Herpes simplex virus type 1 (HSV-1) is a prevalent viral pathogen that infects most of the world’s population (Rowe et al., 2013). When infecting the cornea by interacting with host cell surface receptors through its glycoproteins, the HSV-1 virus induces a chronic immune-inflammatory response known as herpes simplex virus keratitis (HSK) (Koujah et al., 2019). The direct pathogenicity of the virus and the potent immune response triggered by viral proteins induce inward vascular growth, leukocyte infiltration, and corneal stromal and endothelial damage, leading to corneal clouding, edema, and neovascularization (Fukuda et al., 2008). Moreover, recurrent HSK can lead to progressive and irreversible corneal scar formation, which can ultimately cause blindness (Rowe et al., 2013). HSV-1 infections can be divided into primary and recurrent. After initial infection, HSV-1 spreads to the trigeminal ganglion (TG), where it becomes latent. Subsequently, after being activated by stimuli such as fever, menstruation, exertion, trauma, ultraviolet (UV) radiation, and some immunodeficiency diseases (Al-Dujaili et al., 2011), the virus migrates from the TG to the corneal area, inducing recurrent corneal infections that can affect vision.

Recurrent HSK is associated not only with the probability of viral reactivation, but also with the expression of viral and host proteins, the accumulation and the kinetics of specific innate and adaptive immune effectors, and the activation of inflammatory responses (Perng et al., 2016). In particular, immune responses triggered by the activation of latent HSV-1 are the main cause of recurrent HSK corneal lesions (Wang et al., 2020). After reactivation, HSV-1 viruses begin to replicate in the infected corneal epithelium and bind to Toll-like receptors (TLRs) on the cell surface, inducing a series of immune responses and signaling pathways that stimulate the production of inflammatory cells; these, in turn, trigger progressive infiltration of pro-inflammatory factors and chemokines into the stroma, thereby aggravating corneal lesion, and even causing blindness. Therefore, the corneal inflammatory response caused by HSV-1 ocular infection and chronic lesions induced by effector T cells can lead to blindness (Bhela et al., 2017). Unfortunately, the efficacy of anti-HSV therapy is limited (Sumbria, et al., 2021). In fact, no specific treatment for recurrent HSK has yet been developed, and long-term antiviral therapy based on acyclovir only reduces recurrence rates by approximately 40% (Ziyaeyan et al., 2007). Moreover, hepatic and renal toxicity associated with long-term consumption of oral antiviral drugs should not be ignored. Thus, prevention and repair of corneal damage after recurrence may be a promising new approach for the treatment of recurrent HSK.

Nevertheless, repairing corneal lesions and scars is a challenging task, since immune regulation and homeostasis are critical to determine the corneal response to treatment (Foulsham et al., 2018). In addition, harmful T-cell effectors that lead to corneal scarring (CS) deserve in-depth investigation (Jaggi et al., 2018). A previous study has revealed that HSV-1 infection and anti-HSV-1 antibodies modulate the activity of regulatory T (Treg) cells (Ciurkiewicz et al., 2020). However, little research has been conducted on the role of Treg cells in HSV-1 entry into latency or reactivation from dormancy. Mott et al. (2014) reported that the negative effect of CD8α⁺ dendritic cells on the replication of viral RNA molecules and latency-associated transcripts (LAT) under T-cell depletion led to a large accumulation of latent viral genome in the TG of intraocularly infected mice, and to an increased recurrence rate. Notably, bilateral disease, recurrent infections, and CS are observed more frequently in immunocompromised patients (Liesegang, 2001). Moreover, a recent study showed that treatment of virus-induced inflammatory responses with anti-IL-27 antibodies increased the number of CD4⁺CD25⁺Foxp3⁺ Treg cells and improved corneal tissue damage during relapses (Xia et al., 2019). Overall, previous research has demonstrated that Tregs play a protective role by maintaining homeostasis in vivo, enhancing immune tolerance, improving corneal damage, and preventing the onset of autoimmune diseases. Therefore, Tregs are considered protective immunomodulatory mediators that control the release of inflammatory factors and chemokines against viral invasion into the cornea (Veiga-Parga et al., 2012). Thus, it is important to study the role of CD4⁺CD25⁺Foxp3⁺ regulatory T cells in the modulation of corneal lesions during HSK relapse.

Treg cells, expressing the transcription factor Foxp3 as a characteristic molecular marker, play an essential role in maintaining immune homeostasis and preventing the disruption of peripheral self-tolerance (Georgiev et al., 2019). CD4⁺CD25⁺Foxp3⁺ T cells, a subpopulation of suppressor T cells, primarily mediate peripheral immune tolerance (Deng et al., 2019). The identity of Tregs and the production of their major regulatory factors are determined by Foxp3 expression; in particular, the expression of CD25, a direct target of Foxp3, is critical for Treg proliferation, survival, and Foxp3 expression (Cheng et al., 2011). In addition, the activation of Foxp3 depends on the STAT5 signaling pathway (Whitehouse et al., 2017; Apert et al., 2018; Fan et al., 2018), a key regulatory pathway for the differentiation of parental CD4⁺ T cells into Tregs (Shi et al., 2018). Therefore, in this study we aimed to test whether the Yiqi Jiedu (YQJD) formula plays a role in activating Tregs through the STAT5 signaling pathway to help reduce corneal lesions via overall immune regulation.

The YQJD formula is composed of Huang Qi (Astragalus mongholicus Bunge.), Bai Zhu (Atractylodes macrocephala Koidz.), Fang Feng [Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk.], Jin Yin Hua (Lonicera japonica Thunb.), Pu Gong Ying (Taraxacum mongolicum Hand.-Mazz.), Da Qing Ye (Isatis tinctoria subsp. Tinctoria.), Zi Cao [Arnebia euchroma (Royle ex Benth.) I.M.Johnst.], Chan Tui (Cryptotympana pustulata Fabricius.), Fu Ling {Wolfiporia extensa (Peck) Ginns [syn. Poria cocos (Schw.)]}, Sheng Di Huang [Rehmannia glutinosa (Gaertn.) DC.] and Chai Hu (Bupleurum chinense DC.). YQJD is a prescription drug based on clinical experience, consisting of Yupingfeng (YPF) powder, which promotes a healthy body Qi, supplemented by Lonicera japonica Thunb. and Taraxacum mongolicum Hand.-Mazz. for heat clearing and detoxification, and Yin-nourishing drugs such as Wolfiporia extensa (Peck) Ginns (syn. Poria cocos (Schw.)), Rehmannia glutinosa (Gaertn.) DC., and Bupleurum chinense DC. to harmonize the Yin-Yang balance. YPF, an ancient Chinese herbal decoction, was developed from Dan-Xi Xin Fa by Zhu Dan-Xi of the Chinese Yuan Dynasty, and has been used in clinical practice for the treatment of cold, flu, and inflammation-related diseases for several centuries, due to its immunomodulatory properties (Fan et al., 2017). Moreover, heat-clearing and detoxifying drugs such as Arnebia euchroma (Royle ex Benth.) I.M.Johnst., Isatis tinctoria subsp. Tinctoria. supplemented with Lonicera japonica Thunb. and Taraxacum mongolicum Hand.-Mazz. (from the Dictionary of Tumor Prescriptions) can exert anti-inflammatory, antibacterial, and antiviral effects (Muluye et al., 2014; Su et al., 2021), as well as regulate immune inflammatory responses. Finally, Cryptotympana pustulata Fabricius., Wolfiporia extensa (Peck) Ginns [syn. Poria cocos (Schw.)], Rehmannia glutinosa (Gaertn.) DC. (Zhou et al., 2019), and Bupleurum chinense DC. have immunomodulatory (Li et al., 2019), anti-inflammatory, and antiviral properties (Yang et al., 2017). Early clinical studies in our department and clinical observations by other researchers have shown that the YQJD formula, prescribed to strengthen body resistance and eliminate pathogenic factors, was safe and effective for the treatment and prevention of recurrent HSK. Indeed, the YQJD formula could relieve the symptoms of patients, improve vital signs, and repair the cornea to a certain extent. However, no studies have yet been conducted to reveal its mechanism of action. Nevertheless, in-depth investigation of the mechanism by which the YQJD formula alleviates corneal lesions in mice with recurrent HSK is important for its application in clinical treatment.

Recurrent HSK ophthalmopathy can be fatal to the cornea, leading to visual damage. However, our previous clinical study revealed that the administration of YQJD formula before the onset of recurrence could improve the ocular surface symptoms of patients. In this study, we assessed whether the YQJD formula could activate the STAT5 signaling pathway in mice, induce CD4⁺CD25⁺Foxp3⁺ Treg cells to exert immune tolerance, inhibit the effects of systemic immune inflammation on corneal damage, and stimulate Tregs to repair corneal damage. Our results provide a scientific basis for the use of the YQJD formula to prevent and treat recurrent HSK.

The ocular surface is a unique mucosal immune zone with anatomical, physiological, and immunological features that act in synergy to establish a particularly tolerant microenvironment. It has been reported that ocular infection by HSV-1 can lead to chronic T cell-mediated inflammatory lesions in the cornea (Bhela et al., 2017). The severity of these lesions is influenced by the balance of different CD4⁺ T-cell subsets, since these are the main coordinators of immunopathological damage (Lobo et al., 2019). Notably, the severity and duration of lesions depend to a greater extent on the balance of T-cell functional subsets rather than on the presence of non-lymphocytes such as neutrophils and macrophages or impairments in neoangiogenesis and neurological function (Jaggi et al., 2020). In particular, it has been observed that lesions are reduced in presence of cells with predominant regulatory functions (Rajasagi and Rouse, 2019). In contrast, lesions are more severe when pro-inflammatory Th1 and Th17 cells are predominant owing to an impaired Treg cell balance (Rouse and Suvas, 2004). In summary, CD4⁺ T cells, CD8⁺ T cells, and Tregs, the major coordinators of immunopathological lesions, play essential roles in the pathogenesis of HSK and corneal injury.

In this study, the corneas of mice were assessed daily for signs of lesions after relapse induction. Compared to those of mice in the Re group, the corneas of mice in the YQJD group were more transparent and presented less neovascularization and lower ocular surface scores, indicating that the YQJD formula mitigated macrophage infiltration in the cornea after HSV-1 infection. IL-17, a key pro-inflammatory cytokine, promotes the immune-inflammatory response to HSK by stimulating the production of pro-inflammatory factors and neutrophil-derived chemokines through the regulation of the secretion of corneal stromal fibroblasts (Maertzdorf et al., 2002). Consistently, anti-IL-17 antibodies can effectively suppress such delayed-type hypersensitivity response and significantly reduce lesion severity in HSK mouse models (Xia et al., 2013). Conversely, IL-4 secreted by Tregs downregulates the expression of inflammatory factors and prevents the formation of HSV-1-induced corneal scars, and, more importantly, reduces viral replication in the cornea (Osorio et al., 2002). Altogether, these findings suggest that YQJD may modulate the expression of anti-inflammatory factors to mitigate the corneal immunopathological response in recurrent HSK mice.

The therapeutic effects of pharmacotherapy on the ocular surface have been well documented. In fact, a series of studies have verified the protective role of Tregs against HSV-1-induced immune-mediated inflammatory processes (Veiga-Parga et al., 2012). In particular, Foxp3⁺ Tregs are central regulators of immune homeostasis and tolerance (Do et al., 2016). Compared to downregulating the number of CD4⁺ T cells to attenuate Th1 cell responses (Xia et al., 2010), stabilizing Tregs via Neuropilin-1 treatment has been shown to be more effective in curbing the development of HSK lesions (Sidaway, 2017). In addition, CD4⁺CD25⁺Foxp3⁺ Tregs protect the cornea against severe damage, whereas their depletion accelerates HSK progression (Suvas et al., 2004). Moreover, CD4⁺CD25⁺Foxp3⁺ Tregs can secrete IL-10, and IL-10 treatment has been shown to reduce neutrophil infiltration into the cornea and improve the degree of corneal damage (Keadle and Stuart, 2005). Consequently, some researchers have proposed to treat HSK by upregulating IL-10 expression (Rajasagi et al., 2012), which can significantly reduce the incidence of HSK and control the degree of inflammation (Tumpey et al., 1998; Sarangi et al., 2008). These results suggest that the YQJD formula promotes the differentiation of CD4⁺CD25⁺Foxp3⁺ Tregs and the expression of anti-inflammatory cytokines in the cornea of mice with recurrent HSK, thereby reducing the severity of the lesions.

The differentiation of Tregs mainly depends on the activation of STAT5 signaling (Cheng et al., 2013). In fact, STAT5 can directly bind to the conserved regions of the Foxp3 gene to promote Foxp3 expression. In this study, we observed that YQJD could effectively promote the activation of the STAT5 signaling pathway, which may subsequently induce the differentiation of CD4⁺CD25⁺Foxp3⁺ Tregs, indicating a suppressive immunopathological effect of the YQJD formula. Similarly, a single administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin by injection has been reported to significantly reduce the severity of HSK lesions in mice (Veiga-Parga et al., 2011). Corneal scar formation and angiogenesis are the result of a chronic inflammatory response against HSV antigens, and intraperitoneal administration of galectin-1 significantly reduced the severity of HSK lesions as well as neointimal formation (Rajasagi et al., 2012). Moreover, systemic administration of Tregs has been applied as a treatment for uveitis (Gregoire et al., 2016), suggesting that this exerts significant effects on the cornea.

HSK, a chronic immunopathological lesion, has been shown to severely impair visual quality (Rajasagi et al., 2017). In particular, over the course of blinding inflammation of the ocular surface, an inadequate immune response increases disease susceptibility, whereas an overreaction induces corneal clouding through inflammatory cell damage; this indicates that balanced immunopathological responses are crucial for preventing corneal clouding, scar formation, and permanent vision loss (Xia et al., 2019). Tregs and the cytokines IL-10 and TGF-β play a protective role by maintaining homeostasis, enhancing immune tolerance, and preventing the onset of autoimmune diseases. Tregs can also reduce susceptibility to disease throughout the body, control the release of inflammatory cytokines and chemokines, and prevent viral replication (Veiga-Parga et al., 2012). Conversely, anti-inflammatory cytokines such as IL-4 can adjust the ratio of anti-inflammatory cells and their activity to reduce the severity of corneal lesions (Varanasi et al., 2017). In summary, our results demonstrated that YQJD can enhance Treg differentiation and reduce corneal lesions; these effects are potentially mediated by the STAT5 signaling pathway. Early administration of YQJD formula can activate the STAT5 signaling pathway to increase the proportion of Tregs while inhibiting the accumulation pro-inflammatory factors and inducing that of anti-inflammatory factors, thereby leading to improved immune tolerance and reduced severity of corneal lesions.

The mechanism which the YQJD formula alleviates corneal inflammatory damage caused by recurrent HSK has been elucidated in this study. The prevention and treatment of recurrent herpes simplex keratitis provide new immune ideas and strategies. We will explore the influence of YQJD on aqueous humor, limbal region blood vessels and the corneal immune microenvironment. It is helpful to clarify the mechanism of drug action. At the same time, further research is made on the main active ingredients in the YQJD prescription.