Khilji Muhammad Saad, Faridi Pouya, Pinheiro-Machado Erika, Hoefner Carolin, Dahlby Tina, Aranha Ritchlynn, Buus Søren, Nielsen Morten, Klusek Justyna, Mandrup-Poulsen Thomas, Pandey Kirti, Purcell Anthony W, Marzec Michal T
Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3168, Australia.
Biomedicines. 2022 Mar 30;10(4):814. doi: 10.3390/biomedicines10040814.
How immune tolerance is lost to pancreatic β-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin chaperone glucose-regulated protein (GRP) 94 from the endoplasmic reticulum (ER) leads to mishandling of proinsulin, ER stress, and activation of the immunoproteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered β-cell major histocompatibility complex (MHC) class-I bound peptidome and inflammasome activation, sensitizing β-cells to immune attack. We used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20 µM), or exposed to proinflammatory cytokines interleukin (IL)-1β or interferon gamma (IFNγ) (15 pg/mL and 10 ng/mL, respectively) for 24 h. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC), followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein (NLRP1), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and (pro) IL-1β expression and secretion were investigated by Western blotting. GRP94 KO increased RT1.A expression in β-cells, as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptide diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NLRP1 expression was upregulated in GRP94 deficient cells along with decreased IκBα content while proIL-1β cellular levels declined, coupled with increased secretion of mature IL-1β. Our results suggest that limiting β-cell proinsulin chaperoning enhances RT1.A expression alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress associated with impeding proinsulin handling may sensitize β-cells to immune-attack.
胰腺β细胞肽引发自身免疫性1型糖尿病时免疫耐受是如何丧失的，这一问题尚不清楚。我们已经表明，内质网（ER）中胰岛素原伴侣葡萄糖调节蛋白（GRP）94的缺失会导致胰岛素原处理不当、内质网应激以及免疫蛋白酶体的激活。我们推测，相对于生物合成需求而言，内质网胰岛素原折叠能力不足可能会导致β细胞主要组织相容性复合体（MHC）I类结合肽组改变以及炎性小体激活，从而使β细胞对免疫攻击敏感。我们使用有或没有GRP94基因敲除（KO）的INS-1E细胞，或在有或没有GRP94抑制剂PU-WS13（GRP94i，20 μM）的情况下，或将细胞暴露于促炎细胞因子白细胞介素（IL）-1β或干扰素γ（IFNγ）（分别为15 pg/mL和10 ng/mL）中24小时。使用流式细胞术评估RT1.A（大鼠MHC I）的表达。对通过反相高效液相色谱（RP-HPLC）分级分离的细胞裂解物进行总RT1.A结合肽组分析，随后进行液相色谱-串联质谱（LC-MS/MS）分析。通过蛋白质免疫印迹法研究含核苷酸结合寡聚化结构域、富含亮氨酸重复序列和吡啉结构域的蛋白（NLRP1）、B细胞中κ轻链多肽基因增强子的核因子抑制剂α（IκBα）以及（前体）IL-1β的表达和分泌情况。与相关对照相比，GRP94基因敲除增加了β细胞中RT1.A的表达，细胞因子暴露也有同样的效果。免疫肽组分析显示，GRP94基因敲除/抑制的细胞以及暴露于细胞因子的细胞中，RT1.A结合肽库增加。GRP94基因敲除/细胞因子暴露组在其肽库中有部分重叠。值得注意的是，在GRP94基因敲除/抑制以及细胞因子暴露的情况下，总RT1.A肽组中胰岛素原衍生肽的多样性增加。在GRP94缺陷细胞中，NLRP1表达上调，同时IκBα含量降低，而前体IL-1β细胞水平下降，同时成熟IL-1β的分泌增加。我们的结果表明，限制β细胞胰岛素原伴侣功能会增强RT1.A表达，改变包括胰岛素原肽在内的MHC-I肽组，并激活炎症途径，这表明与胰岛素原处理受阻相关的应激可能会使β细胞对免疫攻击敏感。
