**3. Etiology and pathogenesis of SJS/TEN**

#### **3.1 Etiology**

Several microorganisms are considered to be the cause of SJS/TEN. For example, SJS/TEN can occur after vaccination with chickenpox, measles [8], *Mycoplasma pneumonia* [9], dengue virus [10], it is also associated with cytomegalovirus reactivation [11], currently, SJS may be related with Covid 19 [12] or Covid 19 vaccination [13, 14]. Other causes include serum injection, host graft rejection [15]. Chung's study showed that a novel variant of *Coxsackievirus A6* can induce severe mucosal bullous reactions, mainly mediated by granulysin-expressing T lymphocytes and NK cells. The clinical symptoms in that group of patients resembled severe EM or SJS [16]. A classic example of the role of viruses in drug response: in DIHS, human herpesvirus 6 (HHV6) plays an important role. HHV6 reactivation in DIHS patients may increase T-cell activity after the onset of drug response, induce the synthesis of

proinflammatory cytokines including TNF-α and IL-6 that are capable of regulating T cell-mediated responses [17].

The majority of cases of SJS/TEN were related to drug hypersensitivity. Some studies show that in people infected with HIV, the prevalence of the disease is about 1 in 1000, which is a thousand times higher than in the population without HIV [6]. In sub-Saharan Africa, where HIV prevalence is high, there is an association between SJS/TEN and HIV due to the use of antiretroviral and anti-tuberculosis drugs [18]. The drugs most commonly causing SJS/TEN are nevirapine, lamotrigine, carbamazepine, phenytoin, phenobarbital, cotrimoxazole, sulfonamides, sulfasalazine, allopurinol, and non-steroidal anti-inflammatory drugs (oxicam group) [6, 19]. Other less common drugs are aminopenicillin, cephalosporin, quinolone. In Asian countries, traditional medicine can also be a causative drug of SJS/TEN [20]. Currently, SJS/TEN is reported to be associated with pembrolizumab [21], and itraconazole [22]. Patch test, lymphocyte transformation test, and enzyme-linked immunospot can be used to detect culprit drugs in SJS/TEN but none of them is considered as standard [1, 23].

Sassolas et al. developed an algorithm for drug causality for epidermal necrolysis (ALDEN). This algorithm rates each drug with a score of −12 to 10 based on six criteria: (1) delay from initial drug component intake to the onset of reaction (index day); (2) drug present in the body on the index day; (3) pre-challenge/ rechallenge; (4)dechallenge; (5) type of drug (notoriety); (6) other cause. Allergenic possibilities are given based on the following total score: ≥ 6: very probable; 4–5: probable; 2–3: possible; 0–1: unlikely; < 0: very unlikely [19].

Stevens-Johnson syndrome and TEN are equally common in men and women. The frequency of the disease increases with age, peaking in people over 50 years of age [24, 25]. In the population of people living with HIV/AIDS, the prevalence of SJS/TEN is higher than in the general population [6].

### **3.2 Pathogenesis**

In SJS/TEN, keratinocytes are necrotic to varying degrees. The pathogenesis of SJS/TEN is related to the mechanism of death of keratinocytes [26–28]. These cells undergo apoptosis or necroptosis, causing the entire epidermal structure to necrosis, detachment, form blisters [26, 28].

### *3.2.1 Immune mechanisms in SJS/TEN*

Toxic epidermal necrolysis is a T-cell-mediated disease, TCD8+ lymphocytes are found in bullous fluid [29–31], perivascular in the superficial dermis [6, 32]. TCD8+ lymphocytes together with NK cells are considered to be the main agents of apoptosis of keratinocytes [29–31]. TCD4+ lymphocytes and other immune cells such as dendritic cells and mast cells also play an important role in TEN [6]. Caproni's study of cell infiltration in the skin of TEN patients showed a high density of CD40 ligand (CD40L) staining cells in the dermis, some infiltrating the epidermis [32]. CD40L is a molecule expressed on the surface of activated CD4+ T cells, and is a co-stimulator of macrophages, dendritic cells, B lymphocytes, and endothelial cells, leading to the release of tumor necrotic factor-alpha (TNF-α), nitric oxide (NO), interleukin (IL)-8, and cell adhesion molecules. Soluble CD40L is elevated in the serum of TEN patients. The TCD4+ lineage in the epidermis and

dermis of TEN patients has a balance between T helper (Th) 1 and Th2 as well as cytokine levels from these two cell types [33].

Macrophages, neutrophils, and NK cells are also involved in the pathogenesis of TEN, and studies have shown that macrophages infiltrate in predominant numbers in skin samples [32, 33]. Tohyama noted the presence of CD14+ CD16+ monocytes in the epidermis and the dermal-epidermal junction in skin lesions of SJS/TEN patients. This reinforces the proliferation and cytotoxicity of TCD8+ lymphocytes through the CD137/CD137L system. Monocytes and macrophages contribute to apoptosis through the production of TNF-α, TNF-related apoptosis ligands [34]. Neutrophils and dendritic cell factor XIIIa+ were also found in these skin samples, but their role in TEN has not been elucidated [32, 33, 35]. NK cells are present in the bullous fluid along with highly cytotoxic T cells that express the NK cell CD56 receptor. Both of these cell types are considered to be major contributors to keratinocyte apoptosis [30]. The important role of TCD8+ lymphocytes in the pathogenesis of TEN has been recently demonstrated with the murine model of TEN, which tends to resemble that of humans [36].

T cells are hyperactive due to decreased T regulatory (Treg) cell function and upregulation by monocytes. CD8+ T cells themselves are not specific for TEN; they are also present in other drug-induced reactions. Treg's function in increasing TCD8+ activity is an important factor in TEN, which causes epidermal injury. The mechanism by which Treg cells degrades function is unknown, but the loss of TCD8+ inhibition has been documented. Treg cells from the peripheral blood of TEN patients do not inhibit T cells. Treg cell counts in TEN patients do not differ from that in normal subjects, but their function is impaired during the acute phase of TEN [37].

Th17 cells, which are subtypes of TCD4+ cells, are present in SJS/TEN at a higher rate than other ordinary drug skin reactions (ODSRs), producing IL-17 and IL-22. In SJS/TEN patients, there are more IL-17-producing CD4+ T cells than in EM patients and healthy subjects. As the disease improves, the Th17 cell count decreases. They can regulate the mobilization of neutrophils and other inflammatory leukocytes, causing inflammation and skin damage. Furthermore, neutropenia, a cause of death in TEN, may be due to the action of Th17 [37].

#### *3.2.2 Role of human leukocyte antigen (HLA) class I in SJS/TEN and SCARs*

Many studies show an association between HLA class I and SCARs. In Han Chinese patients with SJS/TEN, there is a strong association between antiepileptic aromatics such as carbamazepine, phenytoin, oxcarbazepine, and lamotrigine with HLA-B\*15:02 [38], and between allopurinol and HLA-B\* 58:01 [39, 40]. An association between HLA-B\*15:02 and carbamazepine was also seen in Thai, Vietnamese, Malaysian, and South Indian SJS/TEN patients, but not in Japanese, Korean, or European populations [41]. In patients of European descent, there was an association between HLA-B\*57:01 and abacavir-induced SJS/TEN [42], and between HLA-A\*31:01 and carbamazepine-induced SJS/TEN [43]. A study in Japan showed that the HLA-B\*15:11 allele is a risk factor for carbamazepine-induced SJS/TEN, with an association between HLA-A\*02:06 and acetaminophen-induced SJS/TEN [44]. The significant association between SJS/TEN and certain HLA haplotypes has led to speculation that these haplotypes play a role in the pathogenesis of TEN [45]. This concept was proposed when investigating the role of HLA-B\*57:01 in the pathogenesis of abacavir-induced DIHS [46]. From the above studies, it is recommended to screen for some HLA alleles before prescribing some drugs that cause allergies,

related to HLA. For example, HLA-B\*58:01 allele carriers should not take allopurinol, and HLA-B\*15:02 allele carriers should not take carbamazepine [41, 43].

#### *3.2.3 Mechanism of death of keratinocytes in SJS/TEN*

#### *3.2.3.1 Mechanism of apoptosis of keratinocytes*

In SJS/TEN, keratinocytes undergo massive, widespread necrosis. The main reason is due to the apoptosis mechanism. There are many cytotoxic proteins and molecules involved in apoptosis initiation in SJS/TEN, of which granulysin has been shown to play a major role. Other factors including FasL, TNF-α, perforin, granzyme B, and NO also play a certain role [6, 26, 47].

#### *3.2.3.1.1 Granulysin*

Granulysin is a molecule found in the granules of cytotoxic cells (along with granzyme B and perforin) such as TCD8+, NK, and natural killer T cells, that act as a tumor killer and kills bacteria. When there is an interaction between the drug and the specific HLA and T cell receptor of TCD8+, granulysin is released from the granules of TCD8+, causing apoptosis of keratinocytes. Granulysin can cut through the target cell membrane, causing ion imbalance, damage to mitochondria, releasing oxidants and caspase cascade, causing apoptosis [48].

To investigate the role of granulysin in TEN, Chung et al. compared the gene expression of bullous fluid cells. The results showed that the expression of the granulysin gene of bullous cells increased 10–20 times, granzyme B increased 8 times, perforin increased 3 times, serum FasL increased 2 times [26]. When measuring granulysin concentrations in bullous fluid following a similar pattern, granulysin levels were 2–4 times higher than perforin, granzyme B, and soluble FasL, which correlated with disease severity. On immunohistochemical staining, the skin tissues of patients with TEN were strongly stained with granulysin, while the skin tissues of patients with ODSRs were weakly stained [26]. Abe showed that serum granulysin levels were elevated in 4 out of 5 patients with SJS/TEN before epidermal detachment or mucosal lesion. Meanwhile, this concentration increased only in 1 in 24 patients with ODSRs [49].

The above studies demonstrate that granulysin is an important cause of apoptosis in TEN, it is also a marker for early diagnosis and prognosis of disease severity [24, 26].

#### *3.2.3.1.2 Fas-FasL*

Fas ligand (FasL) is a transmembrane protein of the TNF family, found on the surface of cytotoxic T cells, NK cells. When these cells are activated, FasL is expressed, binds to its receptor on the target cell, and activates the intracellular caspase, leading to uncontrolled destruction of the target cell. In addition, Fas can be separated from the cell membrane by metalloproteinase enzymes, producing soluble Fas from FasL, still maintaining the ability to bind to Fas receptors, causing apoptosis [6, 50].

The Fas receptor has a region of repeat cysteines and an intracellular region of 80 amino acids, identical to the region in TNF-R1, named the death domain. This region is required for Fas to induce apoptosis. Mutations in this region destroy apoptosis induction. The only known physical Fas ligand is FasL (CD95L), which belongs to the family of TNF-related cytokines. Like its relatives, FasL is synthesized as transmembrane and trimer-soluble forms, by the enzyme metalloprotease. Fas signaling plays a decisive role in lymphocyte homeostasis. Repeated activation of antigen receptors on T cells induces FasL expression, leading to apoptosis via Fas signaling. When this process fails, due to mutations in Fas or FasL, lymphomas and autoimmune diseases occur [51]. FasL induces apoptosis by binding to the Fas receptor, causing activation of caspases [52]. Fas is expressed mainly on activated T lymphocytes and NK cells. Viard showed that FasL is also expressed in keratinocytes in TEN lesions [27]. Serum soluble FasL levels are elevated in patients with SJS/TEN before skin detachment, mucosal lesion, or both [53].

#### *3.2.3.1.3 Granzyme B and perforin*

Granzyme B and perforin have roles in the apoptosis of keratinocytes and endothelial cells [52]. Cytotoxic T cells, once activated, secrete perforin and granzyme B, create channels on target cell membranes, and activate apoptosis-inducing caspase [6]. In TEN, monocytes from bullous fluid induce apoptosis in the presence of anti-Fas antibodies, but not apoptosis in the presence of perforin/granzyme B inhibitors, indicating perforin/granzyme B is the causative agent of apoptosis [30, 31, 50]. The concentrations of these molecules correlate with the severity of the drug reactions. Therefore, testing for perforin and granzyme B may help differentiate TEN from other drug reactions [47]. Recent studies on skin biopsies of TEN patients showed that endothelial cells were apoptosis, and immunohistochemical staining showed granzyme B and TNF-α around the dermis vessels. Although not found on biopsies, the possibility that soluble FasL is the cause of apoptosis of endothelial cells cannot be excluded. The reason is that the biopsy samples were taken 2–4 days after the onset of the disease when the soluble FasL concentration was greatly reduced [54, 55].

#### *3.2.3.1.4 Other factors*

Other molecules, cytokines such as TNF-α and NO have certain roles in apoptosis. TNF-α acts on the "death receptor" TNF-R1, causing activation of caspases, causing cell death. TNF-α is elevated in the bullous fluid, skin, and serum of patients with TEN. Its role, though, is unclear. In addition to its ability to induce apoptosis, TNF-α also has a protective role by activating the anti-apoptosis pathway with nuclear factor-kappaB (NF-κB). This may explain why the mortality of TEN is increased with treatment with anti-TNF-α (thalidomide) [56]. NO induces apoptosis through its effect on the *p53* gene. Skin samples before blistering of patients with SJS/TEN had increased NO synthase (inducible NO synthase, iNOS) enzyme. IFN-α and TNF-α secreted from activated T cells can induce iNOS expression, which downregulates NO-dependent FasL, especially apoptosis in keratinocytes. Thus, IFN-α, TNF-α, and iNOS are potential molecular bridges between the immune response to drugs and the expression of proapoptotic molecules. The combination of IFN-α, NO, and an increase in reactive oxygen species causes oxidative stress, disrupts the intracellular machinery and cell membranes, leading to apoptosis [6].

#### *3.2.3.2 Mechanism of necroptosis of keratinocytes*

Cell death resembles necrosis but is regulated by a specific intracellular program known as necroptosis [57]. Experimental studies by Saito et al. showed that apoptosis is *Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis DOI: http://dx.doi.org/10.5772/intechopen.102794*

not the only mechanism of keratinocyte death in SJS/TEN. When pan-caspase inhibitors were added to the supernatant cultures of peripheral blood monocytes (PBMCs) of patients with SJS/TEN, incubated with keratinocytes, cell death still occurs. Thus, the PBMCs cultures of patients with SJS/TEN contain a specific molecule that causes the death of keratinocytes, which is independent of the apoptosis mechanism. The authors found that the concentration of annexin A1 protein in PBMCs cultures was significantly higher in SJS/TEN patients than in ODSR patients. Anti-annexin A1 antibody inhibits the death of keratinocytes [28]. Annexin A1 is a member of the family of 13 annexin proteins, which bind to acidic phospholipids with high affinity in the presence of Ca2+ ions [58]. When annexin A1 binds to the formyl peptide receptor 1 (FPR1) receptor on the cells, it causes necroptosis. The level of FPR1 expression was significantly different between the SJS/TEN group and the ODSR group. Therefore, it can determine the probability of SJS/TEN or ODSRs. Although there is no gene difference in the FPR1 promoter region between SJS/TEN, ODSRs, and healthy subjects, the annexin A1-FPR1 interaction may predict the occurrence of SJS/TEN and hold promise for targeted therapy in which necrosulfonamide inhibits the necroptosis pathway related to the annexin A1-FPR1 complex [28, 52].
