Expression and regulation of alarmin cytokine IL-1α in human retinal pigment epithelial cells

Abstract

Human retinal pigment epithelial (hRPE) cells play important immune-regulatory roles in a variety of retinal pathologic processes, including the production of inflammatory cytokines that are essential mediators of the innate immune response within the ocular microenvironment. The pro-inflammatory "alarmin" cytokine IL-1α has been implicated in both infectious and non-infectious retinal diseases, but its regulation in the retina is poorly understood. The purpose of this study was to elucidate the expression and regulation of IL-1α within hRPE cells. To do this, IL-1α mRNA and protein in hRPE cells was assessed by RT-PCR, qPCR, ELISA, Western blot, and immunofluorescence following treatment with a variety of stimuli and inhibitors. ER stress, LPS, IL-1β, and TLR2 activation all significantly increased intracellular IL-1α protein. Increasing intracellular calcium synergized both LPS- and Pam3CSK4-induced IL-1α protein production. Accordingly, blocking calcium signaling and calpain activity strongly suppressed IL-1α protein expression. Significant but more moderate inhibition occurred following blockage of TLR4, caspase-4, or caspase-1. Neutralizing antibodies to IL-1β and TLR2 partially eliminated LPS- and TLR2 ligand Pam3CSK4-stimulated IL-1α protein production. IFN-β induced caspase-4 expression and activation, and also potentiated LPS-induced IL-1α expression, but IFN-β alone had no effect on IL-1α protein production. Interestingly, all inhibitors targeting the PI3K/Akt pathway, with the exception of Ly294002, strongly increased IL-1α protein expression. This study improves understanding of the complex mechanisms regulating IL-1α protein expression in hRPE cells by demonstrating that TLR4 and TLR2 stimulation and exposure to IL-1β, ER stress and intracellular calcium all induce hRPE cells to produce intracellular IL-1α, which is negatively regulated by the PI3K/Akt pathway. Additionally, the non-canonical inflammasome pathway was shown to be involved in LPS-induced hRPE IL-1α expression through caspase-4 signaling.

Keywords: Caspase-4; IL-1α; Interleukin-1α; Non-canonical inflammasome; RPE; Retinal pigment epithelium; TLR2; TLR4.

Fig 1.

IL-1α mRNA synthesis and protein production in hRPE cells. mRNA levels were assessed for cells (A) treated with LPS (1000 ng/ml), tunicamycin (10 μM) or IL-1β (2 ng/ml) for 6 hr. IL-1α protein production was analyzed by ELISA in hRPE cells treated with LPS (B, 1000 ng/ml), tunicamycin (C, 10 μM) or IL-1β (D, 2 ng/ml) for 24 or 48 hr in the presence or absence of caspase-4 inhibitor (LEVD, 4 μM), caspase-1 inhibitor (YVAD, 2 μM), anti-IL-1β antibody (Ab IL-1β) or isotype serium (Ab Ctrl). The p values were calculated by comparing treatment to Ctrl (A), to LPS only (B), to tunicamycin only (C), or to IL-1β only (D). *p<0.05; **p<0.01; ***p<0.001. In order to compare IL-1α mRNA levels under different conditions, expression of the house keeping gene β-action was used to monitor gel loading. Ab, antibody; Ctrl, control; LEVD, Ac-LEVD-CHO; YVAD, Ac-YVAD-cmk.

Fig. 2.

Immunofluorescence analysis of IL-1α expression in hRPE cells. hRPE cells seeded in chamber slides were incubated with LPS (A, 1000 ng/ml), tunicamycin (B, 10 μM) or IL-1β (C, E, F, 2 ng/ml) for 20 hr. Controls include untreated cells (D), IL-1β-treated cells without primary antibody (E), and IL-1β-treated cells with an isotype antibody control (F). The cells were fixed as described in Materials and Methods. The expressed IL-1α is shown in green. The nuclei were stained with 400x bisBenzimide (blue) (A-F, bottom panels). Experiments were repeated twice. Typical images were taken at 400X magnification.

Fig. 3.

Effects of IFNs on caspase-4 expression/activation and LPS-induced IL-1α production. mRNA levels of IL-1α and caspase-4 were analyzed by RT-PCR (A-C) and qPCR (D, G). IL-1α protein was analyzed by ELISA (E and F). Caspase-4 protein cleavage in hRPE cells was shown by Western blot analysis (H). hRPE cells were treated with LPS (1000 ng/ml), IFN-α (1000 U/ml), IFN-β (1000 U/ml), IFN-γ (1000 U/ml), IFN-γ priming or in combination for 6 (A-D, G), 24 (E-F) or 0, 16 and 24 hr (H). In E and F, hRPE cells were pre-incubated with IFN-α, -β and -γ at 1000 U/ml for 16 hr and then switched to fresh growth media with or without LPS (1000 ng/ml) for an additional 24 hr. The p values were calculated by comparing treatment with LPS alone (D, E, F), treatment with LPS plus caspase-4 inhibitor (F), or treatment with control (G). *p<0.05; **p<0.01; ***p<0.001. Ctrl, control; IFN, interferon; LEVD, Ac-LEVD-CHO; pre-, pre-incubation with.

Fig. 4.

Modulation of IL-1α expression through TLR4 and TLR2 signaling. hRPE cells were cultured with LPS (1000 ng/ml) (A, B) or Pam3CSK4 (Pam 300 ng/ml) or LPS plus 100 ng/ml Pam3CSK4 (B, C) for 24 hr. In some experiments, hRPE cells also were pre-incubated with TAK-242 (75 μM) (A), caspase-4 inhibitor LEVD-CHO (4 μM) (C), TLR2 blocking antibody, or isotype control antibody (C). After stimulations, whole cell lysates were harvested and subjected to ELISA. *p<0.05; **p<0.01; ***p<0.001, as compared with inducers alone. Ab T2, anti-TLR2 antibody; Ab Ctrl, isotype control; LEVD, LEVD-CHO; Pam, Pam3CSK4; TAK, TAK-242.

Fig. 5.

Ca²⁺ signaling regulates IL-1α expression. hRPE cells were cultured with or without LPS (1000 ng/ml) (A and F), tunicamycin (Tu, 10μM) (B), IL-1β (2 ng/ml) (C), or Pam3CSK4 (Pam, 100 ng/ml) (D) for 6 (F) or 24 hr (A-D) with or without PD150606 (PD, 100 μM) or BAPTA-AM (BAPTA, 5 μM). hRPE cells were incubated with or without ionomycin (3 μM), LPS, and Pam3CSK4 in presence or absence of caspase-4 inhibitor, LEVD (4 μM), and inhabited with ionomycin plus LPS or ionomycin plus Pam3CSK4 for 24 hr (E). After incubation, the whole cell lysates were harvested and subjected to ELISA. Steady-state IL-1α mRNA was analyzed by qPCR (F). *p<0.05; **p<0.01; ***p<0.001, as compared with inducers alone (A-E), or with control (B).

Fig. 6.

The effect of CU-CPT22 on IL-1α expression. hRPE cells were cultured with or without CU-CPT22 or co-cultured with LPS (1000 ng/ml) or Pam3CSK4 (100 ng/ml) for 24 hr (A-C). Unless specified, the concentration of CU-CPT22 was 10 μM, TAK-242 100μM, BAPTA-AM 5 μM, caspase-4 inhibitor, LEVD 4 μM, Ly 294002 75 μM. After incubation, the whole cell lysates were harvested and subjected to ELISA. *p<0.05; **p<0.01; ***p<0.001, as compared with inducers alone (A-C) or control (A, B). Ab T2 anti-TLR2 antibody; Ab Ctrl, isotype control; CU, CU-CPT22; BAPTA, BAPTA-AM; LEVD, LEVD-CHO; Ly, Ly 294002; Pam, Pam3CSK4; TAK, TAK-242.

Fig. 7.

Effects of PI3K inhibitors on IL-1α expression. hRPE cells were cultured with or without LPS (1000 ng/ml) in the presence or absence of CAL-101, PI-103, Wortmannin and MK 2206 for 6 hr to measure mRNA levels (A, C) or 24 hr to measure protein (B, D). ***p<0.001, as compared with inducers alone.

Fig. 8.

A schematic view of pathways regulating IL-1α expression in hRPE cells. LPS induced IL-1α expression is mediated by two signal pathways: the TLR4 receptor pathway and the non-canonical inflammasome pathway. After stimulation with LPS, TLR4 binds the sorting adaptor TIRAP, forming an early-acting TIRAP/MyD88 complex, which activates NF-kB, leading to IL-1α expression. An alternative pathway, also involving TIRAP/MyD88, appears to mediate pro-IL-1α expression through TRL2 binding by its agonist, PAM3CSK4. However, the pro-inflammatory response by LPS is subject to negative regulation by PI3K, which stimulates dissociation and degradation of TIRAP and triggers a late-acting anti-inflammatory phase (dotted line), in which TLR4 recruits the TRIF/TRAM complex and induces endosomal-internalization. In this process, LPS may also to be internalized. The intracellular [LPS]_in, as with ER-stress and IFNs, mediates non-canonical activation of the inflammasome leading to production of IL-1α. The roles of calcium-dependent calpain in IL-1α production may be due to its stimulation of IκBα degradation, which promotes pro-IL-1α maturation. The inflammasome-mediated release of IL-1β further induces IL-1α expression via an autocrine mechanism. The numbers represent corresponding references: 1 (Aksoy et al., 2012; Siegemund and Sauer, 2012); 2 (Husebye et al., 2006); 3 (Shi et al., 2014); 4 (Bian et al., 2009); 5 (Kayagaki et al., 2015); 6 (Schaecher et al., 2004); 7 (Kavita and Mizel, 1995); 8 (Cui et al., 2013).