Adaptive and innate immune pathogenesis of bullous pemphigoid: A review

Bullous pemphigoid (BP) is an autoimmune blistering disease that primarily affects elderly individuals. The presentation of BP is heterogeneous, typically manifesting as microscopic subepidermal separation with a mixed inflammatory infiltrate. The mechanism of pemphigoid development is unclear. B cells play a major role in pathogenic autoantibody production, and T cells, type II inflammatory cytokines, eosinophils, mast cells, neutrophils, and keratinocytes are also implicated in the pathogenesis of BP. Here, we review the roles of and crosstalk between innate and adaptive immune cells in BP.

The pathogenesis of pemphigoid is unclear, but autoantibodies to the hemidesmosome are implicated. Subepidermal blister formation with obvious inflammatory-cell infiltration is a hallmark of BP but not pemphigus disease (5). The pathogenesis of BP involves various immune cells and factors, including B cells (6), T cells (7), complement cells (5), mast cells (8), neutrophils (9), and eosinophils (10). The pathogenetic role of interactions among the aforementioned types of inflammatory cells is unclear. Here, we review the immune cells and cytokines implicated in the pathogenesis of BP.

.1.1 B cells and autoantibodies
B cells are thought to play a critical role in the pathogenesis of BP, which is confirmed by the efficiency of B-cell depletion therapy for refractory pemphigoid (11). It can also be supported by the increased expression of BAFF (B-cell activating factor) in BP (12,13) and that lower peak serum BAFF levels after rituximab treatment in BP patients predict relapse and a need for earlier intervention (12,13). Most BP patients have serum autoantibodies to the BMZ, which are termed BP180/type XVII collagen/BPAG2 and BP230/BPAG1; these are key components of hemidesmosomes, which mediate adhesion of the epidermis to the dermis. The pathogenic autoantibodies implicated in BP are produced by B cells. The mechanism by which autoreactive B cells are activated to produce autoantibodies has been extensively investigated.
BP180 is a transmembrane protein of the hemidesmosomes in basal keratinocytes. The extracellular domain of BP180 contains 15 interrupted repeated collagenous domains, and its structure consists of a globular head, central rod, and flexible tail. BP180 is inserted into the lamina densa via the rod domain and loops back through the lamina densa via its N-terminal tail (14). Several epitopes of BP180 have been identified, and differential epitope recognition is associated with clinical severity (15). The extracellular portion of the 16th non-collagenous domain (NC16A) of BP180 is the main epitope targeted by autoantibodies. IgG autoantibodies deplete BP180 in cultured normal human keratinocytes, thereby reducing their adhesion (16). The injection of mice with rabbit anti-mouse BP180 antibody induces blisters (17). Anti-human BP180 IgG produced by immunized mCol17 +/mother mice can induce BP lesions in their neonates whose skin expressed human but not mouse COL17 (18,19

T cells
Autoreactive T cells have been detected in the peripheral blood of BP patients with active disease but not in the blood of those in remission (45, 46). T-cell activation by an autoantibody molecule can induce a variety of responses of B cells to a cross-reactive version of the original epitope (47). In BP, CXCL12, which is derived from infiltrated CD3 + T cells in lesions, induced the chemotaxis and accumulation of CXCR4 + B cells by activating the transcription factor c-Myc, thus promoting B-cell differentiation into autoantibody-secreting cells and facilitating autoantibody production (48). CD3 + T cells are categorized as cytotoxic (CD8 + ) or helper (CD4 + ) T cells (Th cells). CD4 + Th cells play a central role in activating immune cells in BP. CD4 + T cells are classified as Th1, Th2, Th17, T follicular helper (Tfh) cells, or regulatory T (Treg) cells depending on the inflammatory reaction (49). The Th1/Th2, Th17/Treg, and Tfh/Treg ratios are important for immune tolerance (50)(51)(52)(53).
Th2 cells and IL-4 play a role in the pathogenesis of BP by promoting autoantibody production by B cells (54). B-cell activation by Th2 cells or surface-clustered immunoglobulins bound to the epitope of the antigen initiates this process (55). P2 (492-506 aa, VRKLKARVDELERIR) and P5 (501-515 aa, ELERIRRSILPYGDS), which are both peptides of BP180 NC16A (the main antigen in BP), are important for IL-4 production by Th2 cells and autoantibody production by B cells (54). The activation of Th2 cells in BP is consistent with predominant IgG4 autoantibody production: IL-4 regulates IgG isotype switching, thereby amplifying IgG4 production (56). IL-4 also promotes IgE isotype switching to stimulate IgE production (57,58). IL-4 and IL-13 are mainly secreted by Th2 cells. The efficacy of dupilumab (autoantibody against IL-4 and IL-13R) in BP implicates type II inflammation in its pathogenesis (59). Moreover, autoreactive Th2 and Th1 cells regulate the autoantibody response to the immunodominant sequences of BP230 (46). In experimental BP models, CD4 + T cells were crucial to promoting the production of pathogenic anti-hCOL17NC16A IgG, leading to active disease (60).
Treg cells maintain peripheral immune tolerance by suppressing autoreactive T cells (61). The contribution of Treg cells to BP is controversial. In a mouse model, Tregs alleviate pemphigoid lesions by altering the migratory capabilities of myeloid cells (62), and an absence of Treg cells leads to pemphigoid lesions (63). In another mouse model, Treg cells suppressed steady-state autoimmune reactions to BP230 and COL17 (64). CD4 + CD25brightFOXP3 + Treg-cell expression is increased in peripheral blood and skin lesions from BP patients (65,66). In conventional BP patients, the expression levels of total Tregs and Treg subsets were increased before, and decreased after, systemic corticosteroid treatment. The expression of CD45RA − Foxp3 hi effector Treg cells is positively correlated with disease severity in conventional BP, and CD45RA + Foxp3 lo -naive Treg cell expression is positively correlated with disease severity in DPP-4i related BP (67). Differences in results among studies may be explained by the use of different markers of Tregs.

Innate immunity 2.2.1 Eosinophils
Eosinophilic infiltrates and peripheral eosinophilia are features of BP and are associated with disease severity and outcome (82,83). Eosinophil degranulation is prominent in early BP lesions and is essential for blister formation (84). The localization of eosinophils to the BMZ is dependent on IgG and complement fixation (85). However, the interaction of eosinophils with IgE may induce their degranulation (86). Eosinophils highly express FcϵRI, which promotes their interaction with BP IgE autoantibodies (which results in eosinophil degranulation and blister formation) (28, 40). Eosinophils also promote initiation of the coagulation cascade (87). In BP patients treated with omalizumab, an autoantibody targeting IgE, disease severity was closely related to peripheral eosinophils, but not IgG (29).
Eosinophils exposed to eotaxin, GM-CSF, IL-5, IFN-g, and thymic stromal lymphopoietin cytokine (TSLP) promote the release of toxic granule proteins, including major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and MMP-9; in turn, these induce a local inflammatory response (84, 88-91). Eosinophil extracellular traps (EETs), which have a web-like structure containing nuclear DNA and proteins, were also discovered in BP lesions (92). IL-5, which is elevated in blister fluid, is essential for toxic protein release by eosinophils and the separation of keratinocytes (93). ECP, MBP, and EPO are increased in BP lesions and plasma and directly promote the separation of keratinocytes (94). In addition, ECP and eosinophilderived neurotoxin (EDN) are decreased in plasma after immunosuppressive treatment, suggesting that these markers are associated with disease activity (95). An initially low level of ECP may promote remission within the first year (95). Benralizumab, a humanized IgG1k monoclonal autoantibody against the IL-5R a subunit, and bertilimumab, a humanized monoclonal autoantibody targeting eotaxin-1 (CCL-11), are currently being evaluated in clinical trials as treatments for BP (NCT02226146 and 04612790).

Neutrophils
Neutrophils infiltrate BP skin lesions and release proteolytic enzymes and reactive intermediates to promote inflammation. A BP model suggests that neutrophils are a determinant of disease phenotype (96, 97). The cytokines and proteases secreted by neutrophils include myeloperoxidase (MPO), neutrophil elastase (NE), MMP-9, and neutrophil-derived nicotinamide adenine dinucleotide phosphate (NADPH). These cytokines degrade the extracellular matrix and split dermal-epidermal junctions, thus exerting an immunomodulatory effect in autoimmune diseases (81,98,99). The formation of neutrophil extracellular traps (NETs), like EETs, is increased in BP peripheral blood and lesions and correlates with disease activity (100,101). In vitro, BP180-NC16A antigen-antibody complexes can induce NETosis, releasing NETs through a cell death process (9). Elevated NETs in BP patients boost autoantibody production by inducing B-cell differentiation into plasma cells, an effect mediated by MAPK P38 cascade activation (9).

Mast cells
Mast cells accumulate and degranulate in early BP lesions (8). The role of mast cells in pemphigoid is debated (102, 103). FcϵRI, an IgE receptor expressed on mast cells, may induce IgE-mediated inflammation, urticarial plaques, skin edema, and eosinophil accumulation and activation (8). Mast cells with IgE and BP180 peptides are present in BP lesions and induce mast cell degranulation (104). Multiple inflammatory cytokines and proteases are released from mast cell granules following their activation. Tryptase, a marker of mast cells, is increased in blisters of BP and related to the BP autoantibody titer to the BMZ. Moreover, the plasma level of tryptase is related to BP autoantibodies (105). IL-5, released by mast cells, promotes eosinophil accumulation and activation in BP. In BP models, activated mast cells release mouse mast cell protease-4 (MCP-4), a homolog of human chymase, which activates MMP-9 and cleaves BP180 (106). MCP-4 also activates NET release by neutrophils, thereby stimulating autoantibody production by B cells (9,107).

Keratinocytes
Keratinocytes are implicated in the pathogenesis of BP. The separation of keratinocytes, induced by BP autoantibodies via Rac1/ proteasome activation, is critical for blister formation (110). Keratinocytes secrete thymus and activation-regulated chemokine (TARC/CCL17), a ligand for CCR4 and CCR8 important for the migration of these receptor-expressing cells (111). TARC is increased in BP plasma and lesions (112, 113). Keratinocytes express tissue plasminogen activator (tPA) after BP180 autoantibody activation (114). tPA, a component of the plasminogen/plasmin system, may interact with MMP-9 or NE to promote inflammation (115,116).

Complement
Linear complement deposition along the dermal-epidermal junction occurs in >80% of BP patients (117). A role for the classical and, to a lesser degree, alternative complement pathways in BP blister formation has been reported (118).
The anti-BP180 NC16A IgG serum level is significantly higher in patients with C3 deposition, and patients without blisters have significantly fewer C3 deposits (117). Antigen-IgG1 autoantibodies binding to the BMZ trigger complement activation (21). C3 activation at the dermal-epidermal junction leads to the formation of chemotactic peptides (activated third component of complement [C3a] and activated fifth component of complement [C5a]) and the recruitment of neutrophils, eosinophils, and macrophages to this site (85, 108, 118, 119). The activated fifth component of complement (C5a), along with C5a receptor 1 (C5aR1), but not C5aR2, plays a role in the early phase of BP by promoting neutrophil standstill and leukotriene release in the endothelium; in turn, this induces neutrophil migration to the interstitial space via an autocrine/paracrine circuit (120,121). C5and C4-deficient mice showed no blisters after mCol17 IgG injection (118). Also, no BP lesions appeared in non-C1q-binding anti-hCol17 IgG1 mutation COL17 humanized mice (21). A targeted C1s inhibitor is under evaluation as a BP treatment in a clinical trial (122).

Interactions among immune cells 3.1 Clinical heterogeneity may be associated with different types of pathogenesis
The mechanism of blister formation in BP is unclear. Some BP patients primarily show eczema lesions for several years (123), and others have BP autoantibodies but not lesions (124, 125). Some BP patients present with blisters and bullous without obvious erythema, whereas others show patchy erythema with few or no blisters (126). In most BP patients, autoantibodies can be detected using commercial products, although in a small proportion of patients, the tests are negative (125). Most infiltrating immune cells in BP are eosinophils, along with some neutrophils (127,128) and other cell types. Some BP patients respond well to topical steroids, whereas others need systemic steroids and immunosuppressants. Also, refractory BP patients respond differently to rituximab, dupilumab, and omalizumab (11, 129). Different immune cells induce inflammation in various BP models (89,96,130). Whether autoantibodies or inflammatory cells are more important in the pathogenesis of BP is unclear. The pathogenesis of BP may involve several immune pathways and infiltrating cell types; clinical presentations and the response to different treatment regimens vary (131-133).

Crosstalk among immune cells
Autoantibody binding to pathogenic antigen cause the separation of the BMZ in a complement-dependent or -independent manner (5, 21, 85, 118) ( Figure 1). Antigen-IgG1 binding to the BMZ triggers complement activation. C3a and C5a induce neutrophil and eosinophil chemotaxis, as well as mast cell degranulation, which in turn induce inflammation and blister formation (21, 109, 118, 121).
Antigen-IgG4 induced the separation of the BMZ through a complement-independent pathway (5,22,134). The antigenantibody combination leads to the recruitment of neutrophils and eosinophils in BP, and, consequently, to the release of proteolytic enzymes (5). Eosinophils trigger the separation of the BMZ in the presence of IgE or IgG (84, 86,89). The activation of intracellular pathways leads to pyrolytic hemidesmosomes and attracts immune cells, inducing the inflammatory cascade.
Tfh cells promote the production of high-affinity autoantibodies by B cells in germinal centers. Activated Th2 cells in BP secrete IL-4, which regulates IgG isotype and IgE switching, thereby amplifying the production of IgG4 and IgE (56,135,136). IgE autoantibodies induce BP180 internalization in basal keratinocytes, which reduces their adhesion (30). IgE autoantibodies interact with eosinophils, mast cells, basophils, and B cells via CD23 and FcϵRI (39-42). Mast cells, activated by IgE, degranulate and release IL-5 to promote eosinophil accumulation and activation (8, 104). Eosinophils are attracted to the BMZ by IgG autoantibodies and complement fixation (85) and degranulate after interacting with IgE (86).
In conclusion, a variety of immune cells and cytokines are implicated in the pathogenesis of BP, including T cells, B cells, eosinophils, mast cells, neutrophils, complement, and plasminogen/plasmin. However, the underlying pathways require further investigation.

Author contributions
TY wrote the manuscript, and ZZ revised the article for important intellectual content. All authors contributed to the article and approved the submitted version.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Innate and adaptive immunity in bullous pemphigoid: B cells produce IgE, IgG1, and IgG4 autoantibodies to bind antigens to the BMZ. Antigen-IgG1 binding to the BMZ triggers complement activation. C3 activation at the dermal-epidermal junction leads to the formation of chemotactic peptides (C3a and C5a), which recruit neutrophils and eosinophils and induce mast cell degranulation, thereby contributing to blister formation. Antigen-IgG4 binding leads to the recruitment of neutrophils and eosinophils and, consequently, to the release of proteolytic enzymes. BP180-specific IgG autoantibodies modulate IL-6, IL-8, and tPA expression in human keratinocytes. TARC/CCL17 secreted by keratinocytes can recruit and activate Th2 cells. IgE autoantibodies amplify the inflammation in BP by interacting with eosinophils, mast cells, and B cells. IgE autoantibodies could also induce BP180 internalization in basal keratinocytes, thereby suppressing their adhesion. Tfh cells promote the production of high-affinity autoantibodies from B cells via regulation by IL-21 and ICOS-ICOSL. Activated Th2 cells secrete IL-4, which regulates IgG isotype and IgE switching. Mast cells activated by IgE degranulate and release IL-5 to promote eosinophil accumulation and activation. Eosinophils are attracted to the BMZ by IgG autoantibodies and complement fixation, and degranulate after interacting with IgE. Eosinophils secrete EETs and toxic granule proteins, including ECP, MBP, EPO, and MMP-9, which are involved in the local inflammatory cascade. In addition, activated mast cells release MCP-4, which activates neutrophils. Activated neutrophils release cytokines and proteases, including NE and MMP-9, which degrade the extracellular matrix and split dermal-epidermal junctions. Neutrophils also release NETs and stimulate autoantibody production by B cells. Neutrophils and mast cells release IL-17 and IL-23, thereby significantly enhancing MMP-9 and NE production by neutrophils. tPA, a component of the plasminogen/plasmin system secreted by keratinocytes, may interact with MMP-9 or NE to promote inflammation. What's more, tPA, MMP-9, NE and eosinophils all could lead to the activation of coagulation system, inducing thrombotic and bleeding risk of skin. BMZ, basement membrane zone; C3, complement 3; EETs, eosinophil extracellular traps; MMP-9, matrix metalloproteinase-9; NE, neutrophil elastase; NETs, neutrophil extracellular traps; MCP-4, mast cell protease-4; C3a, activated third component of complement; C5a, activated fifth component of complement; C3aRs and C5aRs, C3a and C5a receptors, respectively; RBC, red blood cell; EOS, eosinophil; Th, helper T cell; Tfh, T follicular helper cell; Neu, neutrophil; B, B cell; Mast, mast cell; MBP, major basic protein; ECP, eosinophil cationic protein; EPO, eosinophil peroxidase; TARC, thymus and activation-regulated chemokine; tPA, tissue plasminogen activator.

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