Primary adrenal insufficiency (PAI) is characterized by inadequate cortisol secretion, due to the destruction of the adrenal gland and a consequent lack of glucocorticoid and mineralocorticoid productions. Autoimmune adrenalitis, or Addison’s disease (AD), is the most common cause of PAI. PAI can occur in isolation or as part of a group of autoimmune disorders known as autoimmune polyendocrine syndrome (APS).

Loss of tolerance to self-antigens in the adrenal cortex progressively leads to AD, primarily driven by self-reactive Th1 cells (1). Autoimmune disorders, including endocrine autoimmune diseases, are often associated with an imbalance between T helper type (Th)-17 and regulatory T lymphocytes (Tregs) (2). Specifically, Treg cells, due to their insufficient numbers or loss of function, fail to limit autoreactivity and establish peripheral self-tolerance (3). However, the role of Tregs in PAI remains a subject of ongoing debate. Recent studies have reported that patients with PAI exhibit similar percentages of Tregs, but with a higher turnover rate that could impair their functionality (2).

Fully functional Treg cells, initially identified as CD4⁺CD25⁺ T cells, are characterized by the expression of the transcription factor Forkhead box P3 (Foxp3). Their surface phenotype is defined by several membrane clusters of differentiation (CDs), with differential expressions closely related to the composition of the extracellular microenvironment (4, 5).

The modulation of proinflammatory cytokines and immunomodulatory factors, notably interleukin (IL)-6 and IL-17A, cyclooxygenase (COX)-2, heat shock protein (HSP)-70, indoleamine-2,3-dioxygenase (IDO), and programmed death-ligand 1 (PD-L1), may differently affect CD4⁺ and CD8⁺ expansion and initiate Treg expansion by different CD4⁺ or CD8⁺ T-cell conversion (2, 4–9). The multiplicity of phenotypes likely reflects the heterogeneity in the efficacy of Treg to counteract CD8⁺ expansion. Furthermore, this variability contributes to the difficulty in elucidating the clinical involvement of Tregs in the progression of pathology (10). Exploring Treg biology and the molecular process involved in their differentiation and activation could provide a valuable opportunity for developing immunotherapy approaches in PAI.

Glucocorticoids (GCs), generally, exert anti-inflammatory and immunosuppressive effects by regulating the expression of genes encoding various inflammatory and immunomodulatory proteins and by modulating the lymphocyte pattern, including Treg induction. However, high doses of glucocorticoids can have immunosuppressive effects, increasing susceptibility to infections and promoting a proinflammatory state (11).

Conventional GC replacement regimens (cortisone acetate and hydrocortisone) remain the cornerstone of treatment for patients with PAI, typically administered in two or three daily doses. However, overexposure to replacement GCs can lead to adverse effects, including an increased risk of infections, diabetes mellitus, and cardiovascular disease, which result in higher mortality rates compared to the general population (12). In recent years, the innovative dual-release hydrocortisone (DR-HC), a novel formulation designed to mimic the physiological circadian rhythm of cortisol secretion, has been associated with improvements in quality of life (QoL), metabolic parameters, and the proinflammatory state in AI patients (12–23).

Unlike other autoimmune disorders, such as type 1 diabetes mellitus and autoimmune thyroid disorders (e.g., Graves’ disease), studies on the immune system and circulating T-cell phenotypes in autoimmune PAI are limited and often controversial (24, 25). Understanding which immune cells, factors, and pathways are involved in the progression of the disease is crucial for identifying new therapeutic strategies. We believe that highlighting potential differences in responses to distinct therapeutic strategies could help identify novel mechanisms underlying disease progression and provide valuable insights for the prevention of PAI.

Based on this evidence, we aimed to investigate whether conventional GC replacement regimens or switching to DR-HC elicits different responses in terms of anti-inflammatory and immunomodulatory effects on peripheral circulating lymphocytes and serum parameters.

The primary outcome of the current study was the evaluation of the difference in CD4⁺/CD8⁺ T-cell frequency and CD4⁺/CD25⁺ Treg population in patients with PAI at baseline and after 12 months of GC replacement treatment (conventional and DR-HC).

Secondary outcomes included the evaluation of Treg promotion in terms of the percentage of the Foxp3⁺ gated subset within CD4⁺/CD25⁺ T cells, as well as the assessment of biochemical inflammation parameters and gene expression levels of IL-6, IL-17A, COX-2, HSP-70, IDO, PD-L1, inducible nitric oxide synthase (iNOS), and thioredoxin (TXN)-1.

In humans, data regarding the modulation of the immune response and the inflammatory status in PAI patients compared to healthy controls are limited. In our study, we first analyzed the circulating T lymphocyte subsets in patients with autoimmune PAI and healthy controls and subsequently the changes during different replacement regimens.

We found a higher percentage of CD4⁺ and CD8⁺ T cells and a lower CD4⁺/CD8⁺ ratio in patients with PAI compared to healthy controls, confirming that these cells play a crucial role in the evolution of adrenalitis. Consistent with our findings, Nowotny et al., who evaluated the immune cell subsets in patients with PAI of different etiologies, showed that in AD, T helper, cytotoxic, and natural killer cell subsets were higher compared to controls (32).

Characterizing the different T-cell subsets is crucial for elucidating the pathogenetic process of PAI. It is well documented that Tregs are involved in the maintenance of self-tolerance, and when they are deficient or dysfunctional, this can favor the development of autoimmune disorders. According to several studies, the development and persistence of autoimmune diseases, such as type 1 diabetes mellitus and rheumatoid arthritis, are associated with a decrease in the Treg population or a defect in the suppressive activity of Treg cells. An increase in the number of fully active Treg cells may be beneficial in autoimmune disorders (33).

In autoimmune PAI, there is a gradual destruction of adrenal glands, resulting in poor and inadequate hormone production (34, 35). In patients with AIRE mutations, defective and reduced Tregs have been observed, while conflicting data are available for patients with isolated PAI (36).

In the present study, we compared the T-cell profile between patients with PAI after 12 months of conventional GC replacement therapy and DR-HC treatment. Following 12 months of DR-HC therapy, we observed a decrease in the number of CD8⁺ T cells and a normalization of the CD4⁺/CD8⁺ ratio to greater than 1, aligning more closely with a physiological condition and indicative of improved immune function. Although there was a significant increase in the CD4⁺ T-cell population, this was not associated with a promotion of the CD4⁺/CD25⁺ subpopulation. Additionally, we identified a higher CD4⁺/CD25⁺ Treg subpopulation in patients with PAI compared to controls; however, no significant changes were observed in the CD4⁺/CD25⁺/Foxp3⁺ Treg population between CSR and DR-HC after 12 months.

These findings are consistent with those of Sjogren et al., who did not observe differences in the number and metabolic capacities of Tregs in patients with PAI compared to healthy controls, suggesting that functional impairments may contribute to the autoimmune setting (37). Several studies have also investigated the effects of GC replacement therapy on the immune system in patients with adrenal insufficiency, although the results are quite controversial, likely due to varying study methodologies and the different parameters assessed. Bancos et al. demonstrated that patients with adrenal insufficiency receiving a hydrocortisone replacement regimen three times daily experienced a selective impairment of natural killer cell cytotoxicity (38). Isidori et al. studied the changes in the immune system in patients with adrenal insufficiency after switching from multiple daily doses to a once-daily hydrocortisone regimen. In this single-blind randomized controlled trial, patients on conventional replacement therapy exhibited an increased number of classical monocytes (CD14⁺CD16⁻) and a reduced number of CD16⁺ NK cells. This immune pattern was reversed after switching from the multidose regimen to the single-dose hydrocortisone regimen, likely correlated to a resynchronization of circadian rhythm and clock genes (39). The frequency of infections also improved in patients who switched to once-daily dosing, suggesting that the most harmful metabolic and cardiovascular effects of glucocorticoid replacement therapy are associated with a low-grade inflammatory state, which can be reversed by modifying the timing of administration (20). However, data regarding the immune and inflammatory response adaptation to different therapies remain missing.

The modulation of the immune response is primarily mediated by the change in the expression of several modulator factors, which are also released by T cells, including cytokines and inflammatory proteins. Among these, PD-L1 interacts with its receptor PD-1 to inhibit Th1 lymphocyte proliferation and promote their conversion into Treg cells (4, 5). PD-L1 is also expressed on human PBMCs, and its expression has been identified in several autoimmune disorders, functionally protecting against autoimmune disorders and preventing disease progression (40). Indeed, the use of anti-PD-1/PD-L1 inhibitors, as pharmacological agents, has been reported to cause adrenal insufficiency (41).

Similar to PD-L1, IDO released by PBMCs is involved in immunological tolerance, including the regulation of Treg function (6). IDO is a signaling protein that reflects the immunological microenvironment in vivo, playing a crucial role in inhibiting Th17-like effector cells while simultaneously inhibiting IL-6 secretion and reducing infiltrating CD8⁺ T cells (7).

Once the self-tolerance collapses, autoreactive Th1 lymphocytes release several cytokines, including IL-6 and IL-17A, which contribute to the inflammatory state of the microenvironment, altering the balance of COX-2, HSP-70, iNOS, and TXN-1 (6, 8, 26). HSP-70 is an inducible protein whose expression is upregulated under conditions of cellular stress, involved in cell repair, survival, and the prevention of protein aggregation. Under cellular stress, HSP-70 rapidly accumulates first in the nucleus and then in the cytoplasm of cells. Extracellular HSP-70 exhibits proinflammatory activity when acting as an element of immune signaling pathways (8). COX-2 is an inducible enzyme whose upregulation is associated with a proinflammatory immune response, generating and maintaining inflammatory reactions through the production of prostaglandins, prostacyclin, and thromboxane (42, 43). TXN-1 was recently described as a novel key player in the expansion of T cells (9).

Our data revealed that, at baseline, patients with PAI on conventional GC replacement therapy exhibited an increased proinflammatory status, with elevated levels of inflammatory markers (IL-6, IL-17A, COX-2, HSP-70, TNX-1) and immunomodulatory parameters (PD-L1, IDO, and INOS), compared to healthy controls. After switching to DR-HC treatment, a significant improvement in the immune and inflammatory response was observed. Interestingly, 12 months of DR-HC treatment led to a decrease in the expression of proinflammatory IL-6 and IL-17A (which are able to induce the differentiation of CD4⁺ T cells to Th17 cells and reduce Treg cell differentiation) (44) and was associated with a significant downregulation of COX-2, HSP-70, and TNX-1.

Additionally, we demonstrated that DR-HC was associated with an improvement in BMI and a significant decrease in WC. An interconnection between obesity and the immune system has been suggested. Schmitz et al. showed a significant inverse correlation between CD4⁺ Treg populations and some anthropometric parameters including BMI, WC, relative visceral adipose tissue, and waist-to-hip ratio, suggesting that obesity affects the immune system (45). In the present study, we did not find any correlation between anthropometric parameters and CD4⁺ and CD8⁺ T cells. Few studies have examined the link between obesity, body composition, and Tregs, with inconsistent results. Some found reduced Tregs in obesity and improved insulin sensitivity with Treg induction, while others reported body mass-dependent Treg depletion in adipose tissue (45–48).

Our comprehensive PPI analysis highlighted a potential relationship between inflammatory factors (COX-2, HSP-70, TXN-1) and sphingolipid biosynthesis, including serine palmitoyl transferase long-chain base subunit-1 (SPTLC1) and glucocerebrosidase-1 (GBA). This supports the hypothesis that a different CD4⁺/CD8⁺ T regulation could be established. Beyond their role in structural function, sphingolipids also act as key second messengers in various cell types, including T cells (49). Abimannan et al. suggested a potential role of de-novo sphingolipid biosynthesis in altering intracellular oxidative balance, leading to an enhancement of Th17 cell frequency in autoimmune diseases (50). Moreover, the bioactive role of sphingolipids in glucocorticoid metabolism and their involvement in various endocrine disorders have been described (51, 52).

In summary, our results suggest that the improvement in immune response is independent of Treg promotion or may be mediated by a Treg subset with a different phenotype. This could support the hypothesis that DR-HC restores a physiological rhythm of Treg expansion, making it more aligned with normal physiological conditions.

Furthermore, we also observed a significant decrease in serum inflammatory parameters after 12 months of treatment with DR-HC compared to conventional GC therapy, which to our knowledge represents an innovative finding. The regulation of inflammatory indices is crucial for maintaining immune homeostasis. Based on our results, it could be also hypothesized that the restoration of circadian rhythm and the resynchronization of the clock gene, likely induced by DR-HC treatment as suggested by Isidori et al. (20), could contribute to the improvement of the inflammatory response, independent of Treg promotion.

The current study has some limitations. First, we only investigated the subsets of circulating lymphocytes mentioned and did not include other subsets that were studied in other research. Additionally, the panel of cytokines we evaluated was not exhaustive. We focused on IL-6 and IL-17A as the main proinflammatory cytokines, excluding other potential cytokines. Similarly, we evaluated Foxp3⁺, in conjunction with CD4⁺CD25⁺, although these are not the only markers for identifying Tregs. Second, we only investigated two time points (i.e., baseline and 12 months after the switching). Future studies should employ a repeated measures design to provide further insight into the changes in circulating lymphocyte subsets. Another limitation concerns the correlation between adrenal-infiltrating and circulating lymphocytes. A paired analysis of circulating T lymphocytes and corresponding adrenal-infiltrating subtypes should be conducted in future studies to identify the cell subsets that could serve as biomarkers in peripheral blood. Fourth, our results may be at risk of bias due to the non-randomization of the study. Fifth, a small number of patients was included in the study, although adrenal insufficiency and, notably, APS are rare conditions affecting 1 in 5,000–7,000 individuals in the general population. Finally, there is heterogeneity among the patients, as we included both patients with isolated PAI and those with APS, despite a similar disease duration. Among the APS patients, only one had APS-1, a condition that could affect the balance between CD4/CD8 or CD4/CD25 cells.

Sixth, all patients included in the study were treated with a twice-day conventional steroid replacement therapy. We acknowledge that HC has a shorter half-life compared to cortisone acetate, and some patients may require a thrice-daily dosing schedule to adequately mimic the physiological cortisol profile. In our study, we aimed to standardize treatment with twice-daily HC dosing, which is commonly used in clinical practice. However, we recognize that this regimen may not be sufficient for all patients, particularly those with higher metabolic demands or variable absorption rates. Despite these limitations, the strength of the study lies in the fact that the switch from multiple daily doses of immediate-release cortisone or hydrocortisone to DR-HC treatment was able to change T lymphocyte phenotype and CD4⁺/CD8⁺ ratio, inducing a significant improvement in the immune and inflammatory pattern in adult patients with autoimmune PAI, not at the onset of the disease. Specifically, to our knowledge, this is the first time that the panel of inflammatory and immune modulators (TXN-1, HSP-70, and COX-2 and IL-6, IL-17A, PD-L1, and IDO) we have selected has been analyzed in patients with autoimmune PAI.

Patients with autoimmune PAI exhibit a lower CD4⁺/CD8⁺ ratio compared to healthy controls. Treatment with DR-HC is associated with a normalization of the CD4⁺/CD8⁺ ratio, in contrast to patients treated with conventional GC treatment, who maintain a low CD4⁺/CD8⁺ ratio.

The use of DR-HC promotes an increase in PD-L1 expression and a decrease in TXN-1, which inhibits lymphocyte proliferation, as well as a reduction in the expression levels of COX-2 and HSP-70. However, this is not accompanied by enhanced production of Th2-associated cytokines. Although a significant increase in IDO was observed, no significant differences were found in the percentage of Foxp3⁺ Tregs in patients treated with DR-HC compared to those on conventional GCs, suggesting that the role of Tregs in the maintenance of adrenal insufficiency remains unclear and warrants further investigation.

The findings of our study suggest that improvement in immune response is independent of Treg promotion, or possibly mediated by a different Treg subset, with a different phenotype. This supports the hypothesis that DR-HC restores a rhythm of Treg expansion, aligning it to a more physiological condition, potentially due to the resynchronization of the circadian cortisol rhythm and clock genes. We also hypothesize that the normalization of the cortisol rhythm halts or prevents the progression of adrenalitis. Finally, our work suggests that the CD4/CD8 ratio might serve as a potential biomarker for immuno-monitoring in adult patients with autoimmune PAI. However, larger studies are needed to confirm our preliminary findings and identify new therapeutic targets.