RBP4 activates antigen-presenting cells, leading to adipose tissue inflammation and systemic insulin resistance

Abstract

Insulin resistance is a major cause of diabetes and is highly associated with adipose tissue (AT) inflammation in obesity. RBP4, a retinol transporter, is elevated in insulin resistance and contributes to increased diabetes risk. We aimed to determine the mechanisms for RBP4-induced insulin resistance. Here we show that RBP4 elevation causes AT inflammation by activating innate immunity that elicits an adaptive immune response. RBP4-overexpressing mice (RBP4-Ox) are insulin resistant and glucose intolerant and have increased AT macrophage and CD4 T cell infiltration. In RBP4-Ox, AT CD206(+) macrophages express proinflammatory markers and activate CD4 T cells while maintaining alternatively activated macrophage markers. These effects result from direct activation of AT antigen-presenting cells (APCs) by RBP4 through a JNK-dependent pathway. Transfer of RBP4-activated APCs into normal mice is sufficient to induce AT inflammation, insulin resistance, and glucose intolerance. Thus, RBP4 causes insulin resistance, at least partly, by activating AT APCs that induce CD4 T cell Th1 polarization and AT inflammation.

Figure 1. Elevated serum RBP4 levels cause glucose intolerance, insulin resistance and AT inflammation.

(A) Glucose tolerance test (left panel) and insulin tolerance test (right panel) (n=13-15/group). (B) Serum insulin levels (n=13-15/group). (C) Body weight and (D) fat mass (n=13-15/group). (E) Serum triglycerides and free fatty acid (FFA) levels (n=11-13/group). (F) Serum adiponectin levels (n=11-13/group). (G) Flow cytometry representation of gated ATMΦ (CD11b⁺F4/80⁺). ATMΦ numbers in perigonadal fat (right panel) (n=8/group). (H) TNF and IL-1β intracellular staining in ATMΦ (n=5/group). (I) Flow cytometry representation (top panel) and number (botton panel) of CD11c⁺ and CD206⁺ ATMΦ (n=8/group). Studies were performed at 12-16 weeks of age. Values are means±standard error. *P<0.05. AUC: area under the curve. AAC: area above the curve. Pg: perigonadal.

Figure 2. Elevated serum RBP4 levels activate the adaptive immune system in AT

(A) Total CD4⁺ AT T cell numbers. (B) Flow cytometry representation of gated AT Tregs (left panel) from WT and RBP4-Ox mice. CD4⁺CD25⁺Foxp3⁺ AT Treg percentages and numbers (right panel). (C) Flow cytometry representation of gated CD4⁺Foxp3^-CD44⁺ (memory) and CD4⁺Foxp3^-+CD69⁺ (activated) T cells (upper panel). Memory and activated CD4 T cell numbers (bottom panels). (D) Flow cytometry representation of gated AT CD4 T cells stained intracellularly with IFN-γ and IL-17 (left panel). Percentage and number of CD4⁺IFN-γ⁺ T cells (right panel). (E) Total CD11b⁺F4/80⁺ ATMΦ from WT and RBP4-Ox were evaluated for the expression of MHCII and co-stimulatory molecules (CD40, CD80 and CD86) by flow cytometry (n=5/group). (F) MHCII and co-stimulatory molecule (CD40, CD80 and CD86) expression in gated CD11c⁺ (F) and CD206⁺ (G) ATMΦ. (n=5-6/group). Values are means±standard error. *P<0.05. Pg: perigonadal.

Figure 3. Elevated serum RBP4 levels increase liver macrophage with no effect on CD4 T cell activation.

(A) mRNA expression of Tnf, Il-6, Cd11c and Arg-1 was determined by qPCR. (B) Flow cytometry representation of liver macrophages (CD11b⁺F4/80⁺) gating and macrophage numbers in liver (right panel). (C) Flow cytometry representation (left panel) and number (right panel) of CD11c⁺, CD11c⁺CD206⁺ and CD206⁺ liver macrophages. (D) Flow cytometry representation of TNF and IL-1β intracellular staining in liver macrophages (left panel) and percentage and total number of TNF⁺ macrophages (right panel). (E) MHCII and co-stimulatory molecule (CD40, CD80 and CD86) expression in gated CD11c⁺ and CD206⁺ liver macrophages. (F) Flow cytometry representation of gated AT CD4 T cells stained with IFN-γ and IL-17 (upper panel). Percentage and number of CD4⁺IFN-γ⁺ T cells (bottom panel). (G) Western blot of transgenic human RBP4 (hRBP4) in AT and liver (left panel) and quantification of blot (right panel). (H) Levels of endogenous mouse RBP4 (mRBP4) in serum, AT and liver of RBP4-Ox and WT mice using an anti-serum that does not cross-react with human RBP4. n=4-6/group. Values are means±standard error. *P<0.05. Pg: perigonadal. MFI: median fluorescence intensity.

Figure 4. RBP4 overexpression increases proinflammatory markers in CD206⁺ (alternatively-activated) ATMΦ resulting in Th1 polarization

mRNA expression of classically-activated (A) and alternatively-activated (B) macrophage-related markers in FACS-sorted (98%-purity) CD11c⁺ and CD206⁺ Pg ATMΦ from RBP4-Ox and WT mice (n=3 experiments pooling AT from 10-12 mice/group in each experiment). (C) Flow cytometry representation of CD4 T cell proliferation induced by co-culture with CD11c⁺ and CD206⁺ ATMΦ. Proliferation was evaluated by cell trace dilution. (D) Expansion index representing the degree of CD4 T cell proliferation. (E) Flow cytometry representation of IL-4 and IFN-γ intracellular staining in CD4⁺ T cells co-cultured with CD11c⁺ or CD206⁺ ATMΦ. (F) Percentage of IFN-γ⁺CD4⁺ T cells in the co-culture assay. (G) IFN-γ levels in media from co-culture of CD11c⁺ or CD206⁺ ATMΦ with CD4 T cells. (H) mRNA expression in the co-culture assay of CD4 T cell lineage transcription factors. Co-culture assays were performed with FACS-sorted CD11c⁺ and CD206⁺ ATMΦ pooled from 6-10 WT or RBP4-Ox mice and incubated with syngeneic splenic CD4 T cells. Values are means±standard error. *P<0.05 versus all other groups or as indicated. ^#P<0.05 vs control. Pg: perigonadal.

Figure 5. RBP4 directly activates dendritic cells (DCs) which induce CD4 T cell proliferation and polarization

DCs were generated from bone marrow (BMDCs) of 8 week old male WT mice. (A) DC activation was demonstrated by increased expression of CD40, CD80, CD86 and MHCII determined by flow cytometry. (B) Dose response effect of RBP4 on secretion of TNF, IL-6 and IL-12 from BMDCs. (C) Increased production of IL-12 by RBP4- or LPS-activated BMDC was confirmed by intracellular staining of CD11c⁺MHCII⁺ (BMDCs). (D) RBP4- or LPS-activated BMDCs were co-cultured with splenic syngeneic cell trace-labeled CD4 T cell and CD4 T cell proliferation was demonstrated by cell trace dilution. (E) Expansion index representing the degree of CD4 T cell proliferation. (F) IFN-γ secretion on day 5 of co-culture. (G) RBP4-activated BMDCs induce IFN-γ and TNF production by CD4 T cells, visualized by intracellular staining using flow cytometry. (H) mRNA expression in the co-culture assay of CD4 T cell lineage transcription factors. Data represent 3 experiments performed in triplicate, each using pools of bone marrow cells from 2-6 mice. *P<0.05 versus dialysate. ^#P<0.05 versus LPS. MFI: median fluorescence intensity. Dia: Dialysate control.

Figure 6. Transfer of RBP4-activated APCs into WT recipient mice causes insulin resistance, glucose intolerance and AT inflammation

DCs were generated from the bone marrow (BMDCs) of WT mice. BMDCs were treated with dialysate (not activated; iDC) or activated with RBP4 (mDC). PBS without cells (control), iDCs or mDCs were injected i.p. in WT mice once a week for 6 weeks. (A) Insulin tolerance test (ITT). (B) Area above the curve of the ITT. (C) Glycemia following food removal. (D) Glucose tolerance test. (E) Area under the curve of the GTT. (F) Serum insulin levels. (G) Body weight and serum adiponectin levels. (H) Flow cytometry representation of gated ATMΦ (upper panel) and total number of ATMΦ and ATMΦ expressing TNF or IL-1β (bottom panel). (I) Flow cytometry representation of gated CD11c⁺ and CD206⁺ ATMΦ (left panel) and percent of CD11c⁺ and CD206⁺ ATMΦ (top right). Total number of CD11c⁺ and CD206⁺ ATMΦ (bottom right). (J) Flow cytometry representation of gated CD4⁺, CD4⁺IFN-γ⁺ and CD4⁺TNF⁺ AT T cells. (K) Total AT CD4 T cell numbers (right panel) and CD4⁺IFN-γ⁺ and CD4⁺TNF⁺ AT T cell numbers and percentages. (n=8-10/group for all experiments). Values are means±standard error. *P<0.05 versus all other groups. ^#P<0.05, mDC versus iDC. Pg: perigonadal.

Figure 7. RBP4-induced activation of adipose tissue macrophages and resulting CD4 T cell proliferation and Th1 polarization are JNK-dependent

(A) Histograms representing pJNK staining (pT183/pY185) in CD45⁺CD11b⁺ AT cells from RBP4-Ox and WT mice (upper panel) and normalized pJNK levels (bottom panel). (B) CD11c⁺ and CD206⁺ ATM were sorted from WT mice and stimulated with RBP4 (50 μg/mL for 24h) in the presence or absence of JNK inhibitor (5μM). Levels of TNF, IL-6, IL-12 and IL-1β were measured by ELISA. (C) CD11c⁺ and CD206⁺ ATM were sorted from macrophage specific JNK1/2 knockout (JNK KO) and WT mice and treated with RBP4 (50 μg/mL for 24h). Levels of TNF, IL-6, IL-12 and IL-1β were measured by ELISA. (D) CD11c⁺ and CD206⁺ ATM were sorted from JNK KO and WT mice and treated with RBP4 (50 μg/mL for 24h). Next, CD4 T cells plus anti-CD3 antibody were added and CD4 T cell proliferation and IFN-γ⁺CD4⁺ T cells were measured. Flow cytometry representation of CD4 T cell proliferation (left panel) and IFN-γ and IL-17 intracellular staining in gated CD4 T cells (right panel). (E) Expansion index representing the degree of CD4 T cell proliferation (left panel) and IFN-γ⁺ secretion in the co-culture assay (right panel) (n=4/group). Values are means±standard error. *P<0.05 versus dialysate control or RBP4 treated. ^#P<0.05, versus RBP4 treated. Pg: perigonadal. (F) Elevation of RBP4 in serum activates resident ATMΦ and DC and induces pro-inflammatory cytokine secretion and expression of MHCII and co-stimulatory molecules in AT through a JNK-dependent pathway. The pro-inflammatory molecules and cytokines directly contribute to AT inflammation and insulin resistance. These AT APCs induce CD4 T cell proliferation and Th1 polarization resulting in increased levels of TNF and IFN-γ that can activate ATMΦ and further increase AT inflammation and systemic insulin resistance.