Molecular Hydrogen Reverses Sepsis-induced Immunoparalysis: Insights from Longitudinal Deep Immunophenotyping

Abstract

Background/Aim

Refractory septic shock and post-cardiac arrest syndrome (PCAS) can induce lethal immunoparalysis. However, longitudinal evidence on the effects of molecular hydrogen on deep human immunophenotyping remains scarce.

Case Report

We report the case of a 49-year-old man with severe tricuspid regurgitation and Child-Pugh class B alcoholic cirrhosis who developed severe pneumonia, refractory septic shock, and PCAS after high-energy thoracic trauma. At a therapeutic impasse marked by high levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP) (>35,000 pg/ml) and profound immune depletion, adjuvant hydrogen inhalation was initiated, which led to a 64% reduction in myocardial stress and temporary clinical stabilization. Deep longitudinal flow cytometry unveiled system-wide immunological reprogramming. In the T-cell compartment, hydrogen induced a biphasic resolution of exhaustion in T-helper cells marked by an immediate decline in expression of FAS cell surface death receptor (FAS; also known as CD95) and asynchronous normalization of expression of programmed cell death protein 1 (PD1) and T-cell immunoglobulin and mucin domain 3 (TIM3), alongside bidirectional restoration of physiological immune checkpoints in cytotoxic T-cells. Furthermore, we observed a sequential, compensatory recovery of the regulatory T-cell (Treg)/type 1 regulatory T-cell (Tr1) immune-braking system. In the humoral compartment, despite prolonged bone marrow suppression and loss of transitional/regulatory B-cells, hydrogen therapy was associated with a V-shaped recovery of plasma and double-negative (for both cluster of differentiation 27 and immunoglobulin D) B-cells. It also appeared to protect switched memory B-cells by markedly reducing FAS expression, suggesting an anti-apoptotic effect.

Conclusion

Although the patient ultimately succumbed to irreversible chronic comorbidities on day 86, this case of severe tricuspid regurgitation and Child-Pugh class B alcoholic cirrhosis demonstrates profound yet transient immunological rescue. By disrupting the cycle of immune exhaustion and exerting anti-apoptotic effects, molecular hydrogen may represent a promising immunomodulatory adjunct in severe sepsis.

Introduction

The clinical management of severe polytrauma combined with refractory septic shock remains one of the most formidable challenges in contemporary intensive care. Traumatic flail chest, characterized by multiple rib fractures and paradoxical chest wall movement, frequently precipitates acute respiratory distress and carries a significantly high mortality risk due to compromised pulmonary mechanics (1). The complexity of such cases is often compounded by post-cardiac arrest syndrome (PCAS), a multifaceted condition following successful resuscitation that triggers a systemic inflammatory response akin to sepsis, leading to profound multi-organ dysfunction and persistent hemodynamic instability (2). In the context of severe sepsis and shock, as defined by the Sepsis-3 consensus (3), the host immune system often undergoes a rapid transition from a hyper-inflammatory cytokine storm to a prolonged state of immunoparalysis (4). This state is primarily marked by T-cell exhaustion, characterized by the upregulated expression of inhibitory receptors such as programmed cell death protein 1 (PD1) and T-cell immunoglobulin and mucin domain 3 (TIM3) on naive and effector T-cell compartments (5, 6). Recent studies emphasize that recurrent inflammatory insults further exacerbate exhaustion of cluster of differentiation 4 (CD4)⁺ and cluster of differentiation 8 (CD8)⁺ T-cells, contributing to a dangerous loss of physiological immune checkpoints (7, 8). Concurrently, severe sepsis induces a vacuum effect within the humoral immune system, leading to the massive apoptosis of mature B-cell subsets and a total halt in terminal plasma cell function (9, 10), which leaves the patient vulnerable to secondary opportunistic infections (11).

Traditional therapeutic interventions often fail to address the underlying immune dysregulation in refractory cases. Molecular hydrogen has emerged as a promising medical adjunct with potent selective anti-oxidative, anti-inflammatory, and anti-apoptotic properties (12, 13). Preclinical study has demonstrated that hydrogen therapy can ameliorate sepsis-induced organ damage by modulating the nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase-1 (HO1) signaling pathway and protecting against endothelial dysfunction (14). Furthermore, recent proteomics research suggests that hydrogen may target specific proteins such as apolipoprotein A2 (APOA2) to mitigate sepsis-related lung injury (15), These findings are supported by primary experimental studies demonstrating that hydrogen administration modulates oxidative stress related protein expression and inflammatory signaling pathways in sepsis models, thereby attenuating pulmonary damage and improving histopathological outcomes. Such mechanistic evidence provides a biological basis for the radiographic improvements observed in clinical settings.

Despite the wealth of preclinical evidence, longitudinal clinical data on hydrogen therapy and deep immune phenotyping in sepsis remain scarce. We report a complex case of refractory septic shock and PCAS after severe polytrauma, in which adjuvant hydrogen therapy was associated with transient immunological recovery and clinical stabilization. Through comprehensive flow cytometric monitoring, we documented the sequential reversal of T-cell exhaustion (5, 6), restoration of the regulatory immune-braking system involving regulatory T-cells (Treg) and type 1 regulatory T-cells (Tr1) cells (16, 17), and successful reconstitution of the humoral immune compartment, including the repopulation of double-negative [cluster of differentiation 19 (CD19)⁺, cluster of differentiation 20 (CD20)^low, cluster of differentiation 27 (CD27)⁻ and immunoglobulin D (IgD)⁻] B-cells (18). The dramatic decline in N-terminal pro-B-type natriuretic peptide (NT-proBNP) level further underscores the systemic benefits of hydrogen in reducing myocardial stress during the recovery phase (19, 20). This study was approved by the Institutional Review Board of Tri-Service General Hospital, National Defense Medical Center, Taiwan (IRB no. B202105106; August 11, 2021). All procedures complied with institutional guidelines and the ethical standards of the Declaration of Helsinki and its amendments.

Case Report

A 49-year-old male with severe tricuspid regurgitation, hypokalemia, and Child-Pugh class B alcoholic cirrhosis was admitted in April 2023 following high-energy blunt chest trauma. Imaging revealed extensive thoracic injuries, including a left-sided flail chest with fractures of ribs 1-10, a distal left clavicle fracture, and a left lung contusion with secondary myocardial injury. The clinical course was complicated by atelectasis and bacterial infection, leading to suspected left-sided empyema and persistent respiratory failure.

In May 2023, the patient’s condition progressed to severe pneumonia with refractory septic shock, evidenced by a surging C-reactive protein level (17.4 mg/dl) and profound hypoxemia (SpO₂ <80%). The clinical instability culminated in a witnessed in-hospital cardiac arrest (Figure 1A). Following successful return of spontaneous circulation, the patient developed PCAS, characterized by acute-on-chronic heart failure with secondary pulmonary hypertension and multi-organ dysfunction (2, 3). By early June, the patient had reached a therapeutic impasse. Chest radiography on June 9th (Figure 1B) documented extensive bilateral pleural effusions and dense parenchymal consolidations, requiring invasive mechanical ventilation and multiple thoracic drainage systems. Myocardial wall stress was extreme, as evidenced by NT-proBNP levels (19, 20) that remained at a high-risk plateau, exceeding the laboratory’s upper limit of 35,000 pg/ml, for several weeks (Figure 1C). Given the refractory nature of the shock and the poor prognostic indicators, a do-not-resuscitate order was signed by his family on June 13th, 2023. On June 14th, amidst this terminal clinical trajectory, adjuvant therapy with molecular hydrogen (pure hydrogen inhalation, self-administered via a high-flow delivery system) was initiated alongside standard intensive care (12, 13). The initiation of hydrogen therapy marked a clear physiological turning point. Within 5 days (by June 19th), the patient showed rapid improvement in pulmonary gas exchange and hemodynamic stability, enabling successful extubation and transition to high-flow nasal cannula (Figure 1A).

Figure 1.

Integrated clinical course, radiographic evolution, and trend in cardiac biomarkers from critical illness to recovery (May-August 2023). (A) Clinical timeline illustrating the patient’s course, including major symptomatic events such as multiple cardiac arrests and septic shock, together with key diagnostic and therapeutic milestones. After a period of profound instability and the signing of a do-not-resuscitate order, adjuvant pure hydrogen therapy was initiated, followed by successful extubation and subsequent clinical recovery. (B) Sequential chest X-rays demonstrating radiographic progression: June 9th (baseline during intubation), showing significant bilateral pleural effusions and multiple support tubes; July 20th, showing improvement after extubation and placement of pigtail catheters with reduction of left-sided effusion and stabilization of pulmonary infiltrates; and August 13th, showing near-complete resolution of pleural effusions, with pulmonary opacities clearing during recovery. Skeletal injuries remained stable throughout the period. (C) Longitudinal trend of N-terminal pro-B-type natriuretic peptide (pro-BNP) level, reflecting cardiac strain and heart failure severity, showing chronic fluctuations followed by a sharp deterioration in May 2023, with a sustained high-risk plateau corresponding to refractory septic shock. After clinical stabilization and initiation of hydrogen therapy, a marked decline in pro-BNP level beginning in July 2023 indicates reduced myocardial wall stress and improved cardiac compensation. CAG: Coronary angiography; CRP: C-reactive protein; CS: chest surgery; DNR: do-not-resuscitate; EF: ejection fraction; ER: emergency room; Fx: fracture; HFNC: high-flow nasal cannula; PAP: pulmonary artery pressure; RHC: right heart catheterization; ROSC: return of spontaneous circulation; SICU: surgical intensive care unit; SpO₂: peripheral capillary oxygen saturation.

The biological impact of the intervention was further corroborated by longitudinal monitoring. Follow-up imaging on July 20th (Figure 1B) showed a dramatic reduction in pleural effusions and a clearing of the previously dense pulmonary infiltrates, coinciding with the therapeutic effect of hydrogen on endothelial integrity and inflammatory resolution (14, 15). Crucially, this period was characterized by a precipitous decline in NT-proBNP level, dropping approximately 64% from the initial plateau to 12,597 pg/ml by mid-July-signaling a significant alleviation of myocardial strain (Figure 1C) (20). Despite the successful reversal of the acute immune exhaustion and the stabilization of the septic and respiratory crises, the patient’s underlying chronic frailties posed insurmountable long-term challenges. During the recovery phase, the clinical course was further complicated by an episode of Candida parapsilosis fungemia, reflecting the profound state of immunoparalysis typically observed after prolonged critical illness (Figure 1A) (5, 11). While the adjuvant hydrogen therapy provided a critical window of stabilization, allowing the patient to survive the acute phase and be successfully discharged from the Intensive Care Unit on August 15th, 2023, after an 86-day battle, he ultimately succumbed to complications arising from his pre-existing conditions. This case illustrates that while molecular hydrogen can act as a potent immunological rescue by reversing acute exhaustion and stabilizing organ stress, the ultimate outcome remains determined by the cumulative burden of chronic comorbidities, including Child-Pugh class B alcoholic cirrhosis and severe valvular heart disease.

Discussion

This case report provides rare and highly valuable longitudinal immune phenotyping, revealing how adjuvant molecular hydrogen therapy can reverse profound immunoparalysis induced by severe pneumonia with septic shock and PCAS. Our data demonstrate that the microscopic reconstitution of the immune system, comprising biphasic resolution of T-cell exhaustion, sequential restoration of the regulatory immune-braking system and functional normalization of the humoral compartment, tightly correlated with the macroscopic resolution of refractory shock, the clearance of pulmonary infiltrates, and the dramatic alleviation of myocardial stress.

During the progression of severe sepsis, persistent systemic inflammation drives T-cells into a state of deep exhaustion and high apoptosis (5, 8). Our data revealed that during the acute phase, the death receptor FAS (CD95) was aberrantly upregulated on naive T-helper cells. Following the initiation of hydrogen therapy, FAS expression exhibited an immediate and rapid decline (Figure 2), aligning closely with the potent anti-apoptotic properties of molecular hydrogen (12, 13). More importantly, the classic markers of exhaustion, PD1 and TIM3, displayed a biphasic resolution on T-helper cells: a transient, slight increase during the early stabilization phase, followed by a precipitous drop to physiological level. This asynchronous dynamic illustrates the capacity of hydrogen therapy to provide immediate cytoprotection followed by the gradual resolution of immune exhaustion (6). Furthermore, at the onset of shock, basal PD1 expression on naive cytotoxic T-cells diminished abnormally, indicating a dangerous loss of physiological immune brakes (7). Longitudinal profiling confirmed that hydrogen therapy successfully facilitated progressive recovery of PD1 expression, culminating in complete restoration to a healthy level (Figure 2D). This underscores the unique capacity of molecular hydrogen to exert bidirectional homeostatic effects on immune checkpoints.

Figure 2.

Biphasic resolution of immune dysregulation and restoration of checkpoints in naive T-cells. Longitudinal flow cytometric analysis across three critical time points: baseline (June 14th), early stabilization (June 19th), and recovery phase (July 20th). (A) Naive T-helper cells (Th): The abnormally high expression of the death receptor FAS (CD95) showed an immediate and rapid decline following the initiation of hydrogen therapy. (B, C) Classic markers of exhaustion, programmed cell death protein 1 (PD1) and T-cell immunoglobulin and mucin domain 3 (TIM3), exhibited a transient but slight increase during the early stabilization phase, followed by a precipitous drop to physiological level by the recovery phase. This asynchronous dynamic demonstrates the capacity of hydrogen therapy to provide immediate cytoprotection while gradually resolving immune exhaustion. (D) Naive cytotoxic T-cells (Tc): Basal PD1 expression was abnormally diminished at the onset of shock, indicating a loss of physiological immune brakes. Continuous hydrogen therapy led to a progressive recovery of PD1 expression, ultimately restoring the level to a physiological one by the recovery phase. This highlights the bidirectional homeostatic effects of hydrogen therapy on immune checkpoint regulation. HC: Healthy controls, data obtained from healthy volunteers.

The collapse of immune-suppressive mechanisms is a central driver of multi-organ failure. Current immunological paradigms emphasize the critical role of Tregs in re-establishing systemic tolerance (16). Our analysis (Figure 3A-C) unveiled a multi-layered, sequential restoration of this immune-braking system. The activated Treg population was virtually abolished at baseline but demonstrated a rapid and vital recovery following hydrogen intervention, successfully stabilizing the acute hyper-inflammatory state. In contrast, as the patient transitioned into the recovery phase, the memory Treg population exhibited an explosive, compensatory expansion, indicating the establishment of long-term immune tolerance. Concurrently, the Tr1 cell population experienced initial depletion followed by a robust restorative rebound. Together, these trajectories underscore a comprehensive reconstitution of immune suppression (17), creating a protected microenvironment for tissue repair. Sepsis inflicts devastating damage on the humoral immune system (9, 10). Our analysis (Figure 3D-H) illustrates a comprehensive reconstitution and normalization of the B-cell lineages. As treatment progressed, the terminal plasma cell population showed a restorative V-shaped recovery, marking the functional reopening of the antibody-secreting machinery. Simultaneously, the double-negative B-cell pool demonstrated a robust restorative increase from an initial nadir, eventually exceeding physiological levels, correlating with its unique expansion potential during protective immune responses (18). Crucially, longitudinal tracking highlighted the targeted cytoprotection of memory B-cells amidst severe bone marrow suppression (Figure 4). The switched memory B-cell population remained significantly depleted throughout the course, reflecting a lasting immunological scar caused by prolonged bone marrow suppression and subsequent secondary fungemia (11). Despite this depletion, surviving switched memory B-cells showed a dramatic and sustained downregulation of the death receptor FAS, with its expression eventually dropping below the physiological level (Figure 4B). This indicates that hydrogen therapy provides a potent anti-apoptotic shield for these irreplaceable memory cells. Concurrently, transitional and regulatory B-cells remained effectively eradicated, highlighting the total loss of B-cell-mediated tolerance and further emphasizing the vital importance of the surviving, hydrogen-shielded memory B-cells. The miraculous microscopic recovery of the immune system perfectly explains the dramatic improvements in the patient’s macroscopic clinical indices. The patient suffered from pre-existing severe tricuspid regurgitation and secondary pulmonary hypertension; under the overwhelming stress of septic shock, the NT-proBNP level surged beyond 35,000 pg/ml (Figure 1C). However, following hydrogen intervention, it plummeted by 64%. This sharp decline reflects a fundamental reduction of severe myocardial wall stress induced by systemic inflammation (19, 20). Simultaneously, serial chest radiographs documented the rapid absorption of massive bilateral pleural effusions and the clearing of left lung contusion infiltrates (Figure 1B). Pathophysiologically, this radiographic improvement can be attributed to the protective effects of hydrogen on endothelial integrity, likely mediated through the NRF2/HO1 signaling pathway and newly identified targets such as APOA2 (14, 15).

Figure 3.

Sequential and multi-layered restoration of immune homeostasis. Longitudinal analysis of regulatory T-cell and B-cell compartments across three critical time points: baseline (June 14th), early stabilization (June 19th), and recovery phase (July 20th). Regulatory T -cells (Tregs): (A) Activated Tregs were nearly abolished at baseline but rapidly recovered by June 19th, helping stabilize the acute hyper-inflammatory state. (B) Memory Tregs exhibited a compensatory expansion during the recovery phase, indicating the establishment of long-term immune tolerance. (C) Type 1 regulatory T-cells (Tr1) showed initial depletion followed by a robust restorative rebound. Collectively, these trajectories reveal a multi-layered reconstitution of immune suppression. B-Cells: (D) The level of CD21⁺ naive B-cells remained highly stable, serving as an internal control. (E) Overall, naive B-cells were initially overrepresented due to the loss of mature subsets, then their level gradually normalized. (F) Double-negative (DN) B-cells [cluster of differentiation 19 (CD19)+, cluster of differentiation 20 (CD20)low, cluster of differentiation 27 (CD27)− and immunoglobulin D (IgD)−] experienced a strong restorative increase from an initial nadir, eventually exceeding the physiological level. (G) The aberrantly elevated CD21⁺ DN B-cell population returned to a physiological level. (H) Terminal plasma cells displayed a V-shaped recovery, indicating functional restoration of the antibody-secreting machinery. HC: Healthy controls, data obtained from healthy volunteers.

Figure 4.

Targeted cytoprotection of memory B-cells amidst bone marrow suppression. Longitudinal analysis of B-cell subsets under hydrogen therapy. (A) Switched memory (SM) B-cells remained significantly depleted throughout, reflecting a lasting immunological scar. (B) Surviving SM B-cells exhibited sustained downregulation of the death receptor FAS CD95). (C, D) Transitional B-cells and regulatory B-cells (Bregs) were effectively absent across the patient’s course, demonstrating prolonged bone marrow suppression and the loss of B-cell-mediated tolerance. HC: Healthy controls, data obtained from healthy volunteers.

This study has several limitations. As a single-case report, it is inherently limited in generalizability and does not allow causal inference between hydrogen therapy and the observed immunological and clinical changes (21-23). The lack of a control group, together with concurrent standard intensive care, makes it difficult to distinguish the specific contribution of hydrogen therapy. Although longitudinal immunophenotyping provided mechanistic insight, functional immune assays and broader multi-omics validation were not performed. In addition, the patient’s complex comorbidities, including advanced liver disease and severe valvular heart disease, may have influenced both immune responses and clinical outcomes. Further controlled studies are needed to confirm these findings and clarify the role of molecular hydrogen in sepsis.

Conclusion

This case highlights the interplay between the effects of acute intervention and chronic comorbidities in critical care. Hydrogen therapy induced transient system-wide immune reprogramming, resolving T-cell exhaustion, restoring immune brakes, and protecting memory B-cells from apoptosis, thereby disrupting the cycle of inflammation and immune depletion. It provided a critical stabilization window, allowing Intensive Care Unit discharge after 86 days, although the patient ultimately succumbed to chronic comorbidities. These findings suggest early hydrogen therapy as a potential immunomodulatory adjunct in severe sepsis.

Conflicts of Interest

The Authors declare that they have no conflicts of interest or competing interests related to this study.

Authors’ Contributions

CLW: Conceptualization, methodology, writing-original draft, writing review and editing. JWL: Conceptualization, methodology, writing-original draft, writing review and editing. YJH: Conceptualization, methodology, project administration, writing-review and editing. SWL: Conceptualization, methodology, writing-review and editing. TYH: Conceptualization, methodology, writing-review and editing. KYW: Conceptualization, methodology. FCL: Conceptualization, investigation, supervision, writing-review and editing.

Acknowledgements

This study was supported by the National Science and Technology Council, Taiwan (grants NSTC 112-2314-B-016-033, NSTC 113-2314-B-016-052, NSTC 114-2314-B-016-052-MY3 NSTC 114-2314-B-016-052-MY3 and NSTC 114-2313-B-019-012) and Tri-Service General Hospital, Taiwan (grants TSGH-E-112218 and TSGH-E-113238).

Artificial Intelligence (AI) Disclosure

During manuscript preparation, a large language model (Microsoft Copilot, by OpenAI) was used only for language editing and stylistic improvements in selected paragraphs. All research data generation, analysis, and interpretation were performed solely by the Authors. No figures or visual data were created or modified using generative AI or machine learning-based tools.