Granzyme B: A novel therapeutic target for treatment of atopic dermatitis

Introduction

Granzyme B, a multifunctional serine protease thought to be derived from cytotoxic T lymphocytes and natural killer cells, is secreted and expressed along with perforin for the induction of apoptosis.¹ Indeed, granzyme B also plays an important role in the extracellular non-perforin-dependent environment of many inflammatory diseases, including some lung diseases, cardiovascular diseases and skin diseases, promoting inflammatory responses, influencing extracellular matrix remodelling and inducing epithelial-mesenchymal transition and fibrosis.² Atopic dermatitis is a chronic inflammatory skin disease characterised by pruritus and skin lesions and is governed by a variety of genetic, environmental and immunological factors. Epidemiological surveys show that atopic dermatitis affects 5–20% of children and 2–10% of adults worldwide, and the prevalence is increasing.³ The pathogenesis of atopic dermatitis is still not fully understood, and most of the existing treatments have associated side effects. Several recent studies have demonstrated that granzyme B is detected at elevated levels in skin lesions and peripheral blood of atopic dermatitis patients and is involved in the disruption of atopic dermatitis barrier function, the development of inflammatory responses, increased vascular permeability and several other potential pathogenic mechanisms. In this review, we discuss the pathogenic mechanisms of granzyme B in atopic dermatitis, which promises to be a new therapeutic target for atopic dermatitis.

Granzyme B

In the 1980s Masson et al.⁴ identified two serine proteases in cytotoxic T lymphocytes cells, one of which, a 29-kDa protein consisting of a single polypeptide chain, was named granzyme B. For a long time granzyme B was thought to be expressed only by cytotoxic T lymphocytes and natural killer cells, and only since the turn of the century has granzyme B been found to be expressed in a variety of non-immune cells such as smooth muscle cells, keratinocytes and chondrocytes under certain conditions.⁵ Therefore, granzyme B has now been shown to function via two diverse pathways. One is a classical perforin-dependent apoptosis-inducing function, and the other is a non-apoptotic function independent of perforin, which is closely related to the pathogenesis of atopic dermatitis. Gapud et al.⁶ treated human submandibular gland cell cultures with granzyme B in the absence of perforin, and although cleavage of intracellular proteins was still present, no significant induction of death was detected by flow cytometry after 90 minutes, and nuclear morphological changes of apoptosis were similarly not observed by immunofluorescence microscopy. This suggests that granzyme B may perform other potential functions in the presence of perforin deficiency. It has been found that approximately one-third of granzyme B escapes from the immune synapse to the extracellular environment during entry into the target cell. Since the extracellular environment lacks effective granzyme B inhibitors, it can remain active there and then cleave a variety of substrates (e.g., cytokines, proteins, enzymes), resulting in a range of pro-inflammatory responses including protein cleavage and epithelial barrier disruption, fibrosis, inflammation and autoimmunity.⁷ For example, in autoimmune blistering diseases, granzyme B degrades anchoring proteins, induces inflammatory factor secretion and neutrophil infiltration; in cutaneous leishmaniasis, it promotes the production of tumour necrosis factor and interleukin-6 and correlates positively with the size of lesions in the disease; in pemphigus, it cleaves several key proteins and leads to blister formation.⁸–10 Therefore, granzyme B may be found as a key inducer and/or modulator in a variety of inflammation-related skin diseases.¹¹

The role of Granzyme B in the pathogenesis of atopic dermatitis

Yawalkar et al.¹² observed significantly higher expression of granzyme B in skin lesions than in normal skin in eight patients with moderate to severe atopic dermatitis. Kamata et al.¹³ also found by enzyme immunoassay that plasma granzyme B concentrations in atopic dermatitis patients were elevated and positively correlated with indicators of atopic dermatitis severity, and that plasma granzyme B levels were significantly lower after treatment with some conventional drugs compared to the untreated group. These experimental results suggest that granzyme B may play a crucial role during atopic dermatitis. The onset and development of atopic dermatitis are multifactorial, with the following three being the main pathways currently identified: firstly, impaired skin barrier function, secondly, T helper-mediated cellular immune imbalance, and thirdly, reduced skin microbial diversity characterised by an increased proportion of Staphylococcus aureus colonisation.^14,15 It is important to clarify that these mechanisms do not cause the disease independently but interact with each other, leading to recurrent episodes and a chronic course of atopic dermatitis. To date, research has found that granzyme B is primarily involved in the pathogenesis of the first two aspects of atopic dermatitis, which are discussed in detail below. A brief summary is presented in Table 1.

Perspectives on Granzyme B for atopic dermatitis

Based on basic research into the mechanism of action of granzyme B in atopic dermatitis, more and more researchers are realising that the treatment of atopic dermatitis by inhibiting granzyme B activity is very promising clinically. Serine protease inhibitors (serpins) work by using their reaction centre ring as a pseudo-substrate and cleavage site for homologous proteases, forming covalent complexes with the corresponding proteases to render them biologically inactive.³⁰ SerpinB9 is a natural inhibitor of intracellular granzyme B, which unfortunately has not yet been found in the extracellular environment. Subsequently, Saito et al.³¹ transplanted OVA-specific CD8+ T (OT-I) cells (transgenic CD8⁺ T cells expressing ovalbumin-specific T-cell receptor) into chicken ovalbumin transgenic mice to construct a mossy tissue reaction/interface dermatitis model and after five days of treatment with serpina3n, a murine extracellular serinease inhibitor, observed not only a significant improvement in the symptoms of skin lesions compared to the control group but also pathological findings showing a reduction in the number of OT-I cell infiltrates. Also, the onset of acute graft-versus-host disease was delayed after prophylactic administration of serpina3n to OT-I cell receptor ovalbumin mice.³¹ However, although this granzyme B inhibitor found in mouse plasma is homologous to human α1-antichymotrypsin, the latter is mainly expressed in the liver and whether they have the same function and mechanism of action has not been clarified.³²

The recent development of a highly selective and potent human granzyme B small molecule inhibitor in Vancouver, namely: VTI-1002, has largely advanced the progress of drug research based on granzyme B as a therapeutic target.³³ Shen et al.³⁴ applied VTI–1002 gel to the skin lesions of burn model mice continuously for a total of 30 days, resulting in faster wound healing and less scar formation in the VTI–1002 gel–treated mice compared to the excipient– only control group. It is important to highlight that the concentration of VTI–1002 in the skin remained virtually unchanged over 4–24 hours, suggesting that it can remain at the application site for a long enough time to be effective.³⁴ Further studies by Turner et al.¹⁶ on the oxazolone-induced atopic dermatitis mouse model exhibiting thinning of ear thickness and reduced signs of skin lesions such as scaling, erythema and erosions following the application of VTI-1002 gel demonstrated that VTI-1002 works by blocking or reducing granzyme B-mediated loss of filaggrin and E-cadherin proteins to improve skin barrier function. So far, although the studies on granzyme B inhibitors are still in the experimental stage in animal models, they have all shown significant therapeutic effects and no adverse effects, which provides a theoretical basis and data reference for future clinical trials.

Conclusions

In summary, granzyme B is an important protein molecule involved in the pathogenesis of atopic dermatitis, and therapies targeting granzyme B have undoubtedly opened new research ideas for its management. However, the specific regulatory mechanisms of granzyme B in atopic dermatitis are not well understood, and whether inhibition of granzyme B expression and reducing its level can improve the inflammatory status and skin barrier function in atopic dermatitis patients, and whether it can be a biomarker for monitoring changes in the disease course and assessing prognosis remains to be investigated in more depth and observed in long-term clinical trials.