Extracellular HMGB1 exacerbates autoimmune progression and recurrence of type 1 diabetes by impairing regulatory T cell stability

Aims/hypothesis High-mobility group box 1 (HMGB1), an evolutionarily conserved chromosomal protein, was rediscovered to be a ‘danger signal’ (alarmin) that alerts the immune system once released extracellularly. Therefore, it has been recognised contributing to the pathogenesis of autoimmune diabetes, but its exact impact on the initiation and progression of type 1 diabetes, as well as the related molecular mechanisms, are yet to be fully characterised. Methods In the current report, we employed NOD mice as a model to dissect the impact of blocking HMGB1 on the prevention, treatment and reversal of type 1 diabetes. To study the mechanism involved, we extensively examined the characteristics of regulatory T cells (Tregs) and their related signalling pathways upon HMGB1 stimulation. Furthermore, we investigated the relevance of our data to human autoimmune diabetes. Results Neutralising HMGB1 both delayed diabetes onset and, of particular relevance, reversed diabetes in 13 out of 20 new-onset diabetic NOD mice. Consistently, blockade of HMGB1 prevented islet isografts from autoimmune attack in diabetic NOD mice. Using transgenic reporter mice that carry a Foxp3 lineage reporter construct, we found that administration of HMGB1 impairs Treg stability and function. Mechanistic studies revealed that HMGB1 activates receptor for AGE (RAGE) and toll-like receptor (TLR)4 to enhance phosphatidylinositol 3-kinase (PI3K)–Akt–mechanistic target of rapamycin (mTOR) signalling, thereby impairing Treg stability and functionality. Indeed, high circulating levels of HMGB1 in human participants with type 1 diabetes contribute to Treg instability, suggesting that blockade of HMGB1 could be an effective therapy against type 1 diabetes in clinical settings. Conclusions/interpretation The present data support the possibility that HMGB1 could be a viable therapeutic target to prevent the initiation, progression and recurrence of autoimmunity in the setting of type 1 diabetes. Electronic supplementary material The online version of this article (10.1007/s00125-020-05105-8) contains peer reviewed but unedited supplementary material, which is available to authorised users.


Flow cytometry
To evaluate surface markers, the cells were stained in PBS containing 2% BSA and FITC-

Cell purification and culture
To induce naïve CD4 + T cell differentiating into FOXP3 + cells, the isolated naïve cells (5 × 10 5 cells per ml) were seeded into plates coated with 10 ng/ml anti-CD3 and 10 ng/ml anti-CD28 antibodies, supplemented with 5 ng/ml TGF-β and 10 ng/ml IL-2 (R&D Systems, Minneapolis, MN, USA) for 3 days, and then the populations of differentiated cells were expanded for up to an additional 2 days in the presence of 5 ng/ml TGF-β and 10 ng/ml IL-2 [7]. The impact of the HMGB1-RAGE/TLRs-PI3K-AKT-mTOR axis on Treg stability was determined by applying inhibitors, including 1μM wortmannin

Real-time PCR and Western blot analysis
Total RNAs were extracted from Tregs using the RNAiso plus reagents (9109; TaKaRa, Japan) as instructed. Real-time PCR was conducted to assess Treg function-related genes using an ABI prism 7500 Sequence Detection System (Applied Biosystems, San Francisco, CA, USA). Actb was used for normalization, and the relative expression levels for each target gene were calculated using the 2 -ΔΔCt method.
For Western blot analysis, Treg lysates were separated on 10% SDS-PAGE gels and incubation with an HRP-conjugated secondary antibody, the reactive bands were visualized using an enhanced chemiluminescence system as reported [8].

In vitro suppression assays and T cell-transfer model of colitis
In vitro suppression assays: CD4 + CD25cells (conventional T cells) and CD4 + CD25 + cells were purified to obtain responder cells and Tregs, respectively. Tregs were preincubated with rHMGB1 (500 ng/ml) for 24 h. CFSE-labeled CD4 + CD25cells were cultured in triplicates in roundbottom 96-well plates along with anti-CD3/CD28 stimulation. The inhibitory effect of Tregs on the proliferation of responder cells was tested by directly addition different ratios of Tregs into 2 × 10 5 responder cells. After 3 days of co-culture, cell proliferation was assessed by flow cytometry following CFSE dilution.
T cell-transfer model of colitis: Colitis in NOD-scid mice was induced. Briefly, CD4 + CD25 + Tregs and naïve CD4 + CD44 low/-CD62L + CD25 -T cells from NOD mice were prepared with isolation kits. Tregs were preincubated with or without rHMGB1 (500ng/ml) for 24 h and then washed in sterile PBS. Each NOD-scid recipient mice was given by intravenous injection of 2 × 10 5 naïve T cells alone or together with 5 × 10 4 Tregs. Body weight of mice was monitored weekly.
The severity of colonic inflammation was graded by two pathologists in a blinded fashion using well-described four-point scales [9]: 0, normal tissue; 1, one or a few multifocal mononuclear cell infiltration in the lamina propria, along with minimal epithelial hyperplasia and slight to no mucus depletion from goblet cells; 2, more frequent lesions, and typical changes including several multifocal, mild inflammatory cell infiltration in the lamina propria.

Purification and confirmation of the HMGB1 neutralising antibody
We first purified the HMGB1-neutralising antibody from peritoneal dropsy obtained from BALB/c mice using protein A columns. The purified antibody was further passed over polylysine columns to remove any contaminating endotoxin. The purity of the antibody was high as determined by SDS-PAGE (ESM Fig. 1a). The purified monoclonal antibody showed high specificity and titers against HMGB1, as shown on the western blots (ESM Fig. 1b). To test its neutralising potency, rHMGB1 was added into the RAW264.7 cell cultures, which increased TNF-α secretion by 20-fold, while addition of above purified antibody 2 h before rHMGB1 stimulation, TNF-α secretion was reduced by 3-fold (ESM Fig.   1c), indicating that the neutralising antibody is highly potent to block the HMGB1 stimulatory effect.