Molecular hydrogen is associated with immunophenotypic modulation and changes in T-cell exhaustion markers in refractory primary Sjögren’s syndrome-associated interstitial lung disease: A case report

Abstract

Sjögren’s syndrome-associated interstitial lung disease is a severe systemic complication that often proves refractory to conventional immunosuppressive and biologic therapies. Molecular hydrogen has recently emerged as a novel therapeutic agent known for its selective antioxidant, anti-inflammatory, and immunomodulatory properties. This report evaluates the therapeutic potential of hydrogen in a patient with progressive Sjögren’s syndrome-associated interstitial lung disease. A 72-year-old female with refractory primary Sjögren’s syndrome-associated interstitial lung disease presented with worsening dyspnea, anemia, and progressive pulmonary fibrosis despite prior treatment with corticosteroids, synthetic disease-modifying antirheumatic drugs, and rituximab. The patient initiated oral molecular hydrogen capsule therapy as an adjunctive treatment. Follow-up assessments revealed marked clinical stabilization: pulmonary function tests demonstrated significant objective improvement in gas exchange capacity, anti-Ro antibody titers significantly decreased, and high-resolution computed tomography showed no further progression of fibrosis. Notably, flow cytometric analysis indicated changes associated with the normalization of immunophenotypic markers suggestive of improved immune homeostasis, evidenced by observed alterations in effector T-cell subsets and a shift in previously aberrant regulatory T-cell populations toward levels comparable to healthy controls. No adverse events were observed. Although the patient had received multiple immunosuppressive therapies, including a single dose of rituximab nearly 3 years prior, these outcomes require a cautious interpretation of causality. While the observed clinical and immunophenotypic changes may represent delayed effects of prior therapies or natural disease fluctuation, the extended timeframe makes a solely delayed effect unlikely, strengthening the potential association with hydrogen therapy. These findings suggest hydrogen therapy as a safe and potentially beneficial adjunctive strategy for refractory autoimmune fibrotic diseases, supporting further clinical validation.

Introduction

Sjögren’s syndrome (SjS) is a chronic systemic autoimmune disease primarily characterized by immune-mediated destruction of exocrine glands, leading to the hallmark sicca symptoms—dryness of the eyes (xerophthalmia), mouth (xerostomia), and occasionally the vagina. ¹ In addition to these glandular features, a considerable proportion of patients experience fatigue, arthralgia, and myalgia, reflecting the systemic nature of the disease. ² Extraglandular involvement is common and may affect multiple organs, including the lungs, kidneys, and nervous system.^3,4 Pulmonary involvement is among the most serious systemic complications, occurring in ~10%–20% of patients.^5,6 When severe, as in New York Heart Association class III–IV interstitial lung disease (ILD), it represents a potentially life-threatening condition and a critical determinant of prognosis. ² Pathogenetically, the histological hallmark of SjS is focal lymphocytic infiltration of the salivary and lacrimal glands, mainly composed of cluster of differentiation 4 (CD4)⁺ T cells and B cells. ⁷ The underlying mechanisms are multifactorial, involving autoantibody production of anti-SjS-related antigen A (anti-Ro/SSA) and anti-SjS type B antibody (anti-La/SSB), epithelial cell dysregulation through abnormal activation of the nuclear factor kappa B (NF-κB), inflammasome, and interferon signaling pathways, and cellular defects such as acinar and ductal cell apoptosis and progenitor cell dysfunction.^8,9 Together, these processes drive chronic inflammation, epithelial injury, and progressive glandular and systemic impairment.

Systemic therapy of SjS centers on immunomodulation tailored to disease severity, guided by the European Alliance of Associations for Rheumatology (European League Against Rheumatism [EULAR]) recommendations, which emphasize individualized management based on systemic involvement. Corticosteroids are used to rapidly suppress inflammation during disease flares, while conventional immunosuppressants help maintain long-term disease control and minimize steroid exposure. ¹⁰ In refractory or organ-threatening cases, biologic agents, particularly B-cell-targeted therapies such as rituximab and belimumab, have shown promising results in improving systemic manifestations and reducing autoantibody production. ¹¹ Emerging studies also highlight adjunctive therapies with selective immunomodulatory effects and minimal toxicity. ¹²

Molecular hydrogen therapy (HT) has recently emerged as a novel adjunctive therapeutic approach with potential applications across various oxidative and inflammatory disorders.^13,14 Due to its small molecular size and high lipid solubility, hydrogen (H₂) can rapidly diffuse through biological membranes and organelles, reaching both cellular and subcellular compartments. ¹⁵ Initially recognized as a selective antioxidant that neutralizes hydroxyl radicals (•OH) and peroxynitrite (ONOO⁻), ¹⁶ subsequent studies have demonstrated broader biological effects involving redox balance, inflammation regulation, and cell signaling modulation. ¹⁷ Immunologically, HT promotes macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype, ¹⁸ enhances regulatory T-cell (Treg; CD4⁺ CD25⁺ forkhead box P3 (FoxP3⁺)) populations, ¹⁹ and mitigates the phenotypic markers of exhausted CD8⁺ programmed cell death protein 1 (PD-1)⁺ T cells by improving mitochondrial bioenergetics. ²⁰ Clinically, more than 2000 studies, including about 100 randomized controlled trials, have reported its potential therapeutic benefits in ischemia-reperfusion injury, metabolic syndrome, neurodegenerative diseases, autoimmune disorders, and cancer supportive therapy.^{21 –26} Various administration routes, such as H₂ inhalation, H₂-rich water, and H₂–saline injection, produce distinct pharmacodynamic and gene-regulatory effects. ²⁷ Overall, accumulating evidence supports HT as a safe and biologically active therapy with selective immunomodulatory and antioxidant properties that merit further clinical investigation.

This report describes a 72-year-old female patient with SjS complicated by ILD who received molecular HT as an adjunct to conventional treatment.^15,28 The patient received PURE HYDROGEN capsules (HoHo Biotech Co., Ltd., Taipei, Taiwan), each containing 170 mg of H₂-enriched calcium, capable of releasing ~1.7 × 10 ²¹ H₂ molecules (200 mL each) of H₂-rich water with a concentration of 1200 ppb (0.6 mM).^{28 –30} Since June 2024, the patient has reported no adverse events. Furthermore, because directly measuring volatile H₂ gas in the bloodstream is technically challenging in standard clinical settings, the therapeutic context was clarified by strictly monitoring downstream clinical and immunophenotypic markers. In addition to tracking anti-Ro autoantibody titers, objective pulmonary function tests (PFTs), functional exercise capacity via the 6-min walk test (6MWT), and structural pulmonary fibrosis via high-resolution computed tomography (HRCT), this study analyzed the dynamic changes in peripheral blood T-cell subsets using flow cytometry to characterize the patient’s immunological responses and evaluate the effectiveness of the H₂-assisted therapy.

Case report

A 72-year-old Taiwanese woman with a history of hypertension and hyperlipidemia under stable control presented with progressive dyspnea. Her illness began in May 2019, when she was hospitalized for community-acquired pneumonia with acute respiratory failure. Despite empirical broad-spectrum antibiotics and antifungal therapy, intermittent low-grade fever persisted, and bronchoalveolar lavage showed cytomegalovirus (CMV) antibody positivity. Autoimmune serologic testing revealed an elevated antinuclear antibody titer of 1:160 (cytoplasmic pattern) and increased anti-Ro antibody levels (123 U/mL). Both the Schirmer’s test and sialoscintigraphy were positive, although salivary gland biopsy was not performed. The diagnosis of primary SjS fulfilled the 2016 American College of Rheumatology/European League Against Rheumatism (EULAR) classification criteria with a total score of 4 (anti-SjS-related antigen A (anti-Ro/SSA) positivity contributing three points, and a positive Schirmer’s test contributing one point). Because the diagnostic criteria were met non-invasively and the patient presented with severe pulmonary complications, a salivary gland biopsy was not performed to avoid unnecessary invasive procedures. Chest CT demonstrated diffuse ground-glass opacities in both lungs, supporting a diagnosis of SjS–ILD complicated by CMV pneumonitis. The patient was subsequently treated with antiviral therapy targeting CMV, in combination with systemic corticosteroids and the immunomodulator hydroxychloroquine, which resulted in gradual clinical and radiologic improvement.

In June 2021, the patient developed worsening dyspnea and persistent low-grade fever. HRCT showed progression of pulmonary fibrosis, and lung perfusion scanning revealed right upper lobe hypoperfusion, leading to a diagnosis of primary SjS flare complicated by pulmonary embolism. Maintenance therapy was optimized with apixaban (5 mg BID, from June 2021 to December 2024), long-term systemic corticosteroids (currently prednisolone 5 mg QD), and conventional immunomodulators (currently azathioprine 25 mg QD, mycophenolic acid 180 mg BID, and hydroxychloroquine 200 mg QD since June 2021). A single dose of rituximab (100 mg) was administered in June 2021, resulting in partial symptomatic improvement. However, follow-up imaging in March 2024 demonstrated cystic changes, ill-defined consolidations, and residual ground-glass opacities in both lungs, with progressive respiratory impairment severely limiting her daily activities.

In June 2024, the patient initiated molecular H₂ capsule therapy (one capsule daily) as an adjunctive treatment. Several months later, biochemical and serologic parameters showed remarkable improvement: anti-Ro antibody levels decreased from 7901 to 933 U/mL. Importantly, this decline was confirmed as a continuous downward trend through multiple repeat measurements. All tests were consistently performed at our institutional laboratory using a fluorescent enzyme immunoassay (FEIA) on a Phadia analyzer, which effectively eliminates inter-assay variability. Alongside the anti-Ro reduction, longitudinal tracking of immune parameters revealed that peripheral B-cell (CD19) counts remained persistently suppressed (ranging from 0.08% to 2.25%) following the 2021 rituximab administration. Conversely, total immunoglobulin G (IgG) levels were consistently maintained within the normal physiological range (913, 948, and 1008 mg/dL across 2021, 2023, and 2024, respectively). Clinically, by November 2024, she reported enhanced exercise tolerance, a noticeable reduction in dyspnea, and overall stabilization of her disease. Follow-up PFTs performed in April 2026, ~22 months after initiating HT, revealed a significant improvement in gas exchange capacity. Her diffusing capacity for carbon monoxide (DLCO) increased from a baseline of 7.3 mL/min/mmHg (47% of predicted) in 2019 to 9.58 mL/min/mmHg (55% of predicted) in 2026. While the total lung capacity showed a restrictive pattern (62% of predicted), this was clinically attributed to progressive moderate scoliosis and exacerbated chronic pain from left hip osteoarthritis, rather than ILD progression. Furthermore, the 6MWT distance decreased from 230 m in June 2021 to 190 m in April 2026, which was also primarily limited by exacerbated chronic pain from her left hip osteoarthritis rather than respiratory desaturation or dyspnea (as demonstrated in the 6MWT performed in April 2026; video available at: https://figshare.com/s/371ba8559e59dde77ef9). No adverse effects were observed. Follow-up chest radiography and HRCT demonstrated no further progression of pulmonary fibrosis or consolidation, indicating effective control of ILD (Figure 1). Although radiographic findings showed no significant resolution of pulmonary fibrosis, the observed symptomatic improvement may be correlated with the suppression of autoimmune-mediated inflammation and the stabilization of the disease course.

Figure 1.

Longitudinal changes in PFT parameters and exercise capacity from August 2019 to April 2026. The graph and accompanying table illustrate the trajectory of predicted percentages for TLC, FVC, FEV1, and DLCO, alongside the 6MWT distance. Vertical dashed lines denote the initiation of molecular hydrogen therapy (June 2024) and the exacerbation of left hip OA (August 2025). Across the three clinical timepoints (August 2019, July 2021, and April 2026), lung volume parameters demonstrated a progressive restrictive decline (potentially associated with gradually worsening moderate scoliosis): TLC (88%, 82%, 62%), FVC (72%, 64%, 42%), and FEV1 (75%, 65%, 45%). Clinically, this restrictive pattern was attributed to the patient’s progressive moderate scoliosis and exacerbated chronic pain from left hip osteoarthritis, rather than ILD progression. Conversely, DLCO showed a gradual and sustained improvement over the observation period (45%, 47%, 55%), with an upward trend continuing after the initiation of molecular hydrogen therapy. Exercise capacity, measured by the 6MWT, decreased from 230 m in July 2021 to 190 m in April 2026. This functional decline was primarily limited by the exacerbated chronic pain from her left hip OA, rather than respiratory desaturation or dyspnea.

Notably, follow-up flow cytometric analysis of peripheral blood performed in February 2025 revealed dynamic immunophenotypic alterations across T-cell subsets. To comprehensively evaluate the patient’s immune status, specific markers were selected: killer cell lectin-like receptor G1 (KLRG1) and PD-1 were utilized to assess T-cell exhaustion and senescence, while CD39 and Helios were chosen to evaluate the suppressive capacity, activation, and stability of Tregs. During the SjS flare-up, the patient exhibited a marked decrease in the proportions of CD4⁺ T helper (Th) KLRG1⁺, effector memory Th KLRG1⁺, effector CD8⁺ cytotoxic T (Tc) KLRG1⁺, and naïve Tc PD-1⁺ subsets (Figure 2), accompanied by a pronounced increase in CD127^low/− FoxP3⁺ Treg, CD39⁺Helios⁺ Treg, CD39⁻Helios⁺ Treg, natural Treg, activated Treg, and non-suppressive effector T (Teff) populations (Figure 3). Following the initiation of molecular HT, the gradual normalization of these immune parameters was associated with the normalization of immunophenotypic markers suggestive of improved immune homeostasis. Overall, the overarching trend reflects a shift from an aberrant, exhausted immunophenotype during the disease flare toward a more balanced, healthy-like immune state. However, it should be noted that these immunophenotypic shifts might also represent a delayed or cumulative effect of prior immunosuppressive therapies or natural fluctuations in the disease course rather than a direct causal effect alone. No adverse events or complications were reported during the period of H₂ capsule administration (Figure 4).

Figure 2.

(a) Schematic overview of the clinical course of immune-related diseases and associated medications. (b) The patient was diagnosed with Sjögren’s syndrome complicated by ILD in May 2019 and began adjunctive molecular hydrogen capsule therapy (one capsule daily) in June 2024. Follow-up chest radiography showed no further progression of pulmonary fibrosis, indicating disease stabilization under the combined therapeutic regimen.

Figure 3.

Immunophenotypic changes of T cells before and after molecular hydrogen therapy (initiated June 10, 2024). Whole blood analysis included an HC group for comparison (far left). Across three timepoints (June 2021 (Sjögren’s syndrome flare-up); July 2023; February 2025 (8 months after molecular hydrogen therapy)), Panels (a–d) show trends toward phenotypic normalization of effector and memory T-cell populations: effector CD4⁺ T helper KLRG1⁺ (14.3%, 2.9%, 26.2%), effector memory CD4⁺ T helper KLRG1⁺ (12.2%, 4.7%, 19.0%), effector CD8⁺ cytotoxic KLRG1⁺ (27.1%, 4.7%, 25.4%), and naïve CD8⁺ cytotoxic PD-1⁺ (2.1%, 12.6%, 23.1%) subsets. Panels (e, f) show stable proportions of naïve CD8⁺ Tim-3⁺ (14.8%, 2.1%, 2.2%) and CM CD8⁺ Tim-3⁺ (52.3%, 8.2%, 6.9%) subsets, suggesting sustained improvement in markers associated with T-cell exhaustion without aberrant activation.

Figure 4.

Immunophenotypic changes of T cells before and after molecular hydrogen therapy (initiated June 10, 2024). Whole blood analysis included an HC group for comparison (far left). Across three timepoints (June 2021 (Sjögren’s syndrome flare-up); July 2023; February 2025 (8 months after molecular hydrogen therapy)), Panel (a) shows that CD127^low/− Treg cells (1.2%, 0.4%, 2.6%) remained relatively stable throughout the observation period. Panels (b–g) show marked increases during the Sjögren’s syndrome flare-up in June 2021, followed by gradual declines after hydrogen therapy in June 2024, ultimately returning to near-baseline levels comparable to the HC group: CD127^low/− Treg (1.2%, 0.4%, 2.6%), CD127^low/− FoxP3⁺ Treg (84.6%, 66.7%, 12.9%), CD39⁻Helios⁺ Treg (36.2%, 41.8%, 10.2%), CD39⁺Helios⁺ Treg (17.4%, 14.3%, 7.2%), natural Treg (52.2%, 33.0%, 5.4%), activated Treg (26.1%, 0.0%, 1.8%), and nonsuppressive Teff (49.3%, 40.7%, 9.6%).

Discussion

This case highlights the potential value of HT as an adjunctive treatment for refractory primary SjS complicated by ILD. Unlike previously reported cases, this patient achieved clinical stabilization despite remaining refractory to achieving sustained remission with multiple conventional synthetic disease-modifying antirheumatic drugs and biologic agents, including rituximab. Following the initiation of HT, the observed clinical stabilization, characterized by reduced dyspnea and improved exercise tolerance, was associated with biochemical improvements and reduced autoimmune activity. While these findings suggest that H₂ may exert multifaceted therapeutic effects by modulating oxidative stress, inflammation, and immune dysregulation, ³¹ the discussion of causality warrants a cautious interpretation. When interpreting the causality of the observed clinical and immunophenotypic improvements, the timeline of preceding treatments must be carefully considered. The patient received only a single 100 mg dose of rituximab in June 2021. Generally, the biological effects of rituximab, specifically B-cell depletion in SjS last for ~6–12 months, with clinical efficacy waning thereafter. The initiation of HT in June 2024 occurred nearly 3 years after the rituximab administration. Furthermore, the onset of marked clinical improvement (reduced dyspnea) in November 2024 and the significant immunophenotypic normalization observed in February 2025 strongly temporally align with the HT window. While the cumulative effects of prior therapies or natural disease fluctuation cannot be completely excluded, this extended interval makes it highly unlikely that the recent, pronounced immunological shifts are solely a delayed effect of the single rituximab dose, thereby strengthening the potential association with the adjunctive HT.

The immunopathological hallmark of SjS–ILD lies in the aberrant interplay between epithelial and immune cells, resulting in sustained activation of CD4⁺ T cells, B cells, and type I interferon signaling pathways. ³² In this biological context, HT potentially acts as a selective antioxidant by neutralizing hydroxyl radicals and activating the erythroid-2-related factor 2 nuclear factor erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 signaling pathway, ³³ which may assist in suppressing NF-κB mediated inflammatory cascades.^34,35 These mechanisms provide a plausible biological rationale for the clinical stabilization observed in this patient.

Following HT, anti-Ro antibody titers decreased markedly from 7901 to 933 U/mL, and this continuous downward trend was verified through repeated measurements. To further clarify the context of this serological response and rule out global immunosuppression, we analyzed the longitudinal trends of peripheral B-cell counts and total IgG levels. While CD19⁺ B-cells remained persistently suppressed (0.08%–2.25%) following the 2021 rituximab administration, total IgG levels were consistently maintained within the normal physiological range (913–1008 mg/dL) throughout the course. This divergence demonstrates the absence of global hypogammaglobulinemia. Crucially, although B-cells had been deeply depleted since 2021, the marked and continuous decline in anti-Ro titers did not manifest until late 2024, closely aligning with the initiation of molecular HT. This temporal relationship, coupled with stable total IgG and the elimination of inter-assay variability via standardized FEIA testing, implies that the selective reduction in autoantibodies was unlikely a mere delayed effect of generalized rituximab-induced humoral suppression or natural fluctuation. Rather, it points toward a potential selective immunomodulatory effect of the adjunctive HT. Clinically, dyspnea improved, and HRCT demonstrated stable pulmonary fibrosis. The therapeutic efficacy was further corroborated by the objective recovery of DLCO, which reached 55% of the predicted value (9.58 mL/min/mmHg) in 2026. The discrepancy between the patient’s symptomatic improvement in dyspnea and the stability of radiological findings is notable. In the context of advanced ILD, the stabilization of fibrotic progression on HRCT rather than resolution is often the primary therapeutic goal, while the reduction in symptoms may correlate with a decrease in systemic oxidative stress and autoimmune-mediated inflammation.

Flow cytometric analysis revealed increased proportions of KLRG1⁺ and PD-1⁺ effector and memory Th/Tc subsets, whereas T-cell immunoglobulin and mucin domain 3⁺ exhausted T cells remained stable. Notably, the previously expanded Treg populations, including CD127^low/− FoxP3⁺ Treg, CD39⁺Helios⁺ Treg, CD39⁻Helios⁺ Treg, natural Treg (CD25^high), activated Treg, and no suppressive Teff subsets—declined to levels comparable to healthy controls. These immunophenotypic shifts were suggestive of improved immune homeostasis. However, we must explicitly distinguish between phenotypic normalization and functional recovery. It remains unclear whether these changes represent true functional immune recovery or merely phenotypic shifts without proven functional correlates, particularly as they may exceed expected intra-individual variability in chronic autoimmune disease. Specifically, our evaluation relied solely on flow cytometric surface markers. Without corroborating functional assays, such as cytokine profiling or T-cell proliferation, to confirm the functional recalibration of exhausted T-cell and Treg populations, the observed immunophenotypic normalization suggests, but cannot definitively prove, functional immune recovery (Supplemental Material).

The normalization of the immunophenotypic profile suggests that HT may assist in modulating Treg populations ³⁶ and mitigating T-cell exhaustion markers. Potential mechanisms involve the activation of the NRF2 pathway to enhance mitochondrial bioenergetics ³⁷ and the modulation of PD-1/KLRG1 immune checkpoints.^38,39 This integrated mechanism is proposed as a potential contributor to the stabilization of autoimmune activity observed in this patient. ⁴⁰ No adverse events were observed, supporting the safety profile of molecular H₂ as an adjunctive strategy. Several limitations of this report must be acknowledged. First, as a single-patient case study, the observed clinical and immunophenotypic improvements cannot be definitively attributed to molecular HT alone; they may represent a delayed or cumulative effect of prior treatments, such as rituximab, or natural disease fluctuation, although the extended 3-year interval since rituximab administration makes this less likely. Finally, these findings are hypothesis-generating and cannot be generalized to the broader SjS–ILD population. Future multicenter randomized controlled trials are required to validate these findings and define optimal dosing.

Conclusion

This case highlights the potential role of molecular H₂ as a hypothesis-generating adjunctive therapy in refractory autoimmune ILD, warranting further controlled investigation. In this patient with refractory primary SjS–ILD, adjunctive molecular HT was associated with clinical stabilization, significant objective improvement in gas exchange capacity, and distinct immunophenotypic changes, including modulation of T-cell exhaustion markers (KLRG1 and PD-1) and the expansion of the Treg population, suggestive of improved immune homeostasis. However, these findings should be interpreted cautiously, as they may reflect delayed or cumulative effects of prior immunosuppressive therapies or natural disease variability rather than a direct therapeutic effect. Nevertheless, the nearly 3-year interval between the prior rituximab administration and the recent clinical improvements renders a solely delayed effect unlikely, supporting a potential direct therapeutic role for molecular H₂. Notably, while symptomatic improvement and functional gas exchange recovery were observed, radiologic fibrosis remained stable, underscoring that disease stabilization, rather than radiologic reversal, may represent a realistic therapeutic goal in advanced ILD. Given the inherent limitations of a single case report, these observations remain hypothesis-generating and are not generalizable. Further well-designed controlled studies, incorporating functional immune assays, are required to confirm the efficacy of molecular H₂ and to elucidate its immunomodulatory mechanisms in autoimmune-mediated pulmonary fibrosis.

Supplemental Material

sj-pdf-1-sco-10.1177_2050313X261454858 – Supplemental material for Molecular hydrogen is associated with immunophenotypic modulation and changes in T-cell exhaustion markers in refractory primary Sjögren’s syndrome-associated interstitial lung disease: A case report

Supplemental material, sj-pdf-1-sco-10.1177_2050313X261454858 for Molecular hydrogen is associated with immunophenotypic modulation and changes in T-cell exhaustion markers in refractory primary Sjögren’s syndrome-associated interstitial lung disease: A case report by Wei-Hung Hsu, Jeng-Wei Lu, Yi-Jung Ho, Shan-Wen Lui, Ting-Yu Hsieh, Kuang-Yih Wang and Feng-Cheng Liu in SAGE Open Medical Case Reports