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

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-1 alpha 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-1 alpha within hRPE cells. To do this, IL-1 alpha 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-1 beta, and TLR2 activation all significantly increased intracellular IL-1 alpha protein. Increasing intracellular calcium synergized both LPS- and Pam3CSK4-induced IL-1 alpha protein production. Accordingly, blocking calcium signaling and calpain activity strongly suppressed IL-1 alpha protein expression. Significant but more moderate inhibition occurred following blockage of TLR4, caspase-4, or caspase-1. Neutralizing antibodies to IL-113 and TLR2 partially eliminated LPS- and TLR2 ligand Pam3CSK4-stimulated IL-1 alpha protein production. IFN-beta induced caspase-4 expression and activation, and also potentiated LPS-induced IL-1 alpha expression, but IFN-beta alone had no effect on IL-1 alpha protein production. Interestingly, all inhibitors targeting the PI3K/Akt pathway, with the exception of Ly294002, strongly increased IL-1 alpha protein expression. This study improves understanding of the complex mechanisms regulating IL-1 alpha protein expression in hRPE cells by demonstrating that TLR4 and TLR2 stimulation and exposure to IL-1 beta, ER stress and intracellular calcium all induce hRPE cells to produce intracellular IL-1 alpha, 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-1 alpha expression through caspase-4 signaling.