Intracellular H₂S generation primarily depends on three enzymes (44): CBS (liver, kidney, brain) can be activated by SAM and translocates to mitochondria under stress, coupling RSS signaling to energy metabolism (45, 46). CSE (liver, kidney, vasculature, immune cells) exhibits Ca²⁺-responsive regulation via calmodulin, enabling rapid H₂S generation during immune activation (47–49). 3-MST shows unique dual cytoplasmic-mitochondrial localization, serving as the primary mitochondrial H₂S source (50, 51). These spatially and temporally regulated enzymes create RSS generation nodes across immune cell types, while cysteine dioxygenase 1 (CDO1) indirectly regulates RSS levels by controlling substrate availability (52, 53).

H₂S oxidative metabolism represents a critical regulatory axis controlling cellular RSS levels. In mitochondria, H₂S undergoes sequential enzymatic oxidation through sulfide quinone oxidoreductase (SQOR), persulfide dioxygenase (ETHE1), thiosulfate sulfurtransferase (TST) and sulfite oxidase (SUOX), ultimately converting to thiosulfate and sulfate while feeding electrons into the respiratory chain (54, 55).

SQOR catalyzes the initial step, oxidizing H₂S and transferring electrons to coenzyme Q, directly coupling sulfide oxidation to ATP production (54). Critically, SQOR uses glutathione (GSH) as the primary sulfur acceptor to form glutathione persulfide (GSSH). This GSSH intermediate serve dual roles: (1) as a substrate for downstream catabolic enzymes, and (2) as a mobile persulfide donor that diffuses from mitochondria to participate in cytosolic RSS signaling. Thus, SQOR functions both as a catabolic enzyme controlling H₂S levels and as a generator of signaling-competent persulfide intermediates. ETHE1 subsequently oxidizes persulfides (primarily GSSH) to sulfite. TST then catalyzes the reaction between another GSSH molecule and SO₃^2- to produce thiosulfate as a stable product. Sulfite can also be further oxidized by SUOX to sulfate (SO₄^2-) (55). The genetic importance of this pathway is underscored by ethylmalonic encephalopathy (ETHE1 mutations), where toxic H₂S accumulation impairs mitochondrial function and immune responses, such as chronic inflammation and neutrophil dysfunction (56).

Collectively, the coordinated action of biosynthetic enzymes (CBS, CSE, 3-MST, CDO1) and catabolic enzymes (SQOR, ETHE1, TST, SUOX) constitutes an integrated RSS metabolic network. The spatiotemporal regulation of their expression, activity, and subcellular localization determines RSS levels and speciation across different immune cell types and physiological-pathological states, providing multiple therapeutic intervention points for immune-metabolic diseases.

NF-κB is a master regulator of macrophage inflammatory responses and M1 polarization (71–73). RSS modulates this pathway through three mechanistic tiers that collectively determine context-dependent outcomes: transcriptional regulation, post-translational modification, and metabolic crosstalk. Understanding these multilevel interactions provides mechanistic rationale for RSS-based interventions in diseases featuring NF-κB hyperactivation coupled with endogenous H₂S deficiency (74).

While NF-κB governs transcriptional inflammatory programs (Section 3.1.1), the Keap1-Nrf2 pathway provides a complementary antioxidant defense mechanism. Nrf2 serves as the master regulator of cellular antioxidant responses. Under physiological conditions, Keap1 sequesters Nrf2 in the cytoplasm for continuous proteasomal degradation. RSS activate this pathway through site-specific Keap1 cysteine modification, mimicking oxidative stress signals to initiate protective responses.

Beyond NF-κB and Nrf2 regulation, RSS coordinate macrophage functional remodeling through mitochondrial quality control, programmed cell death pathways, and mitogen-activated protein kinase (MAPK) signaling cascades. These mechanisms provide additional layers of regulation in sepsis-associated organ dysfunction, burn injuries, and metabolic liver diseases.

PINK1/Parkin-mediated mitophagy protects macrophage phenotypic homeostasis across diverse pathological contexts. In sepsis-associated cardiorenal syndrome type 5, exogenous NaHS and S-propargyl-cysteine (SPRC) activate this pathway to clear damaged mitochondria, restore membrane potential, suppress ROS generation, and promote RAW264.7 macrophage M1-to-M2 transition in cardiac/renal tissues (93). In acute arsenic exposure, endogenous PINK1/Parkin upregulation with elevated LC3-II/I ratio confers hepatoprotection; pharmacological PINK1 inhibition (cyclosporin A) exacerbates arsenic-induced hepatic macrophage inflammation (94). These findings establish mitophagy as a convergent mechanism linking exogenous H₂S donors and endogenous stress responses to macrophage phenotypic homeostasis.

In rat burn wound models, NaHS modulates the expression of three major MAPK family members, including ERK, c-Jun N-terminal kinase (JNK) and p38, in basal skin macrophages at injury sites (95). Given that MAPK pathways govern macrophage inflammatory cytokine production, proliferation, and apoptosis decisions, this modulation pattern suggests RSS integrate pleiotropic immunoregulatory effects through fine-tuning MAPK pathway.

Indirect evidence for RSS involvement in metabolic liver disease emerges from studies on the immune checkpoint molecule Tim-3. In methionine-choline-deficient diet-induced non-alcoholic steatohepatitis (NASH), Tim-3 suppresses macrophage IL-1β and IL-18 secretion, thereby exerting hepatoprotection. Similarly, N-acetylcysteine (NAC), a cysteine derivative and ROS scavenger, ameliorates hepatic steatosis and inflammation (96). Although H₂S levels were not directly measured in this study, NAC’s dual function as both a cysteine donor (H₂S biosynthetic substrate) and ROS inhibitor implies potential crosstalk between sulfur metabolism and the ROS-inflammasome axis, warranting further investigation into RSS roles in metabolic liver pathology.

Collectively, RSS regulates macrophage function through mitochondrial quality control, MAPK signaling, and potential interactions with immune checkpoint-ROS pathways. These mechanisms complement NF-κB and Nrf2 (Sections 3.1.1-3.1.2), constituting a multilayered regulatory network (Figure 3). The therapeutic relevance of these mechanisms is further validated in disease models and innovative biomaterial systems, as examined in the following section.

In addition to maintaining mitochondrial integrity, RSS further regulate immune cell fate by controlling the metabolic switch between glycolysis and mitochondrial respiration.

H₂S targets GAPDH, modifying it via persulfidation at the active site cysteine (Cys150). While initial studies reported that this modification significantly enhances GAPDH enzymatic activity due to the increased nucleophilicity of the persulfide group (97, 98), subsequent in vitro analyses utilizing polysulfide donors have suggested that persulfidation may instead inhibit catalytic function, possibly due to steric hindrance at the active site (99). This discrepancy suggests that the functional impact of GAPDH persulfidation may be context-dependent or sensitive to the specific sulfur donors employed.

A mechanistic breakthrough by Rahman et al. revealed that Mycobacterium tuberculosis infection triggers excessive host CSE expression, leading to supraphysiological H₂S levels. Unlike its physiological role, this surfeit of H₂S acts as a broad metabolic suppressor, inhibiting central carbon metabolism and mitochondrial respiration. The suppression of glycolytic flux is particularly detrimental, as it prevents the stabilization of Hypoxia-inducible factor 1-alpha (HIF-1α). As HIF-1α is required for IL-1β production and NADPH-dependent ROS generation, its destabilization leads to impaired bactericidal capacity (100). Consequently, CSE-deficient macrophages maintain robust glycolytic rates and HIF-1α levels, thereby exhibiting enhanced bacterial control. Our previous work showed that sulfonyl azide effectively scavenges H₂S (101), providing a potential tool to modulate RSS levels. Thus, targeting RSS-producing enzymes or scavenging excess RSS to restore the glycolysis-HIF-1α axis may reverse metabolic immunosuppression.

The mechanistic insights from Sections 3.1.1-3.1.4 have been translated into therapeutic application across multiple metabolic disease models and innovative biomaterial platforms. Rather than reiterating individual studies, this section synthesizes disease-mechanism-intervention relationships and highlights emerging delivery technologies addressing the pharmacokinetic limitations of traditional H₂S donors.

RSS-based interventions demonstrate therapeutic efficacy across diverse metabolic disease contexts (Table 1). In diabetic complications, endogenous H₂S deficiency correlates with chronic NF-κB hyperactivation (wounds, osteoarthritis), impaired Nrf2 signaling (atherosclerosis), and defective mitophagy (nephropathy). Exogenous supplementation consistently restores macrophage homeostasis, thereby reducing tissue infiltration. In obesity-related inflammation, CSE deficiency in adipose tissue macrophages (ATMs) and perivascular adipose tissue (PVAT) drives systemic metabolic dysfunction, reversible by H₂S application or CSE overexpression. Beyond metabolic disorders, targeting RSS-driven immunometabolism shows promise in infectious diseases.

Traditional H₂S donors (NaHS, Na₂S) suffer from rapid release kinetics and poor spatiotemporal control. To overcome these pharmacological limitations, novel polysulfide delivery systems have emerged with superior efficacy. N-acetylcysteine polysulfide (NAC-S2), a cell-permeable sulfane sulfur donor distinct from traditional sulfide salts (105, 106), potently disrupts TLR4 signal and downstream cytokine storms in lethal endotoxin shock models, highlighting the pharmacodynamic advantages of polysulfide donors in managing systemic inflammation. Complementing these molecular advances, recent engineering innovations further address delivery constraints through three technological tiers: (1) Responsive nanocarriers: pH/ROS-triggered release systems, like iron sulfide nanocomposites GOx@FexSy/AZM for infected diabetic wounds (107) that match therapeutic action to microenvironmental cues; (2) Smart hydrogels: microenvironment-adaptive platforms, such as CoS-loaded silk fibroin hydrogels (108), GelMA-ODex@RRHD (109); (3) Multifunctional biomaterials: conductive hydrogels coupling H₂S release with bioelectric signaling to synergistically enhance M2 polarization and tissue regeneration (e.g., PEDOT-alginate/gelatin systems) (110). These platforms demonstrate evolution from single-target intervention toward multidimensional, spatiotemporally precise regulation of macrophage function.

Despite promising preclinical efficacy, clinical translation faces critical challenges. First, long-term safety requires further characterization. Second, hormetic dose-response curves demand patient-stratified dosing based on endogenous RSS levels. Third, delivery routes (topical for wounds, inhalation for lung disease, or systemic) must align with disease pathology. Finally, non-invasive biomarkers for baseline RSS status and treatment monitoring remain undeveloped. Addressing these gaps will enable rational design of RSS-based precision therapeutics.

In type 1 diabetes mellitus (T1DM) rat models, serum IL-1β and IL-8 levels are significantly elevated, accompanied by upregulated neutrophil chemokine receptor CXCR2 expression, indicating diabetes-driven neutrophil activation. NaHS treatment reduces IL-1β and IL-8 expression and secretion while reversing CXCR2 overexpression, demonstrating that RSS inhibits neutrophil chemotactic responses (111). This receptor-level intervention represents a proximal control point for limiting neutrophil recruitment to inflamed tissues.

In LPS-induced acute lung injury (ALI) mouse models, pretreatment with the slow-release H₂S donor GYY4137 significantly reduces neutrophil infiltration in lung tissue and inflammatory mediator accumulation in alveolar spaces, mechanistically linked to downregulated macrophage inflammatory protein-2 (MIP-2/CXCL2) and its receptor expression (112). In vitro experiments further confirmed that GYY4137 directly inhibits neutrophil transendothelial migration and MIP-2 release (112). Similarly, in bisphenol A (BPA)-induced lung injury, NaHS treatment significantly suppresses neutrophil infiltration into bronchoalveolar lavage fluid (BALF) (113), confirming the capacity to attenuate environmental toxicant-induced neutrophil accumulation. In granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced differentiated HL-60 cells, NaHS significantly downregulates oncostatin M (OSM) expression by inhibiting the PI3K-Akt-NF-κB signaling pathway (114), further confirming RSS regulatory effects on neutrophil inflammatory cytokine profiles.

In mechanical ventilation-induced lung injury models, H₂S demonstrates time-dependent protective effects with a relatively wide therapeutic window. Whether administered 1, 3, or 5 hours before ventilation (pretreatment) or at different time points after ventilation (post-treatment), H₂S inhalation effectively reduces pulmonary edema, histological damage, neutrophil infiltration, and ROS generation (115). Mechanistically, H₂S exerts pulmonary protection by synergistically inhibiting stress proteins, including heat shock protein 70 (HSP70), p38 MAPK phosphorylation, NADPH oxidase 2 (NOX2) expression, and ROS generation (116).

Clinical studies found that in asthma and chronic obstructive pulmonary disease (COPD) patients, sputum H₂S levels significantly positively correlate with neutrophil proportions, with elevation consistent with neutrophil-dominated airway inflammatory responses (117, 118). This observation supports H₂S as a potential biomarker for neutrophil-related airway inflammation, revealing its dual role: exerting anti-inflammatory protective effects in acute inflammation models while reflecting neutrophil-mediated inflammatory states in chronic airway diseases.

RSS regulation of basic neutrophil effector functions exhibits marked complexity and donor specificity. In isolated human polymorphonuclear leukocytes (PMNLs), exogenous RSS donors show no significant effects on chemotaxis and phagocytosis but display donor-dependent differences in oxidative burst and apoptosis regulation. For oxidative burst, NaHS enhances phorbol myristate acetate (PMA)- or E. coli-induced responses, whereas diallyl trisulfide (DATS) and cysteine selectively inhibit E. coli’s burst without affecting PMA stimulation. Indeed, garlic constituents have been shown to potently attenuate free radical generation from neutrophils, likely by scavenging oxidizing agents (119). For apoptosis regulation, NaHS, diallyl disulfide (DADS), and cysteine reduce PMNL apoptosis, whereas GYY4137 induces apoptosis via mitochondrial intrinsic pathways (120).

These findings reveal that RSS regulation of neutrophil function exhibits significant donor specificity and context dependence. It primarily exerts anti-inflammatory effects under pathological conditions while sparing basal immune defense, underscoring the need for rational donor selection.

Notably, RSS demonstrates opposite effects in specific pathological contexts. In elastase-induced abdominal aortic aneurysm (AAA) models, sodium thiosulfate (STS, Na₂S₂O₃), significantly enhances neutrophil infiltration in aneurysm walls, especially MMP9⁺ neutrophils, while also observing accumulation of c-KIT⁺ MPO⁺ pre-neutrophil clusters (121). Conversely, CSE-deficient mice (CSE⁻/⁻) exhibit reduced neutrophil infiltration and limited aneurysm expansion under identical AAA induction conditions (121), suggesting RSS may promote neutrophil chemotaxis or activation in this specific vascular pathology. This context-dependent duality underscores the importance of considering disease-specific microenvironments.

NETosis is characterized by chromatin DNA release interwoven with granular proteins (MPO, NE, CitH₃); it plays dual roles in immunity: pathogen capture versus tissue damage amplification (122, 123). In metabolic diseases, excessive ROS generation triggers pathological NETosis that becomes a key driver of complications. RSS inhibits NETosis through three convergent mechanistic axes that collectively suppress the ROS-MAPK-PAD4 signaling cascade underlying chromatin decondensation.

In diabetic wound healing models, CSE expression downregulation and decreased H₂S generation are closely associated with abnormally elevated NETs levels. Both serum from diabetic foot patients and wound tissues from db/db mice show significantly elevated NETs markers, accompanied by delayed wound healing. Exogenous Na₂S or NaHS downregulates PAD4, CitH₃, and NETs component (dsDNA, MPO, NE) release, reduces neutrophil infiltration, and promotes wound healing (4, 87). Proteomic analysis of human surgical wound exudates confirms enrichment of neutrophils and oxidative products (including MPO) in the wound microenvironment (134), providing clinical evidence supporting RSS intervention.

Nutritional intervention studies demonstrate that combined supplementation with vitamin D and L-cysteine (H₂S precursor) reduces neutrophil/lymphocyte ratio and inflammatory marker levels (135). In diabetic peripheral neuropathy models, vitamin D exerts neuroprotective effects by restoring the CBS/H₂S system (136). These findings suggest that enhancing endogenous H₂S generation through nutritional intervention may improve inflammatory status in metabolic disease patients, providing non-pharmacological strategies for clinical application.

Novel H₂S delivery systems provide technical foundations for clinical translation. pH-sensitive donors (JK-1), slow-release donors (GYY4137), and mitochondria-targeted donors (AP39) (112, 130, 137) have demonstrated the capacity to inhibit neutrophil over-activation or/and NETosis in multiple disease models, including diabetic wounds, acute lung injury, and gastric mucosal injury. These controlled-release platforms address the limitations of traditional fast-release donors by achieving spatiotemporally precise H₂S delivery matched to pathological microenvironments, laying groundwork for clinical applications in metabolic disease complications.

Clinical translation requires addressing several key challenges. Donor specificity: the differential effects of NaHS, DATS, and GYY4137 on oxidative burst and apoptosis necessitate rational donor selection based on therapeutic goals. Context dependency: pro-infiltration effects observed in AAA models contrast with anti-inflammatory effects in lung injury, demanding disease-specific risk-benefit assessments. Biomarker validation: while sputum H₂S correlates with neutrophil proportions in COPD patients, this relationship requires prospective validation as a therapeutic monitoring tool. Finally, combination strategies should be explored to integrate H₂S manipulation (using both donors and scavengers) with existing anti-inflammatory therapies such as corticosteroids, potentially achieving synergistic neutrophil regulation.

RSS regulation of DC function operates primarily through control of cysteine availability and GSH homeostasis, rather than solely through H₂S signaling. This mechanistic insight emerges from studies showing that the cystine/glutamate antiporter (system xc⁻, xCT) serves as the gatekeeper of cellular sulfur metabolism (77, 138). System xc⁻ controls cystine uptake and its subsequent reduction to cysteine, the shared substrate for both GSH synthesis and H₂S biosynthesis, creating a metabolic branch point where RSS production and antioxidant defense compete for limited cysteine pools. Blocking system xc⁻-mediated cystine transport creates a dual metabolic crisis: depleted GSH compromises antioxidant defense, while reduced cysteine pools limit endogenous H₂S generation and protein persulfidation. The downstream consequences reveal this axis as a critical redox checkpoint: significantly reduced intracellular GSH levels severely disrupt monocyte-to-DC differentiation without affecting LPS-induced phenotypic maturation, significantly impair exogenous antigen presentation to MHC class II-restricted T cells, and completely block MHC class I cross-presentation (139). These findings establish that GSH homeostasis regulates stage-specific DC differentiation, with GSH and RSS biosynthesis sharing the same cysteine pool.

Beyond basal redox balance, the GSH-RSS axis integrates environmental danger signals through the Keap1/Nrf2 pathway. In human CD34-derived DCs and THP-1 cells, contact sensitizers (nickel, 1-chloro-2,4-dinitrobenzene, cinnamaldehyde) specifically induce Nrf2 target genes (HMOX1, NQO1) at early time points. Pretreatment with NAC significantly inhibits sensitizer-induced NQO1 expression and CD86 upregulation (140), indicating that GSH or RSS levels affect Keap1/Nrf2 activation and directly correlates with DC maturation status. This reveals the cysteine-GSH-RSS metabolic network as an adaptive defense system sensing environmental danger signals.

Critically, RSS-producing enzymes exert non-H₂S-dependent DC regulation via direct cysteine pool control. In rat cardiac allograft models, the CSE inhibitor propargylglycine (PPG) delays rejection and selectively inhibits Th1 factors (T-bet, IL-12, IFN-γ) without affecting antibodies. Mechanistically, CSE inhibition acts directly on monocytes and DCs to reduce IL-12 production capacity through intracellular cysteine pool regulation, independent of NF-κB signaling or H₂S (141). This indicates CSE’s non-canonical function: directly regulating DC immune programming via cysteine availability rather than H₂S signaling. CSE and system xc⁻ constitute a dual-valve system governing intracellular cysteine/GSH homeostasis during DC differentiation. However, the molecular mechanisms linking cysteine availability to IL-12 transcriptional control remain undefined.

Beyond cysteine pool control, H₂S donors selectively inhibit pathological DC subsets in disease-specific contexts. In methionine/choline-deficient diet-induced metabolic dysfunction-associated steatohepatitis (MASH), inflammatory monocyte-derived DCs (moDCs) characterized by dual Ly6C⁺CD11b⁺CD11c⁺MHC-II⁺ expression and continuous TNF-α production undergo 4-fold expansion during late disease. NaHS specifically prevents TNF-α-producing CX3CR1⁺ moDC accumulation, reducing hepatic TNF-α levels and improving transaminase release without affecting normal liver macrophages (104). This selective targeting of pathological subsets reveals the precision immunoregulatory potential of RSS, yet the molecular determinants of this selectivity remain unidentified, including specific metabolic vulnerabilities or surface receptor profiles.

In CNS autoimmune diseases, GYY4137 reshapes DC cytokine balance toward immune tolerance. In CNS antigen-immunized mice, GYY4137 enhances TGF-β expression/secretion in DCs while reducing T cell-derived IFN-γ and IL-17 in lymph nodes and spinal cords. Critically, PBMC from multiple sclerosis patients exhibit significantly lower 3-MST expression compared to healthy controls, with 3-MST levels negatively correlating with pro-inflammatory factors (142), suggesting endogenous H₂S deficiency promotes autoimmune pathology by weakening DC tolerogenic programs. GYY4137’s slow-release kinetics enable sustained low-concentration exposure, potentially more suitable for inducing DC reprogramming compared to fast-release donors.

An unresolved paradox is that H₂S biological effects depend highly on source, chemical form, and concentration. Intestinal microbial protein fermentation-produced H₂S exhibits cytotoxicity to bone marrow-derived DCs at high concentrations in vitro, with unclear effects on cytokine secretion at physiological ranges (143). This contrasts sharply with exogenous donor anti-inflammatory effects, suggesting rapid free H₂S fluctuations produce non-specific toxicity whereas controllable release achieves targeted regulation.

In tumor microenvironments, RSS exhibit profound concentration- and source-dependent duality: exogenous RSS enhance anti-tumor immunity, whereas endogenous CBS/CSE overexpression in tumor cells creates immunosuppression. Understanding this paradox is critical for RSS-based cancer immunotherapy.

Exogenous donors remodel tumor immunosuppression by targeting myeloid-derived suppressor cells (MDSCs). In B16F10 melanoma models, DATS treatment significantly inhibits tumor growth through selective MDSC frequency reduction in spleen, peripheral blood, and tumor tissues, accompanied by increased tumor-infiltrating CD8⁺ T cells and DCs with restored T cell proliferation. Mechanistically, DATS upregulates the expression of antioxidant genes (GCLC, GCLM, HMOX), suggesting that local antioxidant system modulation indirectly promote DC maturation by relieving MDSC-mediated suppression (144) (Figure 5A).

Conversely, elevated CBS/CSE expression in tumor cells correlates with poor prognosis and immune evasion. In ovarian cancer tissues, both CBS and CSE are significantly upregulated, with high CBS expression correlating with shorter progression-free survival (PFS, P = 0.02) and overall survival (OS, P = 0.03). Immune infiltration analysis reveals that CBS/CSE expression significantly correlates with CD4⁺ T cell, neutrophil, macrophage, and DC infiltration scores, suggesting tumor-derived sulfur fosters immunosuppressive milieus (145). In breast cancer, CBS/CSE/3-MST overexpression associates with immunosuppressive phenotypes; inhibiting these enzymes suppresses tumor viability/migration while modulating immunoregulatory molecule expression (GAL3/9, MICA/B, CD155) (146). In tumor microenvironments, tolerogenic DCs are characterized by low CD80/CD86/MHC-II expression, reduced IL-12, and increased IL-10 production, favoring Treg expansion over effector T cell activation (147–149) (Figure 5B).

Mechanistically, endogenous RSS remodels immunosuppression through multiple pathways. They promote Tet1/Tet2 expression by sulfhydrating transcription factor NFYB, catalyzing 5mC to 5hmC conversion at the Foxp3 locus to stabilize Treg differentiation (150). CBS/CSE overexpression in cisplatin-resistant OVCAR8 cells (145) suggests dysregulated RSS metabolism promotes tumor progression through enhanced Treg differentiation, impaired DC maturation, and immunosuppressive microenvironment maintenance.

Comparative analysis reveals this duality arises from four mechanistic distinctions (Figure 5): (1) Temporal kinetics—exogenous donors produce high peak concentrations with short durations possibly preferentially targeting MDSC redox sensitivity, whereas endogenous enzymes continuously produce H₂S enabling chronic immune reprogramming. (2) Spatial distribution—exogenous RSS distributes uniformly throughout tumor microenvironments, whereas endogenous sulfur enriches around tumor cells, forming local immunosuppressive gradients. (3) Target cell selectivity—DATS’s selective MDSC inhibition may stem from MDSC metabolic vulnerabilities, while tumor cell-released H₂S affects multiple immune cells via paracrine mechanisms. (4) Dose-response relationships—RSS effects on immune cells may follow hormetic curves, with low-to-moderate concentrations promoting immunosuppression while high-concentration pulsatile exposure produces cytotoxicity or reprogramming effects.

RSS regulation of DC function reveals a multi-tiered control system: the cysteine-GSH-RSS metabolic axis serves as a foundational redox checkpoint governing DC differentiation (Section: 3.3.1), H₂S signaling provides context-dependent modulation of pathological DC subsets and tolerogenic programming (Section: 3.3.2), and tumor contexts reveal source- and concentration-dependent dual RSS roles (Section: 3.3.3).

However, critical mechanistic gaps constrain clinical translation: First, what mechanisms link cysteine availability to DC transcriptional programs? CSE inhibition reduces DC IL-12 production independent of H₂S (141), yet the cysteine-responsive transcription factors or epigenetic modifiers remain unidentified. Metabolomic profiling of cysteine-depleted DCs coupled with transcriptomic analysis could reveal these regulatory nodes.

Second, what determines donor selectivity for pathological versus physiological DC subsets? H₂S selectively targets CX3CR1⁺ moDCs in NASH without affecting normal macrophages (104), but the molecular basis is undefined, including metabolic signatures, surface receptors, or redox states. Single-cell multi-omics comparing donor-responsive versus resistant DC populations could identify selectivity determinants.

Third, does the RSS-GSH axis apply universally across DC subsets? Different DC populations (cDC1, cDC2, pDCs, moDCs) exhibit distinct metabolic programs, yet subset-specific RSS profiling is lacking. Such data would enable precision targeting of pathogenic DCs in autoimmune and tumor contexts.

H₂S plays an important autocrine regulatory role in T cell activation. Upon T cell receptor (TCR) stimulation, T cells rapidly upregulate expression of endogenous H₂S synthases (CBS and CSE), thereby enhancing endogenous H₂S generation (151). Within physiologically relevant nanomolar concentration ranges, H₂S significantly enhances TCR-mediated T cell activation, manifested by upregulation of early activation markers CD69 and CD25, increased IL-2 expression, and enhanced proliferative capacity. Silencing CBS and CSE expression impairs T cell activation and proliferation, while exogenous Na₂S supplementation completely rescues this defect, confirming the necessity of endogenous H₂S in T cell activation (6) (Figure 6A).

At the molecular level, H₂S optimizes immunological synapse formation efficiency by promoting actin cytoskeleton dynamic rearrangement and microtubule-organizing center (MTOC) reorientation, thereby enhancing TCR signal transduction (152). Additionally, H₂S promotes downstream signaling cascades by enhancing MEK-dependent ERK phosphorylation (153). Notably, due to limited mitochondrial oxidative metabolism of H₂S under hypoxic conditions, its stimulatory activity on T cells is further enhanced in low oxygen environments (152), suggesting potential relevance in hypoxic tumor microenvironments or inflamed tissues.

H₂S’s immunostimulatory effects are strictly constrained by endogenous inhibitory mechanisms. Thrombospondin-1 (TSP-1) functions as the first discovered endogenous H₂S signal inhibitor by binding its receptor CD47 (153). The TSP-1/CD47 signaling axis limits H₂S’s pro-activation effects through two key mechanisms: inhibiting TCR-induced upregulation of CBS and CSE; blocking H₂S-enhanced ERK phosphorylation (153). T cells from TSP-1 knockout mice exhibit heightened sensitivity to H₂S-dependent activation, while exogenous TSP-1 inhibits H₂S responses in wild-type but enhances responses in CD47 knockout T cells, demonstrating CD47’s necessity and sufficiency for TSP-1-mediated H₂S inhibition (152, 153). This precise regulatory balance ensures appropriate T cell activation, preventing excessive immune activation.

RSS-induced immunoregulatory effects exhibit bidirectionality and context dependence. In asthma mouse models, CSE expression levels in lung tissue are closely associated with airway inflammation and hyperresponsiveness. In ovalbumin (OVA)-induced asthma models, airway inflammation accompanies downregulated endogenous H₂S generation, while exogenous H₂S donor supplementation significantly inhibits airway hyperresponsiveness, reduces inflammatory cell infiltration, decreases Th2-type cytokine (IL-4, IL-5, IL-13) expression, and ameliorates airway remodeling (154). This anti-inflammatory effect relates to inhibition of NF-κB-mediated inflammatory and oxidative responses. Furthermore, H₂S alleviates allergic airway hyperresponsiveness by regulating mast cell activation (155). These findings reveal RSS complex regulatory network in different immune contexts: promoting T cell activation in anti-infection immunity while exerting inflammation-suppressing and tissue-protecting effects in excessive immune states.

H₂S has been shown to play a crucial role in maintaining Treg-mediated immune homeostasis through epigenetic regulation of the Foxp3 locus. Stable differentiation of Foxp3⁺ Treg cells relies on specific hypomethylation patterns at the Foxp3 locus, which are established in part through H₂S-mediated regulation of the DNA demethylases Ten-eleven translocation (Tet) 1 and Tet2. At the molecular level, H₂S promotes persulfidation of the nuclear transcription factor NFYβ, enhancing its binding to the promoters of Tet1 and Tet2 and thereby sustaining the expression of these demethylases. Under synergistic TGF-β and IL-2 signaling, activated Smad3 and Stat5 recruit Tet1 and Tet2, respectively, to the Foxp3 locus. These enzymes catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby establishing Treg-specific hypomethylation patterns that stabilize Foxp3 expression. Consistently, dual deletion of Tet1 and Tet2 results in Foxp3 hypermethylation, impaired Treg differentiation and function, and ultimately the development of systemic autoimmune disease (150).

The immunoprotection of CSE/H₂S in CD4⁺ T cells are confirmed in hypertension models. In peripheral blood lymphocytes from hypertensive patients and spontaneously hypertensive rats, CSE-induced H₂S generation is significantly reduced, negatively correlating with blood pressure but positively correlating with serum IL-10 levels. T cell-specific CSE knockout mice exhibit 5–8 mmHg baseline blood pressure elevation and exacerbated angiotensin II-induced hypertension, accompanied by impaired mesenteric artery dilation and arterial inflammation attributed to reduced Treg numbers in blood and kidneys, leading to excessive CD4⁺/CD8⁺ T cell infiltration into perivascular adipose tissue and kidneys (156). Mechanistically, H₂S produced by CSE activates downstream AMPK signaling by inducing persulfidation of liver kinase B1 (LKB1), which enhances its substrate-binding capacity and promotes Treg differentiation and proliferation. Adoptive Treg transfer to CSE knockout mice reverses hypertension, vascular dysfunction, and immune infiltration, demonstrating that CSE deficiency-induced hypertension is partly mediated by reduced Treg numbers and subsequent vascular/renal immune inflammation (156).

Notably, the engagement of the AMPK node by RSS exhibits profound context-dependency. In contrast to this LKB1-mediated homeostatic support in Tregs, our recent work in hepatocellular carcinoma reveals that the exogenous polysulfide donor PSCP activates AMPK via a distinct metabolic route: it competitively binds to the mitochondrial Complex I subunit Ndus3, triggering an ATP crisis that subsequently activates the AMPK-p53 axis to induce tumor cell apoptosis (39). This demonstrates that RSS can dictate opposing cell fates—survival versus death—by engaging the same kinase (AMPK) through divergent molecular mechanisms (direct enzymatic persulfidation versus indirect metabolic stress).

Conversely, endogenous RSS accumulation within the tumor microenvironment can hijack metabolic machinery to drive immunosuppression. In colorectal cancer, elevated H₂S levels promote differentiated CD4⁺CD25⁺Foxp3⁺ Treg activation by sulfhydrating enolase 1 (ENO1 Cys119), constructing immunosuppressive microenvironments. CBS heterozygous knockout (CBS⁺/⁻) or sulfur-amino acid restricted diet (SARD) reduces tumor Treg numbers, increases CD8⁺ T cell/Treg ratios, and enhances anti-PD-L1/anti-CTLA-4 immune checkpoint blockade efficacy (157). This finding supports engineered microbial-nanoparticle hybrid (E.coli@Cu₂O) therapeutic strategies. This system consumes endogenous H₂S at tumor sites, converting Cu₂O to Cu_xS, thereby weakening the tumor’s immunosuppressive shield while enhancing ferroptosis and cuproptosis, which significantly promotes DC maturation and CD8⁺ T cell activation (158).

In summary, this dual role highlights that physiological H₂S promotes Treg differentiation via epigenetic (Tet1/Tet2) and metabolic (LKB1-AMPK) pathways to maintain immune homeostasis, whereas excessive tumor-derived H₂S over-activates Tregs through ENO1 persulfidation, driving pathological immunosuppression (Figure 6B).

RSS exerts multilevel regulatory effects in CD8⁺ T cell-mediated anti-tumor immunity. In sickle cell disease (SCD), a genetic blood disorder, the CD8⁺ T cell three-dimensional genomic structure undergoes rearrangement, causing impaired chromosomal interactions between the anti-ferroptosis gene SLC7A11 and H₂S biosynthesis-related genes, with downregulated expression making CD8⁺ T cells more susceptible to ferroptosis, weakening anti-tumor immune function, and promoting tumor growth (14). Studies show that in mouse and humanized SCD models, restoring H₂S levels rescues SLC7A11 expression, reduces ferroptosis, and enhances anti-tumor immune responses.

In the tumor microenvironment (TME), preferential cystine uptake by tumor cells due to metabolic reprogramming to highly express cystine/glutamate transporter (SLC7A11) has been reported (159). Cystine deprivation causes metabolic stress on tumor-infiltrating CD8⁺ T cells, activating the glutamate-CD36-lipid peroxidation cascade that drives ferroptosis (160). Cystine deficiency disrupts GSH synthesis while interfering with cystine/glutamate exchange, leading to intracellular glutamate accumulation that upregulates scavenger receptor CD36, promoting excessive oxidized lipid uptake and lipid peroxidation. This ferroptotic program manifests as impaired memory formation, loss of stemness, exhaustion marker (PD-1, TIM-3) upregulation, and reduced effector cytokine (IFN-γ, TNF-α, IL-2) secretion, severely weakening anti-tumor immunity (161). In rectal cancer tissues, H₂S level is significantly elevated. It increases the expression of AAK-1 through the persulfidation of cysteine residues in ETS domain-containing protein Elk-4 (ELK4), thereby inhibiting CD8⁺ T cell migration and immunity (157).

Glutamate-cysteine ligase catalytic subunit (Gclc) overexpression confers protection through dual mechanisms: enhancing glutathione synthesis (combating oxidative stress) and consuming excess glutamate (blocking CD36 upregulation), comprehensively resisting cystine deprivation-induced ferroptosis and exhaustion. In adoptive cell therapy, engineered Gclc-expressing CD8⁺ T cells demonstrate superior tumor control, reduced ferroptosis, and enhanced memory phenotype in melanoma models (162). This reveals how cysteine availability indirectly regulates CD8⁺ T cell ferroptosis sensitivity and anti-tumor function through glutathione-mediated redox balance, as cysteine serves as both a precursor for H₂S synthesis and a rate-limiting substrate for glutathione production.

Beyond ferroptosis resistance, RSS signaling protects CD8⁺ T cells by maintaining organelle integrity. Metabolic disorders and oxidative stress in the tumor microenvironment impair Golgi apparatus function, causing Golgi stress and protein modification/transport disorders that inhibit T cell function. Studies show that under TME stress, Golgi structure (e.g., GM130 expression) declines, and this structural disruption is reversed by GYY4137 or CBS overexpression (163). Such interventions enhance T cell stemness, antioxidant capacity, and protein translation, partly through Prdx4 shuttling between ER and Golgi. In melanoma and lymphoma models, CD8⁺ T cells with such interventions exhibit superior tumor control after adoptive transfer (Figure 6C).

Beyond H₂S, other RSS forms, including cysteine persulfide (CysSSH) and glutathione trisulfide (GSSSG), exert regulatory effects on T cell immunity (164, 165). These RSS are generated by cysteinyl-tRNA synthetases (CARS) via their cysteine persulfide synthase (CPERS) activity. Both cytosolic CARS1 and mitochondrial CARS2 contribute to cellular reactive persulfide and polysulfide pools, though with distinct subcellular localization and functional implications (166). CARS1-derived cytosolic RSS may preferentially modulate cytoplasmic signaling pathways and translation-related processes, while CARS2-generated mitochondrial RSS likely influence mitochondrial metabolism and bioenergetics. Collectively, these enzymes maintain cellular reactive persulfide and polysulfide homeostasis, which exerts endogenous proliferation-inhibiting effects in CD4⁺ T cells (5), representing a distinct regulatory mechanism from H₂S’s pro-activation effects (Section 4.1.1) and highlighting RSS family functional diversity.

CARS2/CPERS-dependent RSS metabolism critically limits CD4⁺ T cell proliferation in a cell-intrinsic manner in inflammatory bowel disease (IBD). Under homeostatic conditions, Cars2⁺/⁻ mice exhibit spontaneous effector/memory CD4⁺ T cell accumulation in the colon with aging; under lymphopenic conditions, Cars2⁺/⁻ CD4⁺ T cells show enhanced cell cycle entry with decreased Trp53 expression, triggering severe colitis that is rescued by GSSSG supplementation. Reanalysis of human colon CD4⁺ T lymphocyte datasets confirms that CARS2 downregulation associates with IBD pathogenesis, with GSSSG inhibiting human CD4⁺ T cell proliferation in vitro (5). Together, these findings demonstrate that CARS2/CPERS-dependent RSS metabolism is essential for homeostasis of intestinal effector/memory CD4⁺ T cells, and that dysregulation of this metabolic pathway can lead to development of gut inflammation in both mice and humans (Figure 6D).

H₂S regulation of humoral immunity operates at multiple levels: beyond cellular effects on B cells, H₂S directly modifies antibody molecules, the key effectors of humoral immunity. Antibody function highly depends on abundant disulfide bonds, maintaining correct assembly of heavy and light chains and conformational stability of antigen-binding regions (167, 168). Studies found that pharmacological concentrations of H₂S treatment (which rapidly generates a broad RSS pool, including persulfides/polysulfides) cause disruption of disulfide bonds between antibody heavy and light chains, accompanied by antibody persulfidation modification (169).

Disulfide bond disruption-induced antibody conformational changes produce multifaceted functional impacts. First, H₂S-treated antibodies exhibit significantly reduced antigen-binding capacity. Second, at the functional level, H₂S effectively blocks antibody-mediated red blood cell agglutination reactions and antibody-coated microsphere aggregation. More importantly, H₂S significantly inhibits antibody-dependent multiple effector functions: in glomerular mesangial cells, it substantially suppresses antibody-induced complement-dependent cytolysis (CDCS); in Jurkat cells, it blocks anti-CD95 IgM antibody-triggered cell apoptosis. Furthermore, H₂S significantly inhibits the alternative activation pathway of the complement system (169). These findings reveal that pharmacological concentrations of H₂S can inhibit humoral immune responses by directly sulfhydrating effector molecules.

Beyond direct antibody effects, RSS may indirectly influence B cell-related responses by modulating the immune microenvironment. In benign prostatic hyperplasia (BPH) rat models, diallyl sulfide (DAS) demonstrates significant immunoregulatory effects. Histological changes and immune-inflammatory cascades accompany testosterone propionate (TP)-induced BPH, while DAS or finasteride (Fin) treatment ameliorates these abnormalities (170).

DAS treatment reduces prostate weight by 60%, decreases serum testosterone and dihydrotestosterone (DHT) by 68% and 75% respectively, while androgen receptor (AR) and prostate-specific antigen (PSA) protein expression decline. DAS significantly alleviates the immune-inflammatory microenvironment, manifested by reduced CD4⁺ T cell protein expression and related inflammatory cytokines, while inhibiting insulin-like growth factor-1 (IGF-1), transforming growth factor-β1 (TGF-β1), and phosphorylated ERK1/2 signaling pathways (170). Although this study primarily focuses on T cells and tissue microenvironments, considering that B cells participate in immunopathological processes by producing pathogenic antibodies, and immune microenvironment changes affect B cell activation, differentiation, and antibody generation, these findings provide important clues for exploring RSS roles in B cell-mediated autoimmune diseases and metabolic diseases.

Current understanding of RSS regulation of B cells remains limited. Key unresolved questions include (1): Direct effects on B cell activation and differentiation. Do RSS modulate B cell receptor (BCR) signaling, germinal center formation, or plasma cell differentiation? (2) Class-switch recombination regulation. Do RSS influence antibody isotype switching (IgM→IgG/IgA/IgE) via redox-sensitive enzymes? (3) B cell-T cell crosstalk. How does RSS dysregulation in T follicular helper (Tfh) cells impact B cell responses? (4) Metabolic programming. Does cysteine/GSH availability (Section 3.3.1) similarly constrain B cell proliferation and antibody secretion?

Addressing these knowledge gaps requires systematic investigation using B-cell-specific RSS metabolic enzyme knockout models, proteome-wide profiling to identify B-cell-specific persulfidation targets, and functional assays integrating BCR signaling, germinal center dynamics, and antibody production.