H₂S Serves as the Immunoregulatory Essence of Apoptotic Cell Death

Abstract

Apoptosis supports tissue homeostasis and prevents immune disorders by removing damaged and functionally aberrant cells. Here, Ou et al., 2023 utilized genetic-, pharmacological-, and proteomic-approaches focused on sulfur amino acid catabolism to discover hydrogen sulfide release during apoptosis suppresses Th17 cell differentiation, thus providing therapeutic targets for autoimmune diseases.

Main body:

Historically viewed as an environmental hazard, hydrogen sulfide (H₂S) has become somewhat of a wonder molecule in the past twenty-five years due to the discovery of its malleable cell- and tissue-specific production lending control over its numerous physiological functions¹. Generation of H₂S is primarily via the breakdown of the sulfur containing amino acids cysteine and/or homocysteine by the enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfotransferase (3-MST) or through non-enzymatic catalysis coordinated by iron and vitamin B₆². H₂S imparts its bioactivity as a regulator of redox status and mitochondrial bioenergetics while serving as substrate for protein sulfhydration (aka persulfidation) of thiol (–SH) groups to hydropersulfides (–SS_(n)H) that impact protein structure, function, and/or stability³. Thus, the versatility of this gasotransmitter has rendered it a therapeutic-target in fields such as oncology, transplant surgery, neuro-protection, aging, and as highlighted in the current study by Ou et al.⁴, inflammation and immunology.

Programed cell death through apoptosis is a biological process conserved across evolutionary boundaries and essential to life⁵. By controlling how, when, and where cells die, apoptosis maintains tissue homeostasis by removal of damaged, non-functional, and/or functionally aberrant cells, thus leaving space and resources for new healthy cells to emerge and function. In addition, apoptotic cells actively engage by releasing signaling metabolites regulating gene expression, proliferation, and suppressing inflammation in the healthy neighboring cells⁶. As both endogenous H₂S and apoptotic cells maintain immune homeostasis^7,8, the authors hypothesized that one of the functional immunoregulatory metabolites released by apoptotic cells is H₂S. To test this hypothesis, the authors utilized a number of genetic and pharmacological in vivo mouse and cell-based models to probe the mechanistic roles and interplay between apoptosis and H₂S signaling that drives immune homeostasis (Figure 1).

Figure 1: Apoptosis, endogenous hydrogen sulfide (H₂S) production, and sulfhydration signaling drive immune homeostasis by suppressing aberrant Th17 cell differentiation.

Apoptotic signals including those initiated by staurosporine (STS) but perturbed by genetic mutations MRL/lpr and Bim−/− or the caspase inhibitor drug Z-VAD drive enzymatic H₂S production via the catabolism of cysteine in the resultant apoptotic cells and vesicles. Diminished H₂S production via knocking out the pyridoxal phosphate (PLP)-dependent H₂S generating enzymes cystathionine γ-lyase (CSE) or cystathionine β-synthase (CBS) or treatment with their pharmacological inhibitors propargylglycine (PAG) or hydroxylamine hydrochloride (HA) limits sulfhydration (SSH) of selenoprotein F (Sep15), which can be rescued by exposure to the exogenous H₂S producer sodium hydrosulfide (NaHS). Sulfhydrated Sep15 stimulates nuclear translation and phosphorylation (P) of signal transducer and activator of transcription 1 (STAT1) limiting aberrant Th17 cell differentiation and downstream pathogenesis in immune-related diseases such as systemic lupus erythematosus. Green arrows indicate stimulation, while red crosses indicate inhibition. Figure made using BioRender.

Diminished apoptotic events in mice driven by MRL/lpr and Bim^−/− mutations repressed H₂S levels and/or production in blood, liver, lung, spleen and kidney with abnormal differentiation of Th17 cells. Through manipulating and rescuing apoptosis pharmacologically or with UV radiation, H₂S levels were enhanced in solid tissues and in bone marrow stem cells, peripheral blood mononuclear cells, and in CD4+ T cells. Apoptosis-induced H₂S production was driven by increased mRNA and protein expression of CBS, CSE, and 3-MST in the apoptotic cells themselves as well as in packaged apoptotic vesicles (apoVs) for wider generation and delivery of H₂S. Extending into even more mechanistic territory are the highly impressive results finding CBS-generated H₂S and the H₂S donor NaHS potentiate the enhanced protein sulfhydration occurring during early apoptosis. Sulfhydration of the thioredoxin–like oxidoreductase selenoprotein F (Sep15) specifically on cysteine residue 38, an event that produces the induction of signal transducer and activator of transcription 1 (STAT1) phosphorylation and the inhibition of STAT3 activity, serves as the target in preventing Th17 cell differentiation and its downstream pathogenesis in systemic lupus erythematosus (SLE)-like phenotypes. A finding, which when combined with the previously identified role of H₂S in driving regulatory T cells differentiation⁷ supports the growing body of literature implicating the gaseous signaling molecule in maintaining immune homeostasis. Conversely, the caspase and apoptosis inhibitor drug zVAD dampening these responses, while deficiencies in the H₂S generating enzymes resulted in mice showing hallmarks of SLE such as enlarged spleen weights and tissue damage. These studies deliver a number of novel and exciting findings that will be of high importance to the fields of H₂S biology, immunology, and potential downstream treatments for those that suffer from auto-immune disorders such as SLE.

Moving forward, this study helps raise questions and stimulate potential new avenues of research in the respective fields. Much of the current mechanistic details of H₂S on its downstream signaling focused on Sep15 sulfhydration, yet it is possible the other redox and bioenergetics functions of H₂S play a role in immunomodulation in a tissue-specific manner. Various tissues are exposed to different concentrations of H₂S, with the liver, kidney, and lower GI-track being some of the highest. Thus, does suppression of Th17 differentiation occur more frequently in these tissues, or do tissue-specific resident immune cells counter this with enhanced sulfide oxidation rates via the mitochondrial sulfide quinone oxidoreductase (SQR) complex? It it will be interesting to determine if individuals with autoimmune diseases such as SLE have overactive mitochondrial SQR activity, rendering suppression via H₂S futile. While the authors showed enhanced cysteine sulfhydration in early stages of apoptosis, it was not determined whether increased sulfhydration emerged as a mechanism to resolve other cysteine oxidation events such as sulfenylations or nitrosylations occurring early in the apoptotic process³. Other topics to consider in light of the current work are: 1) Is H₂S release from cells undergoing other forms of cellular death and senescence similar to those undergoing apoptosis?; 2) Is the aging-associated decrease in circulating sulfide levels⁹ due in some part to aging-related declines in apoptosis control?; 3) What mechanisms does persulfidation of Sep15 use to regulate STAT1 phosphorylation? Is it involved in docking of a kinase?; 4) What transcription factors are activated under apoptosis to augment CSE, CBS, and 3-MST expression?; and 5) What therapies based on administration of exogenous H₂S or boosting of endogenous H₂S would have the greatest safety and efficacy for treatment of SLE and other inflammatory diseases?

In summary, apoptotic cells give up the ghost not only in a proverbial sense, but also somewhat literally in the form the bioactive gasotransmitter H₂S traversing aqueous and lipid cellular compartments¹⁰ and enacting its sulfhydration and inflammation-repressing activities long after the aforementioned cell is gone. Thus, apoptotic cells, in their dying moments, embody the words of the late poet Dylan Thomas: “Do not go gentle into that good night, Old age should burn and rage at close of day; Rage, rage against the dying of the light”.