Distinct CD40L receptors mediate inflammasome activation and secretion of IL-1β and MCP-1 in cultured human retinal pigment epithelial cells

Abstract

CD40L signaling occurs in several diseases with inflammatory components, including ocular and retinal diseases. However, it has never been evaluated as a pathogenic mechanism in age-related macular degeneration (AMD) or as an inducer of inflammasome formation in any cell type. mRNA and protein levels of CD40, IL-1β, NALP1, NALP3, caspase-1, and caspase-5 were determined by RT-PCR, qPCR, and Western blot. CD40L receptor (CD40, α5β1, and CD11b) expression was determined by Western and immunofluorescent staining. IL-1β, IL-18, and MCP-1 secretions were determined by ELISA. NALP1 and NALP3 inflammasome formation were determined by Co-IP. Experiments were conducted on primary human retinal pigment epithelial (hRPE) cells from four different donors. Human umbilical vein endothelial (HUVEC) and monocytic leukemia (THP-1) cells demonstrated the general applicability of our findings. In hRPE cells, CD40L-induced NALP1 and NALP3 inflammasome activation, cleavage of caspase-1 and caspase-5, and IL-1β and IL-18 secretion. Interestingly, neutralizing CD11b and α5β1 antibodies, but not CD40, reduced CD40L-induced IL-1β secretion in hRPE cells. Similarly, CD40L treatment also induced HUVEC and THP-1 cells to secret IL-1β through CD11b and α5β1. Additionally, the CD40L-induced IL-1β secretion acted in an autocrine/paracrine manner to feed back and induce hRPE cells to secrete MCP-1. This study is the first to show that CD40L induces inflammasome activation in any cell type, including hRPE cells, and that this induction is through CD11b and α5β1 cell-surface receptors. These mechanisms likely play an important role in many retinal and non-retinal diseases and provide compelling drug targets that may help reduce pro-inflammatory processes.

Keywords: AMD; Age-related macular degeneration; CD40L; IL-1β; Inflammasome; MCP-1; RPE; Retinal pigment epithelium.

Fig 1

Stimulation of CD40 mRNA synthesis and protein production in hRPE cells. CD40 mRNA expression in hRPE cells, unstimulated or with IFN-γ (IFN) for 1.5, 3, 6, 24, 48, and 72 hours (A). FBS had no effect on stimulation. Western blot analysis of CD40 protein expression in hRPE cells at 24 hr of stimulation with IFN-γ (B). Fold changes in mRNA levels were calculated by comparison with unstimulated control after normalization against β-actin. An antibody against actin was used in the Western blot analysis as a loading control. The concentration of IFN-γ used was 500 U/ml. FBS, fetal bovine serum.

Fig 2

CD40L receptor protein expression in hRPE cells. Immunofluorescent protein expression (green) in hRPE cells of CD40 (A), α5β1 (B) and CD11b (C) with and without exposure to IFN-γ (IFN) for 24 hr. The nuclei were stained with bisBenzimide Hoechst 33258 (blue). For CD40, hRPE cells were unstimulated as control (Ctrl) or immunostained with replacement of primary antibody with isotypic control antibody (isotype Ctrl), and omission of primary antibody. For hRPE α5β1 and CD11b immunostaining, cells were left unstimulated (Ctrl) whereas isotypic control antibody (isotype Ctrl), were used in immunostaining of unstimulated and IFN-γ stimulated cells. All images, original magnification of 400×.

Fig 3

CD40L-induced inflammasome activation in hRPE cells. mRNA levels of inflammasome components caspase-1, caspase-5, NALP1 and NALP3 were determined by RT-PCR in hRPE cells treated with or without CD40L for 6 hr (A). The fold changes were calculated by normalization against β actin and comparison with untreated control mRNA levels. Caspase-1 and -5 protein cleavage in hRPE cells was analyzed by Western blot with or without CD40L for 2, 4, 8, or 20 hr (B). An antibody against actin was used in each Western blot analysis as a loading control. Untreated (Ctrl) and CD40L treated hRPE cell lysate were examined for caspase-1 activation by a caspase-1 assay kit (C). Co-IP pulldown of anti-NALP1 (D) or anti-caspase-1 (E, F) followed by Western blotting using antibodies against NALP3, caspase-1, NALP1 or caspase-5. Anti-IgG antibody was used as a control for immunoprecipitation. All concentrations of CD40L in these experiments were 5µg/ml. **P<0.01 as compared with untreated control (C).

Fig 4

Detection of CD40L-induced cleaved, mature IL-1β from hRPE cells through CD40L receptors CD11b and α5β1. mRNA levels measured by RT-PCR and qPCR of IL-1β and IL-18 with or without CD40L (5 ug/ml) or LPS (1 µg/ml) for 6 hr (A). IL-1β secretion from hRPE cells cultured with CD40L (0, 2.5, 5, 10 µg/ml) or LPS (1 µg/ml) for 24 hr (B). CD40L-induced hRPE secretion of IL-18 (C). hRPE cells cultured from 2 donors (627 and 6538) were left unstimulated or stimulated with CD40L followed by Western blot analysis for cleaved IL-1β in the extracellular media (ECM)(D). IL-1β ELISA of conditioned media from hRPE cells cultured with or without CD40L for 24 hr in the presence or absence of antibodies (Abs) against CD40 (5 µg/ml), CD11b (20 µg/ml) or α5β1 (10 µl/m), as well as the corresponding isotype control (E). hRPE cells were cultured with or without CD40L in the presence or absence of IFN-γ (IFN, 500 U/ml), IL-4 (100 ng/ml), or Z-YVAD-FMK (YVAD, 2µM) for 24 hr (F). The concentrations of CD40L were 5 µg/ml (C–F). *p<0.05; **p<0.01; ***p<0.001, as compared with untreated control (A–C) or CD40L-stimulated control (E, F). Ctrl, Control; LPS, lipopolysaccharide; Abs, antibodies.

Fig 5

CD40L induced IL-1β secretion in THP-1 and HUVEC cells through CD40L receptors CD11b and α5β1. THP-1 (A–C) and HUVEC cells (D–E) were challenged by CD40L (1, 2.5, 5 or 10 µg/ml) for 24 hr. Concentration-dependent CD40L-induced IL-1β secretion in THP-1 (A) and HUVEC (D) cells, respectively. Effect of LPS priming plus co-culture (pre-co) on CD40L-induced IL-1β secretion (B). Effects of CD40L-induced IL-1β secretion following treatment with neutralizing antibodies targeting CD40L receptors in THP-1 (C) and HUVEC (E) cells. The antibodies used were anti-CD11b P (from Pharmingen), anti-CD11b B (from Biolegend), anti-α5β1, anti-CD40, and anti-isotype IgG1k control for anti-CD11b (Ctrl) and anti-α5β1. In all experiments, IL-1β protein was detected by ELISA. CD40L concentration was 5 µg/ml. *p<0.05; **p<0.01; ***p<0.001, as compared with untreated control (A, B, D) or between presence and absence of antibodies (C, E). HUVEC, human umbilical vein endothelial cells; LPS, lipopolysaccharide; Ab, antibody.

Fig 6

CD40L-induced MCP-1 mRNA synthesis and protein secretion by hRPE cells. MCP-1 mRNA levels measured by RT-PCR in hRPE cells treated with or without IFN-γ pre-treatment (Pre-IFN) or control (Ctrl) for 24 hr and then switched to serum-free media containing 0 or 5 µg/ml of CD40L for 6 hr (A). The fold changes were calculated by normalization against β-actin and comparison with untreated control. MCP-1 ELISA of conditioned media from hRPE cells pre-treated with or without IL-1β (20 pg/ml) for 24 hr, then replaced with media with or without CD40L for another 24 hr (B). MCP-1 ELISA of conditioned media from hRPE cells treated with or without CD40L (5 µg/ml) in the presence or absence of anti-IL-1β, anti-CD11b, anti-α5β1 or corresponding isotype control antibody for 24 hr (C). MCP-1 ELISA of conditioned media from hRPE cells treated with 10% serum media containing 0 or 5 µg/ml of CD40L in the presence or absence of anti-CD40 or corresponding isotype control antibody for 24, 48 and 72 hr (D). MCP-1 ELISA of conditioned media from hRPE cells treated with 10% serum media with or without IFN-γ for 24 hr, then replaced with CD40L containing or CD40L-free media in the presence or absence of anti CD40, α5β1 or CD11b antibody or isotype controls for an additional 24 hr (E). The concentration of IFN-γ (IFN) was 500 U/ml. *p<0.05; **p<0.01; ***p<0.001, as compared with untreated control (CD40L-treated in C, pre-IL-1 or CD40L-treated in D, and pre-IFN or CD40L-treated in E) or between presence and absence of antibodies (B, CD40Ab in E).