TGF-β: transforming growth factor β; Fn-EDA: fibronectin extra domain A; AD: atopic dermatitis; TSLP: thymic stromal lymphopoietin; SSc: systemic sclerosis; ECM: extracellular matrix.

2.1. TGF-β

As the central cellular effector of fibrotic responses, TGF-β is produced and secreted by inflammatory cells, particularly macrophages, as well as fibroblasts and platelets [1]. Types 1, 2, and 3 are the three subtypes of TGF-β. The levels of type 3 TGF-β are not increased in keloids, but those of types 1 and 2, which activate fibroblasts and are involved in fibrosis and inflammation, are increased ^[20][57]. In keloids, TGFβ-1 has been associated with increased collagen and fibronectin synthesis by fibroblasts. The addition of TGFβ-1 to keloidal fibroblasts leads to increased synthesis of procollagen RNA ^[21][46]. TGF-β is involved in both suppressive and inflammatory immune responses. Under steady-state conditions, TGF-β regulates thymic T-cell selection and maintains homeostasis of the naïve T-cell pool. TGF-β inhibits cytotoxic T-lymphocyte (CTL), Th1, and Th2 cell differentiation while promoting peripheral (p)Treg, Th17, Th9, and Tfh cell generation and T-cell tissue residence in response to immune challenges. Similarly, TGF-β controls the proliferation, survival, activation, and differentiation of B cells, as well as the development and functions of innate cells, including natural killer (NK) cells, macrophages, dendritic cells, and granulocytes ^[22][58]. TGF-β promotes keloid formation by regulating fibroblasts and macrophages. TGF-β works in tandem with IL17A to stimulate IL-6 and CCL2 production in fibroblasts, which are macrophage chemokines and offer a way for macrophages to be recruited to the keloid microenvironment ^[23][27]. Considering macrophages are an important source of TGF-β production, the recruited macrophages produce more TGF-β and further promote collagen synthesis in the keloid microenvironment. In addition, by co-culturing fibroblasts with Tregs, Chen et al. [8] found that Tregs promoted collagen expression, which was more pronounced in keloids than in non-keloid controls, and that the process required TGF-β and anti-CD3/CD28 stimulation. Therefore, targeting TGF-β-related receptors or pathways is a promising method for the treatment of keloids. Current therapies targeting TGF-β/Smad signaling in fibroblasts can be divided into genetic, cellular, and pharmacological therapies ^[24][59]. Several investigational agents that target the TGF-β pathway have entered clinical trials. Clinical trials of the TGF-β neutralizing antibody fresolimumab (GC1008) are underway ^[10][48]. Fresolimumab is a pan-TGF neutralizing antibody that targets all three TGF-β isoforms and is being tested in clinical trials for a variety of fibrotic and cancer disorders ^[10][48]. Phase 1 clinical studies have been conducted in patients with focal segmental glomerulosclerosis (FSGS), idiopathic pulmonary fibrosis (IPF), advanced malignant melanoma, and renal cell carcinoma (RCC). The administration of fresolimumab was well tolerated in three separate human phase 1 clinical trials, and no treatment-related serious adverse effects were reported ^[25][60]. In fibrotic diseases, Rice et al. found that fresolimumab treatment reduced biomarkers and improved clinical symptoms in patients with systemic sclerosis (SSc) ^[11][49]. The expression of several TGF-β and collagen-related genes, including CTGF, SERPINE1, and COL10A1, declined after fresolimumab treatment compared with baseline ^[11][49]. In addition, fresolimumab treatment was associated with a rapid, dramatic decline in fibroblast infiltration of the deep dermis ^[11][49]. Since fibroblasts are the main pathogenic cells of keloids, fresolimumab may be a novel therapeutic strategy for keloids. However, persistent inhibition of TGF-β function may lead to side effects because TGF-β is involved in a variety of biological processes ^[10][48]. It has been shown that systemic reduction in TGF-β activity can be antifibrotic but also blocks its anti-inflammatory function, leading to exacerbation of inflammation ^[26][61]. Adverse effects also include bleeding episodes, such as gastrointestinal bleeding, gingival bleeding, or epistaxis ^[11][49]. Local injection of TGF-β antibodies or inhibitors may be an alternative treatment for keloids. Fibronectin extra domain A (Fn-EDA) is a component of the ECM and is specifically expressed in the fibrotic region. McGaraughty et al. designed a dual-specific antibody targeting Fn-EDA to deliver TGF-β antibody to fibrotic kidney lesion sites ^[12][50]. The molecule partially targets Fn-EDA and partially neutralizes TGF-β using dual variable domain Ig (DVD-Ig) technology. Systemic injection of bispecific antibodies led to notably higher levels of each molecule in obstructed kidneys than in non-obstructed kidneys, ipsilateral kidneys of sham animals, and other tissues. Since Fn-EDA is also enriched in keloids [1], the bispecific molecule targeting Fn-EDA and TGF-β can also be tried in the treatment of keloids. Bispecific antibody treatment may be more effective and have fewer side effects than the anti-TGF-β antibody alone.

2.2. NKG2A/CD94

CD8⁺ T cells can significantly inhibit fibroblast activity and proliferation ^[27][28]. Through scRNA-seq, Xu et al. ^[28][18] found that the downregulation of CD8⁺ CTLs is a feature in the peripheral blood and keloid lesions. In addition, scRNA-seq also showed specific upregulation of the NKG2A/CD94 complex, which may contribute to the reduction in CTLs within the tissue boundaries of keloid lesions. Therefore, it can be inferred that NKG2A/CD94 indirectly promotes the activity and proliferation of fibroblasts in keloids, thereby exacerbating keloid progression. Monalizumab is a novel checkpoint inhibitor targeting the NKG2A/CD94 complex in tumor immunotherapy ^[13][51]. Monalizumab can enhance NK cell activity against various tumor cells and rescue CD8⁺ T-cell function in combination with programmed-cell-death axis (PDx) blockade ^[13][51]. Monalizumab enhances tumor immunity by blocking inhibitory NKG2A receptors to promote CD8⁺ T-cell effector function ^[13][51]. Given that CD8⁺ T cells inhibit fibroblast proliferation and activity, monalizumab is a promising agent for reducing excessive ECM in keloids by restoring the inhibitory effect of CD8⁺ T cells on fibroblasts. Furthermore, a prominent advantage of monalizumab is that blocking NKG2A has little toxicity and, in particular, produces no signs of autoimmunity ^[13][51]. The most common adverse events of monalizumab were fatigue (17%), pyrexia (13%), and headache (10%). Other rare adverse events were interstitial lung disease, colitis, and hypophosphatemia ^[13][51]. In the future, clinical trials of monalizumab in the treatment of keloids may lead to surprising discoveries.

2.3. IL-4/IL-13 Pathway

Th2 cells and their cytokines play an important role in the pathogenesis of keloids. Wu et al. ^[29][32] found that markers of Th2, including IL4R, CCR5, CCL11, and TNFSF4/OX40L, were significantly upregulated in both keloid lesional and non-lesional skin compared to normal skin. IL-4 and IL-13 not only promote ECM deposition, inflammation, and pruritus of keloids but also induce the transformation of monocytes into M2 macrophages ^[30][62], thus playing a pivotal role in the pathogenesis of keloids. IL-4 and IL-13 can also directly stimulate nerve fibers via IL-4 receptors to induce pruritus. Moreover, TGF-β induces the production of IL-31, a Th2-related cytokine that is elevated in skin wound tissue and is significantly correlated with pruritus intensity ^[31][34]. This evidence suggests that Th2 cells and their cytokines, especially IL-4 and IL-13, contribute to keloid development, and the Th2 axis may become a breakout area of research in keloid pruritus treatments. Dupilumab, which was approved for the treatment of moderate-to-severe atopic dermatitis (AD), inhibits type 2 inflammation via the IL-4/IL-13 pathway, also known as the Th2 axis ^[32][63]. Interestingly, AD is an independent risk factor for the development of keloids ^[33][64]. The higher risk of keloid formation in AD patients suggests an association between keloids and the Th2 axis. Diaz et al. ^[14][33] reported a new use of dupilumab in the treatment of keloids. In a patient with both severe AD and keloids, after 7 months of treatment with a 300 mg dupilumab subcutaneous injection every 2 weeks, AD improved significantly, with a 50% reduction in the total size of the fibrotic plaque, shrinking of the large keloid, flattening of the surrounding margins, and full removal of the smaller keloid. Furthermore, dupilumab relieved pruritus in a phase 3 study of patients with moderate-to-severe AD, indicating that it plays a role in blocking signals from itch neurons ^[15][52] and may also improve pruritus of keloids. Injection site reactions are the most common adverse event associated with dupilumab. Other relatively rare adverse events include ocular complications (e.g., dry eyes, conjunctivitis, blepharitis, keratitis, and pruritus), head and neck dermatitis, onset of psoriatic lesions, progression of cutaneous T-cell lymphoma, alopecia areata, hypereosinophilia, and arthritis. Most of these adverse effects can be controlled during continued treatment with dupilumab, but some (such as severe conjunctivitis) may result in discontinuation of treatment ^[16][17][53,54]. Above all, dupilumab, which targets the IL-4/IL-13 pathway, may provide a better treatment choice for keloid patients, especially those with concurrent AD, and is worthy of further large-scale clinical trials.

2.4. TSLP

Thymic stromal lymphopoietin (TSLP) is an epithelial-cell-derived cytokine that directly stimulates Th2 cytokine production, leading to Th2 inflammation ^[34][65], and collagen production, leading to skin fibrosis ^[18][35][36][55,66,67]. Studies have shown that IL-13 induces skin fibrosis in AD via TSLP ^[35][66]. In IL-13 transgenic mice, TSLP neutralization or genetic deletion of TSLPR led to a considerable decrease in fibrocytes and cutaneous fibrosis ^[35][66]. Given that TSLP expression is also greatly enhanced in keloid lesions, extending to uninvolved skin ^[29][32], it is meaningful to investigate whether TSLP also has a role in promoting fibrosis in keloids. Tezepelumab is a human monoclonal antibody that blocks TSLP, which is also implicated in the pathogenesis of asthma by driving Th2 inflammation of the airway. Clinically, tezepelumab is used to treat severe, uncontrolled asthma in adults and adolescents ^[18][55]. The most common adverse events are nasopharyngitis, upper respiratory tract infection, headache, and asthma (which was more frequently observed in the placebo group than in the tezepelumab group) ^[18][55]. Tezepelumab can decrease the enhanced immune cells in keloid tissues. Although there have been no clinical trials of tezepelumab in keloids, it is worth assessing its effectiveness as a treatment of keloids that may produce similar effects to dupilumab.

2.5. IL-6

IL-6, secreted by M1 macrophages and mast cells ^[37][38][10,68], promotes chemotaxis, residency, and activation of macrophages ^[39][26]. Under stimulation of IL-6 and adenosine receptors, macrophages can generate a large amount of IL-10, TGF-β, and VEGF ^[39][26]. IL-6 has been shown to induce collagen and α-smooth muscle actin (α-SMA) expression in dermal fibroblasts ^[19][56]. Previous research has shown that IL-6 expression is increased in keloid fibroblasts ^[40][69]. Inhibiting IL-6 or IL-6 receptor α (IL-6Rα) in keloid fibroblasts revealed a dose-dependent decrease in collagen type I α 2 and fibronectin 1 mRNAs ^[41][70]. Furthermore, the mRNA and protein expression levels of gp130 and several downstream targets in IL-6 signaling (JAK1, STAT3, RAF1, and ELK1) were upregulated in keloid fibroblasts versus normal fibroblasts ^[41][70]. These findings suggest that IL-6 signaling may play an important role in keloid pathogenesis and provide clues for strategies targeting IL-6 for keloid therapy and prevention. Immunotherapies that target IL-6 signaling, such as tocilizumab, sarilumab, and siltuximab ^[19][56], should be further investigated in keloids. Tocilizumab blocks the IL-6 receptor (IL-6R) and is approved in more than 100 countries worldwide for the treatment of rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) ^[19][56]. In fibrotic diseases, the 2018 ACR conference reported the results of a phase III randomized controlled trial of tocilizumab in systemic sclerosis (SSc) treatment ^[19][56], in which 210 patients with SSc received 162 mg subcutaneous tocilizumab treatment. Although the primary endpoint was not met at the end of the experiment, the forced vital capacity of patients taking tocilizumab was superior to that of patients taking a placebo, indicating that tocilizumab significantly inhibited the worsening of respiratory disorders due to SSc-induced pulmonary fibrosis. On this basis, tocilizumab is also a valuable candidate for skin fibrosis treatment. In terms of adverse events, the most common adverse events associated with long-term use (96 weeks) were infections, such as pneumonia, infectious tenosynovitis, sepsis, and otitis media ^[42][71]. A decrease in neutrophils is often observed during treatment with tocilizumab in RA patients; however, neutropenia is not due to the myelotoxic effects of tocilizumab but rather to changes in the localization of circulating neutrophils ^[19][56].