Dissecting the physiological role of the novel lupus-associated C-type lectin-like protein CLEC16A

Abstract

The CLEC16A locus has been identified as a susceptibility gene for multiple autoimmune diseases, including multiple sclerosis, type-I diabetes and systemic lupus erythematosus (SLE), in genome-wide association studies. CLEC16A encodes a novel C-type lectin-like protein, by virtue of a predicted C-type lectinlike domain (CTLD), with unclear function. Studies on the disease-associated SNPs have suggested that CLEC16A polymorphisms affect the expression of neighboring genes, while the effect on its own expression is unclear. Several functional studies have interrogated the physiological role(s) of CLEC16A in disparate directions. The Drosophila ortholog of CLEC16A, Ema, has been reported to regulate endosomal protein trafficking and the autophagic process, while CLEC16A has been found to participate in LPS-induced inflammatory cytokine response in rat astrocytes. Since there is not a consenting role ascribed to CLEC16A, this study was undertaken to investigate the functional involvement(s) of CLEC16A in mammalian cells and the expression of CLEC16A in lupus patients, with the attempt to comprehend the association between CLEC16A and SLE.

By overexpressing in non-immune epithelial cells, CLEC16A was revealed to be an intracellular protein of ~130 kDa in size. CLEC16A displayed a punctated expression pattern, which did not co-localize with endosomes, lysosomes, autophagosomes or endoplasmic reticulum in steady state. When treated with rapamycin or serum-starved, CLEC16A-overexpressing cells exhibited a reduced autophagic response, suggesting that CLEC16A may have an inhibitory role in autophagy. Besides the predicted CTLD, motif prediction has also implicated an immunomodulatory role for CLEC16A. Due to the observed inhibition on autophagy, coupled with recent findings linking autophagy and inflammasome activation, the involvement of CLEC16A in NLRP3 inflammasome was investigated. By knocking down CLEC16A in the human macrophage-like THP-1 cells, CLEC16A was found to potentially regulate NLRP3 inflammasome activation via inhibiting the LPS-induced pro-IL-1aasynthesis. Finally, the expressions of the long and short isoforms, CLEC16A_V1 and CLEC16A_V2 of CLEC16A in PBMCs were compared between healthy controls and SLE patients. A higher CLEC16A_V1 expression was observed in SLE patients, whereas the reverse was found for CLEC16A_V2. The expressions of the isoforms, however, were not correlated with the disease severity and clinical manifestations.

The finding that CLEC16A may inhibit autophagy is in contrast with the reported function of Ema in supporting autophagy, and such discrepancy could be because of the different cell systems used. The finding that CLEC16A may downregulate NLRP3 inflammasome activation has not been previously reported, and the mechanism(s) of such regulation warrant(s) future studies. The molecular basis of how CLEC16A regulates autophagy and inflammasome waits to be delineated. Such knowledge, together with information of where endogenous CLEC16A is expressed, shall incite better understanding of the contribution of CLEC16A to SLE development.