesterol (CHOL) quantified by commercially available kits. (B) Quantitative real-time PCR was performed on liver segments to measure the expression of several genes involved in lipid synthesis and handling. Quantification was performed using the 2^−ΔΔCT method and normalized to littermate controls. Error bars represent standard error. *, **, and **** indicates significance at p <0.05, p <0.01, and p <0.0001 by Student’s t test.

We also processed whole liver segments for RNA to examine potential differences in hepatic lipid production and handling. Transcripts of fatty acid synthase (Fasn), the key enzyme in synthesis of palmitate, were markedly reduced in both female and male CD11c-Cre⁺ Fcgr2b^fl/fl recipient livers compared to controls (Figure 5B). Male CD11c-Cre⁺ Fcgr2b^fl/fl recipients also a reduction in other genes such HMG-CoA reductase (Hmcgr) in the cholesterol synthesis pathway and macrophage scavenger receptor-1 (Msr1) (Figure 5B). Of note, there was significantly elevated hepatic Cd36 transcripts in female CD11c-Cre⁺ Fcgr2b^fl/fl recipients with a slight trend towards reduced Cd36 transcript levels in the males.

Female, but not male, CD11c-Cre⁺ Fcgr2b^fl/fl recipients have increased MHC Class II and CD36 expression on liver DCs.

We next sought to determine if there were differences in immune cells and markers of inflammation within the liver by flow cytometry. We found no differences in the total number of B-cells or CD4⁺ T-cells (data not shown) but did observe an increase in the percent of CD11c^hi CD11b^hi cells in male CD11c-Cre⁺ Fcgr2b^fl/fl recipients (Figure 6A–B, Supplemental Figure 6). Female recipients exhibited the opposite effect with a decrease in percent of CD11c^hi CD11b^hi cells. Given that cells from the liver have been previously shown to relocate to the aorta in Ldlr^−/− mice²⁶, we were curious if the aorta would mimic the liver changes in CD11c^hi CD11b^hi cells. As predicted, we observed an increase in the percent of CD11c^hi CD11b^hi cells in the males and a trend towards decreased CD11c^hi CD11b^hi cells in the females. (Supplemental Figure 7A–B).

Figure 6. Female, but not male, CD11c-Cre+ Fcgr2bfl/fl recipients have increased MHC Class II and CD36 expression on liver DCs.

(A) Percent of CD11c^hi CD11b^hi cells in male (left) and female (right) livers was quantified by flow cytometric staining. (B) Representative contour plots of CD11c^hi CD11b^hi cells, gated on FSC and SSC. (C) Liver CD36 expression on CD11c^hi CD11b^hi cells quantitated by MFI for males and females. (D) Liver MHC Class II expression on CD11c^hi CD11b^hi cells quantitated by MFI for males and females. (E) Percent of T_REGS in male and female livers, quantified by gating on TCRβ⁺ CD4⁺ cells and intracellular FoxP3 staining. Data are representative of N=4–5 mice per group. Error bars represent standard error. * indicates significance at p <0.05 by

Closer examination of the CD11c^hi CD11b^hi subset demonstrated that male mice had significantly less CD36 and MHC Class II expression with knockout of FcγRIIb, whereas females had an increase in the expression of both markers in the liver (Figure 6C–D). CD86 levels trended towards increased expression on CD11c^hi CD11b^hi cells in CD11c-Cre⁺ Fcgr2b^fl/fl recipients but was not significant (data not shown).

Given that one of the major roles of DCs is to present antigen, we analyzed different T-cell populations in the liver. Since we did not see any differences in T_H1 and T_H17 populations (data not shown), we focused on liver-specific T_REGS, as previous studies have shown that hepatic T_REG populations are altered in hypercholesterolemia.²⁶ Furthermore, depletion of FoxP3⁺ populations increases serum cholesterol and TGs.^{26, 27} Livers from male CD11c-Cre⁺ Fcgr2b^fl/fl recipients had a larger percent of FoxP3⁺ T-cells than littermate controls (Figure 6E). In contrast, female CD11c-Cre⁺ Fcgr2b^fl/fl recipients demonstrated no difference in the percent of FoxP3⁺ T-cells.

Discussion

The current study demonstrates sex-specific effects of a CD11c⁺ conditional FcγRIIb KO on atherogenesis. The observation that male CD11c-Cre⁺ Fcgr2b^fl/fl recipients had less atherosclerosis was surprising given the body of literature supporting an immunosuppressive role for FcγRIIb both in vitro and in vivo.^{12, 14, 28, 29} However, these studies do not specify the sex of the mice used during experimentation. Additionally, not all studies have observed a tolerance-inducing role for FcγRIIb.^15,30 Our results suggest that there are major sex differences in the effect of a CD11c-conditional FcγRIIb KO on atherosclerosis, and that this may be more tolerance-inducing in males and inflammation-promoting in females.

Estrogen is thought be protective against atherosclerosis, as females do not usually develop clinical CVD until after menopause. Hepatic lipid metabolism has been implicated as at least a partial causal agent as ovariectomized monkeys demonstrate increased hepatic cholesterol synthesis, hepatic cholesterol storage and consequently, increased atherosclerosis.³¹ However, lipids are only one of the many mediators of atherosclerosis. The role of chronic inflammation in atherogenesis is now more highly recognized and there are marked sex-differences in the rates of autoimmune diseases. SLE, for example, affects ten times more females than males.³² Estrogens are also a potential candidate for this bias as the onset of SLE is more frequent in women of childbearing age.³² A recent study found that estrogens can increase DC maturation, enhance metabolic pathways in DCs, and modulate type I IFN-dependent and type I IFN-independent upregulation of DC activation markers in response to TLR stimulation.³³

Interestingly, estrogens have been shown to affect FcγR expression. Kramer et. al found that 17β-estradiol significantly regulated FcγRIII expression and subsequent cytokine release in monocytes via the estrogen receptor.³⁴ A separate study found that estrogen treatment of guinea pigs enhanced clearance of IgG-sensitized erythrocytes by increasing splenic-macrophage FcγR expression.³⁵ These studies combined with our data warrant further exploration into the ability of estrogen to regulate FcγRIIb.

Given the dramatic near 60% increase in atherosclerosis we observed in female CD11c-Cre⁺ Fcgr2b^fl/fl recipients despite significant lower serum cholesterol and triglyceride levels, we would hypothesize that the protective effect of estrogen on atherosclerosis and hepatic cholesterol metabolism is, in part, through FcγRIIb expression on CD11c⁺ cells. This is supported by our observation that when FcγRIIb is knocked down by even just 50% on CD11c⁺ cells, female mice had increased hepatic cholesterol and TG and increased markers of inflammation (i.e. SAA, MHC Class II expression). In males the role of FcγRIIb on CD11c⁺ cells in maintaining peripheral tolerance may be much less important in the absence of estrogen. Consequently, we would hypothesize that since our male mice did not experience an increase in liver cholesterol, TG or DC activation markers, the atheroprotective effect of the lower serum cholesterol and triglycerides predominated. We also observed increased serum anti-oxLDL IgM levels in male but not female CD11c-Cre⁺ recipients, which in several mouse and human studies was protective against atherosclerosis and is likely contributing to our phenotype.³⁶

Our data suggests that CD36 may be the mechanism by which FcγRIIb on CD11c⁺ cells modulates hepatic cholesterol and TG. Also known as fatty acid translocase, CD36, is a receptor for oxLDL and long chain fatty acids that is abundant in tissues active in fatty acid metabolism such as WAT, skeletal muscle, and cardiac muscle.²⁵ Interestingly, Cd36^−/− mice are protected from atherosclerosis, insulin resistance, and obesity.^37–39 In several other diet-induced obesity studies or human studies of non-alcoholic fatty liver disease, hepatic CD36 levels have been found to be significantly elevated and this elevation correlated with increased liver TG storage.^40,41 Consistent with this, we found increased Cd36 transcripts in whole liver lysates and increased CD36 expression on CD11c⁺ CD11b⁺ cells in female CD11c-Cre⁺ Fcgr2b^fl/fl recipients, which was the group with increased liver TGs and increased atherosclerosis. In addition to its role in metabolism, CD36 is also implicated in promoting inflammation. With cooperation from a TLR4/TLR6 heterodimer, CD36 was found to coordinate NLRP3 inflammasome activation and subsequent IL-1β production.⁴² In DCs specifically, blocking CD36 with a monoclonal antibody inhibited LPS-induced DC maturation.⁴³ Instead of upregulation typical activation markers such as CD86 or secreting IL-12, IL-10 production from these DCs increased significantly. This was particularly interesting, given that we found more T_REGS present in the livers of male CD11c-Cre⁺ Fcgr2b^fl/fl recipients. Intrahepatic T_REGS are particularly important as they are capable of directly migrating to the aorta in Ldlr^−/− mice.²⁶ T_REGS are critical in preventing atherosclerosis development, elegantly shown by Klingenberg et. al when they depleted FoxP3⁺ T_REGS by the diphtheria toxin and observed a 2.1 fold increase in atherosclerosis and a 1.7 fold increase in cholesterol and TGs.²⁷ They reported that liver lipoprotein catabolism was significantly altered by T_REG absence. Therefore, we would hypothesize that the decreased CD36 expression in the livers of male CD11c-Cre⁺ Fcgr2b^fl/fl recipients led to decreased activation markers on hepatic DCs, increased IL-10 production from the same DCs, and a subsequent atheroprotective increase in hepatic T_REGS.

The increase in CD11c⁺ CD11b⁺ cells in male CD11c-Cre⁺ Fcgr2b^fl/fl recipient livers was unexpected given literature reporting increases in liver DCs in obesity in inflammation.^{26, 44} However, Ibrahim et al. discovered two populations of liver DCs – those with high lipid content which were immunogenic and activated T-cells, and those with low lipid content which were tolerogenic and induced T_REGS.⁴⁵ Inhibiting DC fatty acid synthesis reduced DC immunogenicity.⁴⁵ This is consistent with other reports that high cholesterol loading of CD11c⁺ cells can trigger the development of autoimmunity.⁴⁶ Additionally, as MHC II peptide complexes cluster in cholesterol-dependent microdomains on the DC surface, cholesterol depletion often disrupts these clusters as well as the antigen-presentation function of the DCs.⁴⁷ We did not investigate the lipid content of liver DCs in this study but it would be an interesting avenue to pursue in the future as another group reported that liver DCs can contribute heavily to tolerance by active T-cell deletion.⁴⁸

To separate DCs and macrophages in WAT we used CD64, a commonly accepted marker to differentiate ATMs and ATDCs.²³ CD11c⁺ ATMs are M1 like, more inflammatory, and are also more abundant in obese WAT.⁴⁹ A reduction in this population indicates a decrease in WAT inflammation and suggests an improvement in insulin resistance, which often correlates with atherosclerosis risk.⁴⁹ This is consistent with our observed decrease in male ATMs and decreased atherosclerosis. Similarly, females experienced no decrease in ATMs and were more prone to developing atherosclerosis. In contrast to WAT, we realize that there is still considerable overlap in macrophage and DC populations in the liver, blood, and aorta. Accordioning, the differences that we observed in these tissues could be due to macrophages expressing CD11c. This, however, not detract find the findings that highlight the importance of FcγRIIb on CD11c⁺ cells during atherogenesis.

Despite sex-dependent differences in atherosclerosis, we unexpectedly found that both male and female CD11c-Cre⁺ Fcgr2b^fl/fl recipients had lower serum cholesterol and triglycerides. Previous evidence supports a role for CD11c⁺ cells in cholesterol homeostasis. In one study, the DC population was expanded by overexpressing the antiapoptotic gene hBcl-2 under the control of the CD11c promoter.⁵⁰ Despite finding enhanced T-cell activation in these mice, they found no increase in atherosclerosis due to a surprising atheroprotective decrease in serum cholesterol levels. The authors also reversed this effect by conjugating CD11c with the diphtheria toxin receptor to deplete DCs, thereby inducing hypercholesterolemia. However, no explanation has been reported for these effects. Our findings newly implicate FcγRIIb in this cholesterol lowering effect of CD11c⁺ cells, likely through decreased expression of alteration of liver lipid synthesis genes such as Fasn and Hmgcr.

Another potential contributor to this phenomenon is that the cholesterol and TG is stored also being stored in fat. Interestingly, ATDCs have been implicated in influencing differentiation and development of adipocytes and we observed more ATDCs in both our male and female CD11c-Cre⁺ Fcgr2b^fl/fl recipients. However, GM-CSF deficient (Csf2^−/−) mice which had 75% fewer ATDCs had a 30% increase in whole body adiposity.⁵¹ This would indicate that ATDCs actually restrict AT expansion. This is interesting given our observations that CD11c-Cre⁺ Fcgr2b^fl/fl recipients gained less weight overall, which could be in part due to the increase in ATDCs.

In conclusion, we saw two major effects of a CD11c-conditional FcγRIIb KO: a sex-dependent increase in inflammation causing enhanced atherosclerosis and a sex-independent decrease in serum cholesterol and TGs. Our results suggest that increased CD36 expression on hepatic DCs in the female CD11c-Cre⁺ Fcgr2b^fl/fl recipient livers may account for the increase in hepatic TG storage and subsequent increase in atherosclerosis. We found decreased expression of liver lipid synthesis genes, which highlights a novel role for FcγRIIb on CD11c⁺ cells in regulating serum cholesterol and TG levels. Collectively these findings provide better insight into mechanism of FcγRIIb involvement in hypercholesteremia, obesity, insulin resistance, and atherosclerosis.

Highlights.

• Female CD11c-Cre⁺ Fcgr2b^fl/fl recipients have larger plaques than control mice

• Male CD11c-Cre⁺ Fcgr2b^fl/fl recipients have smaller plaques than control mice

• CD11c-specific FcγRIIb alters hepatic inflammation differently in males and females

• A CD11c-conditional FcγRIIb knockout decreases serum lipoproteins in both sexes

• Liver lipid synthesis and handling is altered in CD11c-Cre⁺ Fcgr2b^fl/fl recipients