Thiolutin alleviates acute graft-versus-host disease (aGVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation

Abstract

Acute graft-versus-host disease (aGVHD) is a major, life-threatening complication in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the development of novel molecular-targeted therapies for aGVHD is urgently needed. Thiolutin (THL), a disulfide-containing antibiotic, inhibits the activity of the BRCC3-containing isopeptidase complex and thereby suppresses NLR pyrin domain-containing protein 3 (NLRP3) inflammasome activation. Here, we investigated the effect of THL on aGVHD in a mouse model of allo-HSCT. We found that THL significantly improved survival in aGVHD recipients, reduced weight loss and clinical scores, and attenuated pathological changes in aGVHD target organs, including the liver, small intestine, and colon. Furthermore, THL diminished donor T-cell activation and infiltration into target organs and promoted the expansion of regulatory T cells. THL also inhibited NLRP3 inflammasome activation and reduced the production of proinflammatory cytokines, including tumor necrosis factor (TNF), interferon γ (IFN-γ), interleukin (IL)-2, IL-17A, and IL-1β. This study reveals a previously unrecognized role of THL in regulating aGVHD and highlights its potential as an anti-inflammatory therapeutic agent for clinical development in patients undergoing allo-HSCT.

1. INTRODUCTION

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an established and effective therapy for hematological malignancies, severe acute radiation syndrome, and other hematologic disorders.^1,2 Graft-versus-host disease (GVHD) is a major complication following allo-HSCT and is associated with substantial morbidity and mortality³. Acute GVHD (aGVHD) is primarily mediated by alloreactive donor T cells that are activated after conditioning regimen–induced host tissue damage, which triggers increased secretion of inflammatory cytokines, including interleukin (IL)-6, tumor necrosis factor α (TNF-α), and IL-1β.⁴ Despite prophylactic administration, 34% to 89% of patients undergoing allo-HSCT develop aGVHD.^5,6 Although high-dose corticosteroids and various alternative therapies have been extensively investigated, their efficacy remains suboptimal.⁷ Therefore, novel molecular-targeted therapies for aGVHD are urgently needed.

The NLR pyrin domain-containing protein 3 (NLRP3) inflammasome is a multimeric complex composed of NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1. This complex is essential for the maturation and secretion of IL-1β and IL-18.⁸ Preclinical studies have shown that conditioning regimens administered before allo-HSCT activate the NLRP3 inflammasome and contribute to the development of aGVHD.⁹ Consistent with this finding, NLRP3 deficiency in recipient mice attenuated aGVHD severity,⁹ and pharmacologic inhibition of NLRP3 improved recipient survival,¹⁰ thereby highlighting NLRP3 as a potential therapeutic target for aGVHD. In addition to its role in aGVHD, the NLRP3 inflammasome plays a critical role in sensing diverse pathogenic stimuli and maintaining cellular homeostasis.¹¹ Although direct inhibition of NLRP3 is conceptually attractive, concerns regarding its essential physiological functions and potential adverse effects suggest that indirect modulation of the NLRP3 pathway may represent a more feasible therapeutic strategy.

Thiolutin (THL), a dithiolopyrrolone antibiotic originally isolated from Streptomyces species, exhibits broad-spectrum antibacterial and limited antifungal activity.¹² THL also functions as a zinc-chelating inhibitor of JAB1/MPN/Mov34 metalloprotease (JAMM) family members, including Rpn11, BRCC36 (BRCC3), and CSN5.¹³ Notably, the BRCC36-containing isopeptidase complex (BRISC), a K63-specific deubiquitinating enzyme (DUB) complex, critically regulates NLRP3 deubiquitination, a process that is essential for optimal NLRP3 inflammasome assembly.^14,15 Our previous study demonstrated that THL potently inhibits BRISC DUB activity, thereby blocking NLRP3 inflammasome activation and suppressing NLRP3-dependent inflammatory responses in vitro and in vivo.¹⁶ Given the established role of NLRP3 in aGVHD pathogenesis, we hypothesized that THL might exert protective effects against aGVHD.

Here, we investigated the impact of THL on aGVHD in a murine allo-HSCT model. Our findings demonstrate that THL significantly reduces aGVHD severity by attenuating donor T-cell infiltration and activation and inhibiting NLRP3-mediated production of inflammatory cytokines.

2. MATERIALS AND METHODS

2.1. Mice

C57BL/6 (H-2Kb) and BALB/c (H-2Kd) mice were purchased from SPF (Beijing, China) Biotechnology Co., Ltd. [license SCXK (Beijing) 2019-0010]. All mice were housed at up to five per cage under specific pathogen-free conditions with a 12-hour light–dark cycle, at a temperature of 22 ± 2°C and a humidity of 45% to 65%, and were provided ad libitum access to food and water. Recipient mice were provided sterilized chow and water supplemented with gentamicin (320 mg/L). All animal studies were reviewed and approved by the Animal Care and Use Committee of our institute (Approval No.: IACUC-DWZX-2021-749). All animal experiments were conducted in accordance with the ARRIVE guidelines for reporting experiments involving animals.

2.2. Development of the murine aGVHD model and assessment of clinical features

Recipient BALB/c mice were lethally irradiated on day 0. Six to 8 hours after irradiation, the mice were transplanted via tail vein injection with 1 × 10⁷ C57BL/6 bone marrow (BM) cells and 5 × 10⁶ C57BL/6 splenocytes. Recipients were monitored daily for survival and every 2 days for changes in body weight and clinical signs of aGVHD. Clinical severity of aGVHD was scored based on five parameters (0–2 points for each parameter): weight loss, posture, activity, fur texture, and skin integrity.¹⁷

2.3. Histological analysis

On day 11, samples of the small intestine, large intestine, and liver were collected and fixed in 4% paraformaldehyde (Servicebio, Wuhan, China), paraffin-embedded, sectioned at 5 µm, stained with hematoxylin and eosin, and evaluated by an experienced pathologist who was blinded to the treatment groups. aGVHD severity was graded using a previously established histopathological scoring system.¹⁸

2.4. THL administration

THL was obtained from Cayman Chemical (Ann Arbor, Michigan). Dimethyl sulfoxide (DMSO) was purchased from Innochem Technology Co., Ltd. (Beijing, China). Mice received intraperitoneal injections of THL (1 mg/kg) or vehicle control (DMSO formulated in phosphate-buffered saline (PBS), total volume 200 µL) every other day, beginning 24 hours before transplantation, as previously described.¹⁹

2.5. Western blotting

Cells were lysed in 1% Triton X-100 lysis buffer supplemented with a protease inhibitor (Roche, Basel, Switzerland) on ice for 30 minutes, and the whole-cell lysates were then boiled for 30 minutes. Cell lysates were loaded onto 10-well precast SDS–polyacrylamide gels and electrophoresed at 80 V for 30 minutes, followed by 120 V for 90 minutes. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Massachusetts) at 80 mA for 70 minutes. Membranes were blocked with tris-buffered saline with Tween-20 (TBST) buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween-20) containing 5% skim milk for 1 hour at room temperature. The membranes were incubated overnight at 4°C on a rolling shaker in TBST containing 5% skim milk with diluted primary antibodies. Anti–caspase-1 was purchased from AdipoGEN (San Diego, California), and anti-GAPDH was purchased from ABclonal (Wuhan, China).

2.6. Quantitative reverse transcription polymerase chain reaction

Tissue samples were homogenized, and total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, California). Total RNA was reverse-transcribed using a reverse transcription kit (Accurate Biology, Changsha, China). Specific primer pairs were used for polymerase chain reaction (PCR) amplification. The relative mRNA expression of each gene was normalized to GAPDH using the 2^-ΔΔCt method. Primer sequences used for quantitative PCR were as follows: GAPDH forward, 5′-TGGTGAAGGTCGGTGTGAAC-3′; GAPDH reverse, 5′-CCATGTAGTTGAGGTCAATGAAGG-3′; IL-1β forward, 5′-GAAATGCCACCTTTTGACAGTG-3′; IL-1β reverse, 5′-TGGATGCTCTCATCAGGACAG-3′; IL-18 forward, 5′-GACTCTTGCGTCAACTTCAAGG-3′; IL-18 reverse, 5′-CAGGCTGTCTTTTGTCAACGA-3′.

2.7. Flow cytometry analysis

Cells from the spleen, liver, and small intestine were prepared as single-cell suspensions and filtered through a 40 µm mesh. Cells were counted using counting beads. Counting beads and anti-mouse CD8–PE-Cy7, CD11b–APC, CD25–PE, CD69–APC, and interferon γ (IFN-γ)–PE antibodies were purchased from Invitrogen. Anti-mouse CD4–BV421 and Foxp3–PE antibodies were purchased from BioLegend (San Diego, California). Anti-mouse CD25–APC antibody was purchased from eBioscience (San Diego, California). Anti-mouse H-2Kb–FITC antibody, Fix/Perm kit, and Cytofix/Cytoperm kit were purchased from BD Biosciences (Franklin Lakes, California). The Fix/Perm kit was used for intracellular Foxp3 staining. The Cytofix/Cytoperm kit was used for intracellular IFN-γ staining. Flow cytometric analyses were performed using a BD LSRFortessa X-20 flow cytometer, and data were analyzed using FlowJo v10.

2.8. Enzyme-linked immunosorbent assay

Mouse IL-1β and IL-18 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Abcam (Cambridge, United Kingdom). The levels of IL-1β and IL-18 in serum and tissue samples were measured according to the manufacturer’s instructions.

2.9. Serum cytokine measurements by bead array

The CBA Mouse Th1/Th2/Th17 Cytokine Kit was purchased from BD Biosciences. Serum levels of IL-4, IL-6, IL-10, IFN-γ, TNF-α, and IL-17A in recipient mice on days 6 and 11 after allo-HSCT were measured according to the manufacturer’s instructions.

2.10. Isolation of small intestine lymphocytes

Small intestinal lymphocytes were isolated as previously described.²⁰ In brief, the small intestine was excised, and visible fat and mesentery were carefully removed. Luminal contents were flushed out, and the small intestine was cut into pieces and transferred to medium containing 1 mM DL-dithiothreitol (Sigma, St. Louis, Missouri). The tissue fragments were incubated at room temperature for 40 minutes with continuous shaking, followed by vortexing at medium speed for 3 minutes. The cell suspension was passed through a 100 µm cell strainer, and the flow-through was collected and centrifuged. The cell pellet was resuspended in 4 mL of 36% Percoll and carefully overlaid onto 67% Percoll to generate a discontinuous density gradient (Cytiva Corporation, Massachusetts). Samples were centrifuged at 700×g for 30 minutes at room temperature without brake. Lymphocytes were collected from the interface between the 36% and 67% Percoll layers and washed with warm medium to remove residual Percoll.

2.11. Isolation of liver mononuclear cells

Liver mononuclear cells (MNCs) were isolated as previously described.²¹ In brief, liver tissue was cut into small pieces and passed through a 100 µm cell strainer, and the flow-through was collected. Samples were centrifuged at 50×g for 5 minutes at 4°C, and the supernatants were subsequently centrifuged at 800×g for 7 minutes at 4°C. The cell pellet was resuspended in 7 mL of 35% Percoll and carefully layered onto 75% Percoll to generate a density gradient. After centrifugation at 800×g for 30 minutes without brake, the mononuclear cell layer at the interface was carefully aspirated and resuspended in RPMI-1640 medium containing 2% fetal bovine serum (FBS). The cells were washed twice and incubated in 1× erythrocyte lysis buffer (BioLegend) for 3 minutes at room temperature. MNCs were collected by centrifugation.

2.12. Statistical analysis

All data are expressed as mean ± standard deviation (SD). An unpaired two-sided Student t test was used for comparisons between two groups. Kaplan–Meier survival curves were generated to assess survival in mouse experiments, and differences were analyzed using the log-rank test. Statistical significance is indicated in the figures as follows: *p < 0.050; **p < 0.010; ***p < .001. GraphPad Prism version 7 (GraphPad Software, San Diego, California) was used for statistical analyses and graph generation.

3. RESULTS

3.1. THL significantly alleviates aGVHD in a mouse model

To investigate the protective effect of THL against aGVHD, mice were treated with THL every other day starting on day 1 relative to allo-HSCT. PBS was used as the vehicle control. By day 22, all mice in the control group had died, whereas 60% of THL-treated mice remained alive (p = 0.001 vs control; Fig. 1A). Mice receiving BM transplantation alone exhibited minimal signs of aGVHD and maintained 100% survival until the endpoint. Notably, THL-treated mice exhibited significantly less weight loss from days 10 to 30 after allo-HSCT (Fig. 1B) and showed markedly lower clinical scores compared with vehicle-treated controls (Fig. 1C).

Figure 1.

THL significantly alleviates the severity of aGVHD. BALB/c recipient mice in THL and control group underwent total body irradiation followed by transplantation with 5 × 10⁶ splenocytes and 1 × 10⁷ BM cells from C57BL/6 mice. BMT Group was transplanted with 1 × 10⁷ BM cells from C57BL/6 mice only. Survival (A), weight changes (B), and clinical GVHD scores (C) were monitored, n = 10/group. (D) Mice were sacrificed on day 11 post-transplant, and histology of liver, small intestine, and colon was analyzed. The pathology scores of these organs were analyzed (E), n = 6–7/group. Error bars indicate SD. For all graphs, *p < 0.050, **p < 0.010. aGVHD = acute graft-versus-host disease, BM = bone marrow, BMT = bone marrow transplantation, SD = standard deviation, THL = thiolutin.

3.2. THL attenuates pathological damage in target organs during aGVHD

To evaluate the protective effects of THL against aGVHD-induced organ damage, histological analyses of target organs (liver, small intestine, and colon) were performed on day 11 post-transplantation. Vehicle-treated mice exhibited substantial hepatic inflammatory infiltration accompanied by necrosis, whereas THL treatment markedly reduced liver tissue damage, with minimal necrosis observed. Similarly, the small intestines of vehicle-treated mice showed extensive villus blunting and dense inflammatory cell infiltration within the lamina propria and crypts. In contrast, THL-treated mice maintained normal villus architecture with minimal cellular infiltration. Vehicle-treated mice also displayed severe colonic inflammation characterized by lamina propria destruction, whereas colons from the THL-treated group retained near-normal architecture with significantly reduced crypt damage and inflammatory infiltration (Fig. 1D). Histological scoring confirmed significantly lower aGVHD severity in all target organs of THL-treated mice compared with controls (Fig. 1E). Collectively, these findings demonstrate that THL attenuates aGVHD-induced histopathological damage in key target organs.

3.3. THL reduces donor T-cell infiltration and activation and promotes Treg expansion

We next assessed donor T-cell dynamics in recipient mice. THL- and vehicle-treated recipients were sacrificed on days 6 and 11 post-transplantation. No significant differences were observed in the proportions or absolute numbers of splenic donor T cells between groups (Fig. S1, https://links.lww.com/BS/A174; Fig. 2A–C). However, THL treatment significantly reduced the absolute numbers of infiltrating donor CD4⁺ and CD8⁺ T cells in the liver (Fig. 2D–F) and small intestine (Fig. 2G, H), whereas the proportions of donor T-cell subsets in these organs were similar between the THL-treated and control groups (Figs. S2 and S3, https://links.lww.com/BS/A174). These data suggest that THL inhibits donor T-cell infiltration into aGVHD target tissues.

Figure 2.

THL inhibits donor T-cell infiltration into aGVHD target tissues. BALB/c recipient mice were transplanted with 5 × 10⁶ splenocytes and 1 × 10⁷ BM cells from C57BL/6 mice. THL was administered at a dose of 1 mg/kg. Mice were sacrificed on days 6 and 11 post-transplant. Numbers of donor CD4⁺ and CD8⁺ T cells in SP (A–C), liver (D–F), and SI (G–H) were analyzed. Gating strategy was displayed in (A), (D), and (G), n = 4–5/group. Error bars indicate SD. For all graphs, *p < 0.050, **p < 0.010, ***p < .001. aGVHD = acute graft-versus-host disease, BM = bone marrow, SI = small intestine, SP = spleen, THL = thiolutin.

To evaluate donor T-cell activation, we analyzed CD25 and CD69 expression on donor CD4⁺ and CD8⁺ T cells (Fig. 3A). On days 6 post-transplantation, THL-treated recipients exhibited significantly fewer splenic CD8⁺CD69⁺ T cells compared with controls, whereas activated CD4⁺ T-cell populations (CD25⁺ and CD69⁺) remained comparable (Fig. 3B). By day 11, THL-treated recipients showed reduced numbers of splenic CD4⁺CD25⁺, CD4⁺CD69⁺, and CD8⁺CD25⁺ T cells (Fig. 3C). In the liver, the numbers of donor CD8⁺CD25⁺ and CD8⁺CD69⁺ T cells were markedly reduced in THL-treated recipients on day 6 (Fig. 3D, E). On day 11, the number of donor T cells expressing activation markers was lower in THL-treated recipients than in the vehicle-treated group (Fig. 3F). Similar results were observed in the small intestine (Fig. 3G, H). To determine whether THL inhibits T-cell activation at an early stage, we examined donor CD4⁺ and CD8⁺ T-cell activation 6 hours after transplantation. In the spleen of THL-treated recipients, donor T-cell activation was not significantly altered compared with the control group, which may be due to the possibility that a single administration of THL was insufficient to suppress T-cell activation (Fig. S4a, b, https://links.lww.com/BS/A174). We also examined the number of myeloid cells (CD11b⁺) in the spleens of THL- and vehicle-treated recipients. From days 6 to 11 post-transplantation, the number of splenic myeloid cells significantly increased in both groups; however, no significant difference was observed between THL- and vehicle-treated recipients (Fig. S5, https://links.lww.com/BS/A174).

Figure 3.

THL reduces donor T-cell activation in aGVHD target tissues. BALB/c recipient mice were transplanted with 5 × 10⁶ splenocytes and 1 × 10⁷ BM cells from C57BL/6 mice. THL was administered at a dose of 1 mg/kg. Mice were sacrificed on days 6 and 11 post-transplant. Numbers of donor CD4⁺ and CD8⁺ T cells expressing CD25 and CD69 in spleen (A–C), liver (D–F), and SI (G–H) were analyzed. The gating strategy was displayed in (A), (D), and (G). n = 4/group. Error bars indicate SD. For all graphs, *p < 0.050, ***p < .001. aGVHD = acute graft-versus-host disease, BM = bone marrow, SD = standard deviation, SI = small intestine, THL = thiolutin.

Given the therapeutic relevance of regulatory T cells (Tregs) in aGVHD, we quantified donor-derived CD4⁺CD25⁺Foxp3⁺ Treg cells in the spleen and liver. On days 6 and 11 post-transplantation, the numbers of CD4⁺CD25⁺Foxp3⁺ cells were significantly increased in the spleens of THL-treated recipients compared with control mice (Fig. 4A, B). In the livers of THL-treated recipients, a significant increase in Treg cell numbers was observed on day 11 (Fig. 4C, D).

Figure 4.

THL increases the number of donor Treg cells in spleen and liver. BALB/c recipient mice were transplanted with 5 × 10⁶ splenocytes and 1 × 10⁷ BM cells from C57BL/6 mice. THL was administered at a dose of 1 mg/kg. Mice were sacrificed on days 6 and 11 post-transplant. Numbers of Treg cells (CD4⁺ CD25⁺ Foxp3⁺) in recipient spleen (A–B) and liver (C–D) were analyzed. n = 4/group, error bars indicate SD. For all graphs, **p < 0.010, ***p < .001. BM = bone marrow, THL = thiolutin, Treg = regulatory T cell.

These findings demonstrate that THL ameliorates aGVHD through the concurrent suppression of donor T-cell activation and infiltration and the promotion of Treg expansion.

3.4. THL decreases proinflammatory cytokine production

Proinflammatory cytokines critically contribute to aGVHD pathogenesis.²² We measured serum levels of IFN-γ, TNF-α, IL-1β, IL-2, IL-6, and IL-17A on days 6 and 11 post-transplantation. On day 6, THL-treated recipients exhibited significantly reduced serum concentrations of TNF-α, IL-2, and IL-17A compared with controls, whereas IFN-γ, IL-6, and IL-1β levels remained comparable (Fig. 5A). By day 11, serum levels of IL-2, IL-1β, and IFN-γ in the THL-treated group were significantly lower than those in the control group (Fig. 5B). To determine the cellular source of IFN-γ, we analyzed splenic IFN-γ⁺CD4⁺ and IFN-γ⁺CD8⁺ T cells on day 11. Consistent with these findings, THL significantly decreased both IFN-γ⁺CD4⁺ and IFN-γ⁺CD8⁺ T-cell populations compared with vehicle-treated recipients (Fig. 5C, D).

Figure 5.

THL decreases proinflammatory cytokine production. BALB/c recipient mice were transplanted with 5 × 10⁶ splenocytes and 1 × 10⁷ BM cells from C57BL/6 mice. THL was administered at a dose of 1 mg/kg. Mice were sacrificed on days 6 and 11 post-transplant. Serum levels of IFN-γ, TNF-α, IL-1β, IL-2, IL-6, and IL-17A were measured (A–B), n = 5–7/group. Numbers of donor CD4⁺ and CD8⁺ T cells producing IFN-γ in spleen on days 11 post-transplant were analyzed by flow cytometry (C–D), n = 4/group. Error bars indicate SD. For all graphs, *p < 0.050, **p < 0.010. BM = bone marrow, IFN-γ = interferon γ, IL = interleukin, SD = standard deviation, THL = thiolutin, TNF = tumor necrosis factor.

3.5. THL inhibits NLRP3 inflammasome activation

The maturation of pro-caspase-1 and its cleavage into active caspase-1 are critical steps in NLRP3 inflammasome activation.²³ We analyzed pro-caspase-1 processing in small intestinal tissues and observed significantly reduced caspase-1 activation in THL-treated recipients compared with vehicle controls (Fig. 6A). Furthermore, THL downregulated the expression of key NLRP3-dependent cytokines, IL-1β and IL-18 in small intestine (Fig. 6B-C), splenic macrophage (Fig. 6D) and dendritic cells (Fig. 6E). These results demonstrate that THL suppresses NLRP3 inflammasome activation during aGVHD pathogenesis.

Figure 6.

THL inhibits NLRP3 inflammasome activation. BALB/c recipient mice were transplanted with 5 × 10⁶ splenocytes and 1 × 10⁷ BM cells from C57BL/6 mice. Mice were sacrificed on day 6 post-transplant. (A) The expression levels of pro-caspase-1 and cleaved caspase-1 in small intestine tissue were analyzed by Western blotting analysis. GAPDH was used as internal control. (B) The mRNA levels of IL-1β and IL-18 in small intestine tissue were measured by RT-qPCR. Each mouse was performed in triplicate. Data shown are representative of 3 independent experiments with a total of 3–4 mice per group. (C) The protein levels of IL-1β and IL-18 in small intestine tissue homogenization were measured by ELISA, n = 3/group. (D) Splenic macrophage (CD45⁺CD11b⁺CD11c^-F4/80⁺) and (E) dendritic cells (CD45⁺CD11b^-CD11c⁺MHCII⁺) were isolated using flow cytometry. The mRNA levels of IL-1β and IL-18 were measured by RT-qPCR. Data shown are representative of 3 independent experiments with a total of 3–4 mice per group. Error bars indicate SD. For all graphs, *p < 0.050, **p < 0.010, ***p < .001. BM = bone marrow, ELISA = enzyme-linked immunosorbent assay, IL = interleukin, NLRP3 = NLR pyrin domain-containing protein 3, RT-qPCR = reverse transcription polymerase chain reaction, SD = standard deviation, THL = thiolutin.

4. DISCUSSION

THL is a disulfide-containing antibiotic with reported antiangiogenic properties. Minamiguchi et al demonstrated that THL inhibits endothelial cell adhesion and suppresses wound- and tumor-driven angiogenesis.^24,25 Recent studies have shown that THL targets Rpn11 to suppress esophageal squamous cell carcinoma cell motility and enhance cisplatin sensitivity.^26,27 Our previous study demonstrated that THL inhibits BRISC DUB activity by directly targeting the catalytic Zn2⁺-binding site of BRCC3, thereby suppressing NLRP3 inflammasome activation.^14,19

The present study confirms that THL treatment reduces NLRP3 inflammasome activation, as evidenced by diminished IL-1β production and decreased pro-caspase-1 cleavage. Paradoxically, BRCC3-knockout recipient mice exhibit significantly shortened survival and exacerbated liver damage during aGVHD pathogenesis.²⁸ BRCC3^−/− recipients displayed increased numbers of splenic, hepatic, and intestinal CD8⁺ T cells, as well as CD8⁺CD25⁺ T cells, indicating enhanced donor CD8⁺ T-cell proliferation and activation that aggravates aGVHD.²⁸ These suggest that THL exerts its suppressive effects on aGVHD in a BRISC-independent manner. Notably, THL inhibits multiple JAMM-domain DUBs in addition to BRCC3, including Rpn11, CSN5, and AMSH.¹³ Intriguingly, Rpn11 negatively regulates IL-1β production and inflammasome activity by deubiquitinating pro-IL-1β.²⁹ This polypharmacological profile suggests that THL likely modulates aGVHD through multiple molecular targets. Elucidating the molecular mechanisms underlying THL activity may reveal novel therapeutic targets for aGVHD.

Cytokines critically drive aGVHD pathogenesis. Conditioning regimens (eg, chemoradiotherapy or irradiation) trigger the release of inflammatory mediators, including IL-1, IL-6, TNF-α, and IL-33, which activate antigen-presenting cells (APCs). Activated APCs subsequently stimulate donor T-cell proliferation, differentiation, and effector functions. Ultimately, T cell–derived cytokines (eg, IFN-γ, IL-17A, IL-2, and TNF-α) induce target tissue apoptosis and recruit myeloid cells that amplify aGVHD pathology.^22,30 Notably, randomized trials have demonstrated that single-cytokine blockade targeting TNF, IL-1, or IL-6 fails to significantly improve aGVHD outcomes,^30,31 whereas simultaneous multi-cytokine inhibition effectively ameliorates disease severity.³² These findings establish that concurrent suppression of multiple inflammatory pathways is essential for clinical efficacy. The JAK1/2 inhibitor ruxolitinib exemplifies this principle by potently reducing aGVHD through broad attenuation of cytokine signaling.³³ Our data demonstrate that THL significantly inhibits the production of key cytokines (IFN-γ, IL-1β, TNF-α, IL-2, and IL-17A), thereby positioning it as a promising multi-cytokine–targeting therapeutic for controlling inflammatory cascades in aGVHD.

Our study unexpectedly revealed that THL administration expands Treg populations in allo-HSCT recipients. Tregs are essential for maintaining immune homeostasis and promoting transplantation tolerance. During aGVHD progression, recipient Treg numbers decline, thereby exacerbating disease severity.^34,35 Clinically, reduced Treg-to-effector T-cell ratios correlate with an increased risk of aGVHD.³⁶ Murine models have demonstrated that adoptive Treg transfer ameliorates aGVHD,^37–39 and clinical trials have confirmed the efficacy of recombinant human granulocyte colony-stimulating factor–mobilized peripheral blood– or umbilical cord blood–derived Treg infusions for suppressing aGVHD.^40–42 The mechanism by which THL modulates Treg dynamics in aGVHD remains unclear. Elucidating this regulatory pathway may inform novel strategies to enhance Treg reconstitution.

Collectively, our findings demonstrate that THL mitigates aGVHD severity by suppressing donor T-cell activation and proinflammatory cytokine production in allo-HSCT recipients. These findings indicate that THL represents a promising therapeutic strategy for the clinical management of aGVHD.

ACKNOWLEDGMENTS

This study was supported in part by grants from the Ministry of Science and Technology of the People’s Republic of China (Grant No. 2022YFA1103502), National Natural Science Foundation of China (Grant No. 81222005), and the State Key Laboratory of Proteomics (Grant No. SKLP-K202201, SKLP-O201804).

AUTHOR CONTRIBUTIONS

C.-Y.L. and H.-M.N. conceived the project, designed the research, and supervised the experiment. X.-M.Y. revised the manuscript for important intellectual content. X.L., K.Z., R.G., Z.-C.L., Q.W., G.-M.R., H.-Y.G., S.-S.X., Y.-X.Z., Y.-Q.W., H.-Y.S., H.C., and R.-H.Y. participated in the performance of the research. X.L. and K.Z. participated in data analysis. All authors read and approved the final manuscript.

Supplementary Material

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