Recent Advances in Cutaneous Lupus

Abstract

Cutaneous lupus erythematosus (CLE) is an inflammatory and autoimmune skin condition that affects patients with systemic lupus erythematosus (SLE) and exists as an isolated entity without associated SLE. Flares of CLE, often triggered by exposure to ultraviolet (UV) light result in lost productivity and poor quality of life for patients and can be associated with trigger of systemic inflammation. In the past 10 years, the knowledge of CLE etiopathogenesis has grown, leading to promising targets for better therapies. Development of lesions likely begins in a pro-inflammatory epidermis, conditioned by excess type I interferon (IFN) production to undergo increased cell death and inflammatory cytokine production after UV light exposure. The reasons for this inflammatory predisposition are not well-understood, but may be an early event, as ANA+ patients without criteria for autoimmune disease exhibit similar (although less robust) findings. Non-lesional skin of SLE patients also exhibits increased innate immune cell infiltration, conditioned by excess IFNs to release pro-inflammatory cytokines, and potentially increase activation of the adaptive immune system. Plasmacytoid dendritic cells are also found in non-lesional skin and may contribute to type I IFN production, although this finding is now being questioned by new data. Once the inflammatory cycle begins, lesional infiltration by numerous other cell populations ensues, including IFN-educated T cells. The heterogeneity amongst lesional CLE subtypes isn’t fully understood, but B cells appear to discriminate discoid lupus erythematosus from other subtypes. Continued discovery will provide novel targets for additional therapeutic pursuits. This review will comprehensively discuss the contributions of tissue-specific and immune cell populations to the initiation and propagation of disease.

1. INTRODUCTION

Cutaneous lupus erythematosus (CLE) is an inflammatory autoimmune disease characterized by recurrent, photosensitive skin lesions that can result in scarring and alopecia[1]. CLE lesions are heterogeneous, and drivers of lesion development are not well understood. Recent advances in our understanding of CLE pathogenesis hold promise to improve our treatment options for patients with these often-devastating lesions. This review will discuss current understandings of mechanisms that contribute to CLE, including photosensitivity, and how these have led to development of novel therapeutics (Figure 1).

Figure 1:

Summary of pathogenic interplay between abnormal inflammatory and IFN responses in the skin and the infiltrating inflammatory cell populations. Figure made using BioRender.com.

2. EPIDEMIOLOGY

CLE occurs with and without systemic disease involvement, referred to as SLE-associated CLE and primary CLE, respectively. Cutaneous involvement is one of the most common clinical features of SLE, occurring in up to 70% patients [2]. Progression from primary CLE to SLE occurs in 20% of patients with an initial CLE diagnosis [3]. While connections between systemic and cutaneous lupus are being identified, definitive mechanistic links have remained elusive, and differences between primary and SLE-associated CLE are not well understood.

CLE is estimated to occur at an incidence of around 4 cases per 100,000 people, depending on the population studied, and has comparable occurrence to SLE in some groups [3–5]. Similar to observations in SLE where nine out of ten patients are women[6], CLE more commonly affects females than males, with an incidence ratio of 3:1 [3, 7]. Recent epidemiological studies have additionally described an increased incidence rate of primary CLE in Black Americans relative to White individuals, also mirroring findings in SLE [7, 8]. Racial disparities have been well-described in lupus; while genetic and environmental factors may contribute to these disparities, the effects of systemic racism must remain a central consideration in understanding demographic bias in CLE.

3. CLE CLASSIFICATIONS

CLE lesions are divided into several categories based predominantly on histological findings, time course of lesion development and presence, and clinical features of the patient. The three main diagnostic subsets of CLE are chronic cutaneous lupus erythematosus (CCLE), acute cutaneous lupus erythematosus (ACLE), and subacute cutaneous lupus erythematosus (SCLE) [9]. CCLE is the most common CLE subtype, accounting for up to 75% of all cases [4, 10]. The CCLE subset can be further divided into discoid LE (DLE), LE profundus (LEP), LE tumidus (LET), and chilblain LE (CHLE), where DLE is the most prevalent form. DLE lesions are typically localized to face and scalp, and are rarely more generalized[10]. While slightly less photosensitive than other subtypes[11], DLE lesions often result in scarring, hyperpigmentation, and alopecia [12]. ACLE is most commonly observed as the malar “butterfly” rash, appears in UV-exposed areas, and resolves without scarring or pigmentation changes [12]. ACLE is highly associated with systemic disease involvement, with lesions often preceding systemic disease flares. SCLE also most commonly appears in UV-exposed areas, appearing first with erythema and then typically forming psoriasiform or annular plaques. Alterations in pigmentation are known to occur in SCLE, but lesions resolve without scarring [12].

4. PATHOGENIC MECHANISMS UNDERLYING DISEASE

4.1. Abnormalities in Skin Cells

4.1.1. Keratinocytes

Recent studies have highlighted an important role of “normal-appearing” nonlesional skin in CLE and have provided insight into mechanisms contributing to immune dysregulation and lesion development. Most prominently, an elevated interferon (IFN)-induced gene signature has been identified in both lesional and nonlesional skin, with IFN-κ from keratinocytes being a major contributor to this signature [13–17]. Elevated type I IFN signatures have been observed in both keratinocytes and fibroblasts in nonlesional lupus skin, suggesting that healthy-appearing skin in CLE may be predisposed to lesion formation as a result of elevated basal IFN signaling [18]. In SCLE and DLE patients, systemic type I IFN expression was also found to be increased and correlated with the Cutaneous Lupus Area and Severity Index (CLASI) activity score [19]. Both increased secretion and skewed IFN responses likely contribute to keratinocyte IFN signatures in SLE patients. IFN-κ expression from SLE non-lesional keratinocytes was induced to a higher degree following UV exposure and TLR stimulation [13, 20]. Further, the increase in IFN-response genes was also noted to be more robust in SLE vs. healthy control keratinocytes[21]. Intriguingly, the skewed IFN response may precede disease onset as IFN-κ originating from keratinocytes was found to be increased in an ANA+ patient cohort without CLE or SLE, suggesting a potential role for early IFN dysregulation in disease development [20].

Mechanistically, cutaneous type I IFNs have been shown to promote inflammatory skin responses by amplifying inflammation and cell death in response to UV light and other triggers. In non-lesional SLE keratinocytes, IFN-κ was found to facilitate increased IL-6 expression from keratinocytes following UV stimulation in an autocrine signaling loop [14]. Keratinocyte-derived IFN-κ additionally led to an increase in keratinocyte death following UV exposure [13]. Supernatants from UV-stimulated SLE KCs resulted in activation of dendritic cells in an IFN-signaling dependent manner[22]. Increased cutaneous type I IFNs also disrupt the skin barrier, enabling colonization with S. aureus and culminating to dysbiosis of the skin microbiome [23]. Type I IFN signaling is also known to induce chemokine expression in keratinocytes, notably the chemokines CXCL9, CXCL10, and CXCL11, which can recruit memory and effector T cells and propagate the skin autoimmune response [24].

Keratinocyte-produced type III (lambda) IFNs may also contribute to inflammation in SLE skin. In humans, there are four type III IFNs (IL-29, IL-28A, IL-28B, and IFNλ4). The source of these cytokines is plasmacytoid dendritic cells (pDCs)[25] and keratinocytes[26]. IFN lambda is upregulated in the epidermis of CLE lesions and may participate in a similar autocrine loop as type I IFNs as stimulation of keratinocytes with IFNλ induces production of proinflammatory cytokines and chemokines, including CXCL9, CXCL10, and CXCL11 [27, 28]. Indeed, deficiency of the receptor for IFNλ abrogates immune responses in a TLR7-driven model of lupus, including skin thickening and inflammation at the site of TLR7 exposure[28].

4.1.2. Fibroblasts

In contrast to known defects in DNA damage repair mechanisms in SLE keratinocytes, studies conducted nearly two decades ago described normal DNA repair and survival in SLE fibroblasts[29]. Recently, a unique population of fibroblasts was described in nonlesional CLE skin which had an elevated IFN signature similar to that observed in KCs [18]. In both cell populations, type I and type III interferons were implicated as upstream regulators of the transcriptional profiles unique to nonlesional CLE skin. The phenotypic consequences of these fibroblast changes remain to be studied but are intriguing as IFN-educated fibroblasts were identified to have substantial cross-talk with other potentially pathogenic cell populations such as CD16+ dendritic cells[30]. Other studies have evaluated fibroblasts as bystanders, including fibrosis activation after exposure of fibroblasts to SLE neutrophil extracellular traps (NETs) decorated with tissue factor and IL-17A[31]. Fibroblasts remain an understudied but likely relevant cell population in SLE skin.

4.2. Abnormalities of immune cell populations

4.2.1. B cells and autoantibodies

Among the hallmarks of SLE pathogenesis is the loss of B cell tolerance and aberrant activation of autoreactive B cells, which can be driven by type I IFN signaling (reviewed in [32]). This activation and loss of tolerance leads to production of autoantibodies against self-antigens such as double-stranded DNA and nuclear proteins[33]. Such autoantibodies can be detected at the dermoepidermal junction (DEJ) of even healthy-appearing skin of SLE patients[34], and UV exposure can lead to immune complex deposition at the DEJ[35] which may contribute to further expansion of B cells via toll-like receptor (TLR) stimulation [36]. Indeed, autoantibodies derived from serum of lupus-prone mice led to TNFR1-dependent skin inflammation when intradermally injected into wild-type mice[37]. Antibodies that contribute to skin disease in lupus have classically been thought to be systemically derived; however, recent literature suggests the existence of tertiary lymphoid structures in skin which may provide a source of B cell maturation, local antibody production, and T cell activation[38–40]. Such structures have been identified in pristane-induced murine lupus[41] and human lupus erythematosus profundus biopsies[42, 43], but whether and the extent to which tertiary lymphoid structures contribute to local cutaneous autoantibody deposition in CLE remains under investigation.

In CLE, commonly seen autoantibodies include anti-Ro52 and anti-Ro60[44]. Autoantibodies against these Ro proteins correlate with photosensitivity and can be found in lesional CLE skin[45–48]. Additionally, anti-Ro autoantibodies can cross the placenta and cause neonatal lupus, which presents with a similar CLE-like rash and risk of a fatal heart block in the neonate[49]. Exposure to UV light induces keratinocyte apoptosis[50] and promotes translocation of Ro antigen to the surface of these cells, making it more likely that apoptotic keratinocytes will be bound by anti-Ro autoantibodies[51–54]. Intriguingly, the target proteins of anti-Ro antibodies have a negative regulatory role on IFN generation. Specifically, Ro52 is upregulated in photoprovoked lupus skin[55] and has been shown to ubiquitinate interferon regulatory factors (IRFs), ultimately down-regulating production of inflammatory cytokines and IFNs[56, 57]. Indeed, mice deficient in Ro52 develop CLE-like lesions[58]. Unlike Ro52, Ro60 is an evolutionarily conserved RNA-binding protein[59, 60] which may play a role in responses to UV light; murine embryonic stem cells and bacterial cells lacking Ro60 demonstrated heightened sensitivity to UV exposure[61, 62]. Similarly, mice deficient in Ro60 exhibit a lupus-like phenotype characterized by autoantibody production, glomerulonephritis, and photosensitivity[63]. In one recent study, B and T cells from lupus patients were shown to respond to commensal bacterial Ro60 orthologs in vitro[64]. Taken together, these data suggest that Ro52 and Ro60 have negative regulatory effects on inflammation and type I IFN production, and antibodies against these proteins contribute to the disease pathology in CLE. The links between the role of the autoantibodies and the cellular function of their targets are not known.

Antibody production is not the only mechanism by which B cells are thought to contribute to CLE pathogenesis. Using an MRL/lpr murine model of lupus in which B cells exhibited surface immunoglobulin expression but lacked secreted immunoglobulins, Chan et al demonstrated that lupus-like phenotype was maintained in the absence of circulating antibodies[65]. This suggests that B cells’ contribution to autoimmunity extends beyond autoantibody production and may include antigen presentation to CD4+ T cells[66], though whether this holds true in the skin environment is still unknown.

B cell numbers are known to be elevated in lesional CLE skin, especially DLE[67, 68] and particularly in later stages of the disease[67]. Interestingly, cutaneous B cell gene expression signatures help to distinguish CLE subtypes. Compared to ACLE and SCLE, DLE exhibits a stronger B cell gene signature and increased staining of CD20 and CD27 by immunohistochemistry[69, 70]. This skin B cell signature in DLE is strongest in DLE patients who have isolated cutaneous disease without systemic disease, suggesting that differences in B cell function may bridge cutaneous and systemic disease[69]. Further probing the connections between the roles of systemic versus cutaneous B cells in lupus, Jenks et al demonstrated that there is considerable heterogeneity of B cell phenotypes across different primary CCLE patients[71]. While some B cells from CCLE patients were phenotypically similar to healthy control B cells, others were more similar to B cells from patients with systemic lupus disease[71]. Compared to the former, the latter group was more likely to have increased disease activity and more disseminated disease, suggesting that circulating B cell phenotypes may serve as a prognostic marker for CLE patients at risk for SLE [71]. Taken together, these data raise the importance of clarifying the role of B cell responses in CLE skin.

Further suggesting a role for B cells in CLE pathogenesis, the B cell activating cytokine BAFF has been identified in the peripheral blood and epidermis of DLE, SCLE, and lupus profundus lesions at higher levels than in healthy control skin[72–74]. Furthermore, cultured keratinocytes treated with DNA mimicking immunostimulatory self-DNA in CLE lesions exhibit increased BAFF expression[73]. Indeed, belimumab, an inhibitor of BAFF, has shown clinical benefit in treating CLE[75, 76] and is currently in phase III clinical trials for treatment-refractory CLE[77]. Importantly, BAFF signaling has been shown to be necessary for germinal center maintenance in mice[78]; whether cutaneous BAFF production sustains tertiary lymphoid structures in lesional CLE remains an area for further investigation but is an intriguing hypothesis.

BAFF-targeted therapies are not the only B cell-related therapies tested in lupus. Recent studies in lupus-prone mice treated with chimeric antigen receptor T cells (CAR-T cells) targeting B cells demonstrated improved skin disease[79]. Interestingly, however, other B cell-depleting therapies such as Rituximab, an anti-CD20 monoclonal antibody, have been differentially effective in treating different subtypes of CLE[80, 81]; ACLE patients had marked improvement in their skin disease, patients with CCLE did not experience clinical benefit with treatment, and some even paradoxically sustained skin flares[81]. This may suggest that subsets of B cells are protective in certain subtypes of CLE; indeed, IL-10 producing regulatory B cells (Bregs) have been described in CLE lesions[82] and may be depleted by B cell targeted treatments, precipitating disease flares. Bregs are stimulated by UV light[83], and induction of Bregs suppresses both skin and systemic disease in MRL/lpr lupus-prone mice[84]. Thus, understanding B cell heterogeneity and the role of B cells in skin inflammation in lupus is critical in developing targeted therapies for CLE.

4.2.2. T cells

In SLE, T cells have been shown to contribute to progression of disease through inflammatory cytokine production and providing of B cell help[85–87]. In CLE specifically, T cells are the most commonly observed immune cell type[88], and molecular subtyping analysis suggests the DLE subtype of CLE is characterized by a more T cell-predominant gene signature[89, 90]. Moreover, T cells have been shown to home to the dermal-epidermal (DEJ) junction in lesional skin of SLE patients following UV exposure[91], and UV light has been shown to expand and activate both CD4+ and CD8+ T cells in murine lupus[92]. Human and murine lupus T cells are hyperexcitable via activation of spleen tyrosine kinase (Syk)[93] and transcription factor NFAT[94, 95] downstream of TCR engagement. As such, blocking NFAT with the drug dipyridamole has been shown to alleviate skin manifestations in murine lupus, suggesting a role for T cells in development of cutaneous inflammation[95]. However, dipyridamole has effects on other pathways, such as neutrophil NETs[96], so the specificity of this effect on T cells is unclear. Similarly, inhibitors of spleen tyrosine kinase (Syk) have demonstrated benefit in treating cutaneous manifestations in both the MRL/lpr and BAK/BAX models of lupus, though the authors concede that the effects of these inhibitors are not T cell specific and may also implicate B cells or CD11c+ DCs in disease development[97]. A phase I trial of a topical Syk inhibitor did not show therapeutic benefit for human CLE[98].

More recently, transcriptomic analysis of SLE T cells from both lesional and nonlesional skin revealed striking IFN signatures compared with T cells from systemic sclerosis and healthy control skin[99]. Further suggesting a functional role for T cells in driving CLE pathogenesis, proteomic studies have identified an increase in IL-16, a T cell chemoattractant, in lesional CLE[100]. The specific subsets of T cells and their putative contributions to CLE development are reviewed below.

4.2.2.1. CD8+ T cells:

CD8+ cytotoxic T cells (CTLs) are among the inflammatory infiltrate in CLE[101, 102]. CTLs express granzyme B, which induces caspase-driven apoptosis in target cells[103]. As such, CTLs at the DEJ in CLE are thought to contribute to keratinocyte apoptosis[85, 87, 88, 104]. Granzyme B expression has been identified in lesional CLE skin and correlates with type I IFN expression[85, 104]. Additionally, decreased numbers of circulating CXCR3+ CD8+ cells have been identified in DLE[85, 101] patients, possibly suggesting their recruitment to lesional skin. Similarly, Wenzel et al illustrated a robust CD8+ T cell infiltrate in lesional CDLE skin characterized by the skin-homing markers CCR4 and cutaneous lymphocyte antigen (CLA)[105]. Lending support to a role for CTLs in CLE, one lupus-like mouse model of systemic autoimmunity demonstrated that CD8+ T cells, but not CD4+ T cells, drove CLE-like skin manifestations[106, 107]. However, recent transcriptomic studies identified gene signatures suggestive of lesser cytotoxic activity by T cells from cutaneous lupus samples compared with lupus nephritis[99]. Thus, while evidence suggests a potential role for CD8+ T cells in CLE pathogenesis, the precise mechanisms of involvement warrant further investigation.

4.2.2.2. CD4+ T cells:

Early histologic studies of CLE suggested that CD4+ T cells were slightly more numerous in lesional skin than CD8+ T cells[108, 109], and recent studies suggest that CD4+ T cells are among the most activated cell types in lesional CLE skin[110]. However, the specific subsets of CD4+ T cells contributing to the lesional inflammatory environment remain controversial. Some studies point to CLE lesions being Th1-predominant, particularly in DLE[90, 111–113]. Histologic analysis of CLE lesions demonstrates increased staining of the Th1-recruiting chemokine CXCL10[101, 114]. Type I IFNs, which are abundant in lesional CLE, and type III IFNs stimulate cultured keratinocytes to produce CXCL10; as CXCL10 is a ligand for CXCR3 which is expressed on Th1 cells, this chemokine is thought to recruit Th1 cells to CLE lesions[27, 101, 115, 116]. In accordance with these findings, CXCR3-expressing cells have been shown to be decreased in the circulation of CLE patients, possibly secondary to their recruitment to lesional skin[101]. Furthermore, the ratio of CCR5+ to CCR3+ T helper cells in the circulation of CLE patients – representing the ratio of Th1 to Th2 cells – is increased in CLE and is correlated with cutaneous disease activity[116]. Supporting the role of Th1 cells in CLE, the Th1-associated cytokine IFN-γ drives a lupus-like phenotype and skin lesions with features of CLE in mice overexpressing IFN-γ in the epidermis[117]. Similarly, another murine CLE model demonstrated that development of skin lesions was dependent on IFN-γ-producing CD4+ T cells[118]. Accordingly, deletion of IL-12, which promotes IFN-γ production, leads to long-term prevention of skin pathology in the MRL/lpr mouse model of lupus, although the exact cell populations contributing to this finding are yet undetermined[119].

The predominance of Th1 cells in CLE lesions may be temporally linked; one study identified Th1 cells early in DLE lesion development, but elevated numbers of Th2 cells in DLE lesions later in the disease course[120]. As Th2 cells secrete the pro-fibrotic cytokines TGF-β and IL-13, it is possible that this phenotypic switch portends the scarring observed in some DLE patients[121, 122]. Furthermore, Th2 cells can promote B cell activation through secretion of IL-4 and IL-13, which is in alignment with the observation that, like Th2 cells, B cells are predominant in late DLE lesions[67, 120]. In contrast, murine models of CLE that are dependent on persistent presentation of antigen by keratinocytes suggest a slightly different Th1 vs. Th2 scenario. Here, transfer of Th2, but not Th1, cells into non-lethally irradiated mice is required for initiation of cutaneous disease. Intriguingly, antigen-specific Th2 cells from lesional skin become skewed toward a more Th1-like phenotype with capacity to produce IFN-γ in vivo after transfer[123]. The cytokine environment that directs T cell activation to promote CLE requires further study.

Unlike Th1 cells, the role of Th17 cells in CLE has proven more controversial. Some have identified an increase in Th17 cells in lesional DLE[112], while others have suggested no such increase[111]. However, both murine models and human data argue a role for Th17 cells in CLE pathogenesis. P-selectin-deficient mice exhibit a lupus-like phenotype with spontaneous skin manifestations and demonstrate an expanded Th17 compartment in lesional skin[124]. Furthermore, in both human and murine lupus, T cells exhibit activated calcium/calmodulin kinase IV (CaMKIV), a serine-threonine kinase involved in activation of transcription factors downstream of TCR signaling[125, 126]. Activation of this CaMKIV contributes to Th17 differentiation and production of IL-17[127]. Interestingly, treatment of MRL/lpr lupus-prone mice with a CaMKIV inhibitor leads to suppression of spontaneous cutaneous disease[128], suggesting that Th17 cells may be driving manifestations. In human lupus, type I IFNs prime keratinocytes for heightened production of IL-6, a cytokine known to be important for Th17 cell differentiation[129, 130]. Th17 cells correlate with CLE skin disease activity[131], and Ohl et al have demonstrated a positive correlation between IL-17A expression and IFN-α expression in CLE lesions compared to psoriasis[132]. Similarly, Tanasescu et al have identified increased IL-17 expression in both the skin and serum of patients with CLE[133]. Interestingly, increased IL-17A+ lymphocytes in lesional SCLE skin correlated with the presence of anti-Ro antibodies in the serum of these patients, and deletion of Ro52 in mice leads to Th17-driven cutaneous inflammation[58], suggesting a correlation between the Ro antigen, Th17 activation, and IL-17A production[133].

4.2.2.3. Regulatory T cells:

Foxp3+ CD4+ regulatory T cells (Tregs) are critical in maintaining self-tolerance and have been studied in CLE, though their exact contributions to disease manifestations remain unclear. Tregs are numerous in both healthy human and murine skin[134] where they are recruited by hair follicle epithelial cell-derived CCL20[135]. In contrast to healthy skin, one study of lesional human CLE identified significantly fewer Tregs in CLE compared to other chronic inflammatory skin diseases, while yet another study assessing Treg numbers in CLE subtypes identified the greatest decrease in Treg numbers in lesional SCLE and LET, the most photosensitive subtypes of CLE[136]. While some suggest an increase in circulating Tregs in patients with CLE[137], others identify no significant differences in the number of peripheral blood Tregs between CLE and healthy control patients, suggesting that CLE may be marred by tissue-specific but not systemic regulatory deficits[138]. Additional murine evidence for the role of Tregs in CLE come from the Treg-deficient scurfy mouse model, which develop lupus-like disease and spontaneous skin lesions histologically reminiscent of human CLE[139].

UV exposure in healthy patients is known to activate cutaneous Tregs (Reviewed in ref. [140]). In contrast, in murine studies using the NZM2328 spontaneous model of lupus, chronic UV exposure failed to expand or activate Tregs in skin-draining lymph nodes compared to wild-type mice[92]. Furthermore, compared to wild-type animals, Tregs from UV-irradiated NZM2328 mice exhibited diminished suppressive capacity in a type I IFN-dependent fashion[92]. It is unclear whether this decrease in suppressive capacity is due to Treg-intrinsic mechanisms secondary to type I IFN exposure[141], due to effector T cell resistance to suppression[142], or due to aberrantly activated antigen-presenting cells skewing T cells toward pro-inflammatory phenotypes[22, 142–146]. While the exact mechanisms governing this finding are still under investigation, this suggests that the aberrant upregulation of type I IFNs represses Treg number and function, and therefore may be a promising therapeutic target to prevent skin inflammation after UV exposure.

Additionally, some studies have pointed to the potential therapeutic benefit of Treg-targeted treatments. In a study of 60 SLE patients, treatment with low-dose IL-2 expanded Tregs and led to resolution of skin lesions in 11 of the 13 patients with rash[147]. Another exciting preliminary study of one SLE patient with cutaneous disease treated with adoptive transfer of autologous Tregs demonstrated that adoptively transferred Tregs were recruited to the skin and led to maintenance of stable skin disease for the duration of the study[148]. SLE patients with skin disease refractory to antimalarials demonstrate decreased Tregs in the skin, suggesting that decreased tissue-specific regulation may drive refractoriness and may benefit from repletion of Tregs[110]. Taken together, these data suggest that diminished Treg numbers and function may contribute to the pro-inflammatory CLE lesional environment, particularly in photosensitive skin disease. However, precise mechanisms of Treg dysfunction in CLE and the broad utility of Treg-targeted CLE treatment warrant further investigation.

5.2.2.4. γδ T cells

γδ T cells are a subset of T cells that carry an alternative T cell receptor comprised of a γ and δ chain which can be found in the periphery as well as in the skin[149, 150]. While skin-resident γδ T cells have an important role in wound repair and regulation of the skin immune response in healthy skin, these cells exhibit a variety of functions which may contribute to CLE pathogenesis, including antigen presentation, secretion of pro-inflammatory cytokines, interaction with regulatory T cells, and providing B cell help[151, 152]. γδ T cells were found to be enriched in lesional CCLE[153] as well as grossly normal-appearing skin of SLE patients[154]. Lupus-prone MRL/lpr mice which lacked γδ T cells demonstrated more severe systemic disease manifestations, although the effect of γδ T cell knockout on skin manifestations was not reported[155]. γδ T cells have also been studied in the skin of SLE patients whose disease was refractory to antimalarial therapies. In this study, γδ T cells were identified as high producers of IFNα on a single-cell basis, potentially contributing to the aberrant production of IFNs in CLE skin[110]. In patients who responded to antimalarials like hydroxychloroquine and quinacrine, γδ T cells were found to interact with cytotoxic CD8+ T cells, suggestive of the suppressive role of γδ T cells[110]. However, in patients refractory to antimalarials, no such interactions were noted between γδ T cells and CD8+ T cells, and γδ T cells were negatively correlated with CLASI, indicating an intrinsic defect in immunosuppressive mechanisms[110]. Future studies are required to understand the contributions of γδ T cells to CLE pathogenesis.

4.2.3. Monocytes, Dendritic Cells, and Macrophages

Though adaptive immune cells like T cells have long been associated with CLE disease, recent evidence points to myeloid lineage cells as important drivers of cutaneous inflammation. Studies of UV-irradiated wild-type mice identify monocytes as early infiltrators of irradiated skin and a major source of type I IFN production following UV-mediated injury[156]. Accordingly, keratinocyte production of CSF-1, a monocyte/macrophage chemoattractant, was increased in the MRL-Fas/lpr mouse model of lupus following UV exposure[157]. This CSF-1 production was shown to be necessary for the infiltration of macrophages into irradiated skin and subsequent development of CLE-like lesions[157]. In another murine model of cutaneous lupus-like inflammation, lesional skin demonstrated significantly increased inflammatory monocyte infiltration compared with control mice[118]. Interestingly, one study illustrated that when IgG-containing serum from SLE patients or lupus-prone mice was intradermally injected into wild-type mice, skin inflammation was induced in a monocyte-dependent fashion[37]. Even more intriguingly, IgG from lupus serum promoted differentiation of monocytes into monocyte-derived dendritic cells (moDCs), and lupus serum that was depleted of IgG did not induce skin inflammation[37], suggesting that lesion development may be driven by aberrantly activated myeloid APCs. This is in accordance with data from Sarkar et al illustrating that moDCs cultured with conditioned media from UV-exposed lupus keratinocytes exhibited higher activation than moDCs cultured with media from healthy control keratinocytes[22].

In UV-irradiated human lupus skin, monocytes are among the first respondents to UV-mediated damage, and infiltration of monocytes into UV-exposed skin correlates with type I IFN-stimulated gene expression[158]. Lupus monocytes demonstrate enhanced inflammasome activation compared with healthy control monocytes[159], and inflammasome-derived cytokines like IL-1β and IL-18 are increased in CLE lesions compared to healthy control skin[160–162]. While the role of the inflammasome in CLE is understudied, these inflammasome-related cytokines have been implicated in cutaneous inflammatory responses. In murine lupus models, mice deficient in the IL-1 receptor[163] and the IL-18 receptor[164] are protected from development of skin lesions. IL-18 promotes increased MHC class II expression on keratinocytes, which was shown to have functional relevance in skewing of CD4+ T cell responses[165]. Additionally, IL-18 exposure was shown to promote keratinocyte production of inflammatory cell chemoattractants like CXCL10[165] and the pro-apoptotic cytokine TNF-α[161], which can lead to enhanced keratinocyte apoptosis, inflammatory cell infiltration, and propagation of lesion development.

Further highlighting the importance of myeloid inflammation in human CLE, a subset of CLE lesions is characterized by a monocytic gene signature, including genes relevant to moDCs[89]. Studies of photoprovoked human CLE skin have similarly identified an increase in expression of antigen presentation genes in CLE skin versus healthy control[166]. More recently, Billi et al. identified a highly activated, IFN-educated myeloid DC subset that exhibits a gradient of prominence from healthy control to non-lesional to lesional skin, suggesting a role for myeloid DCs in lesion development and propagation[18]. Interestingly, one study demonstrated an increase in myeloid DCs in the skin of SLE patients who are refractory to antimalarial drugs, which are first-line therapeutics for lupus, suggesting that myeloid DCs may also play a role in treatment refractoriness and/or disease persistence[167].

Macrophages are heterogeneous in function and can be polarized toward proinflammatory M1 macrophages or anti-inflammatory M2 macrophages based on tissue microenvironment[168]. Like monocytes and moDCs, macrophages are thought to participate in CLE pathogenesis. Immunohistochemical studies have shown the presence of FasL-expressing CD68+ macrophages near hair follicles in lesional CLE[169]. CD68+ macrophages were also identified in the immune cell infiltrate in CLE lesions following photoprovokation[170], and CD68+ macrophages may contribute to the IFN signature of lesional skin through production of IFNk[110]. Interestingly, in vitro chemotaxis of macrophages was shown to be induced by keratinocytes incubated with anti-DNA antibodies[171], mimicking conditions identified in CLE lesions and suggesting that infiltrating myeloid cells may be important players in CLE lesion development. In one study, CD14+ CD16+ macrophages, in addition to conventional dendritic cells, were identified as among the most activated cell types in CLE lesions and one of the main cell types producing IFNα and IFNβ[110]. Consistent with the hypothesis that macrophages are pro-inflammatory in CLE, depletion of macrophages through genetic knockout of macrophage chemoattractant protein 1 (MCP1)[172], small molecule inhibition of the CSF-1 receptor[173], and neutralization of the pro-inflammatory cytokine MIF[174] abrogated skin disease in murine lupus. In contrast, one trial of a monoclonal antibody against macrophage-colony stimulating factor (M-CSF) in human SLE patients demonstrated that despite reduction in numbers of peripheral blood monocytes, treatment led to no reduction in skin macrophage numbers or activation[175]. This may indicate that the subsets monocytes blocked are not precursors for the tissue-resident macrophage populations probed in this study, suggesting that M-CSF may not be a clinically relevant mechanism by which myeloid cells contribute to inflammation in CLE[175]. Further studies to understand the precise mechanisms of myeloid-driven cutaneous inflammation will be important for understanding the development and progression of CLE lesions.

4.1.2. Langerhans Cells

Langerhans cells (LCs) are a skin-resident dendritic cell population defined by the expression of Langerin (CD207). LCs localize to the epidermis and have roles in the maintenance of skin barrier integrity, uptake and clearance of apoptotic keratinocytes, initiation of T cell responses to skin infections, and the generation of tolerogenic responses to cutaneous self-antigens [176, 177]. The role of LCs in tolerance to self is especially noteworthy in cutaneous manifestations of lupus given that decreased prevalence, irregular distribution, and altered morphology in LCs has long been described in CLE skin [178]. Following inflammatory stimuli, LCs can traffic to draining lymph nodes and are replenished by monocytic cells[179–181]. However, in the context of lupus skin, LCs fail to migrate to skin-draining lymph nodes in animal models and this correlates with severity of skin disease [182]. Inducible ablation of LCs in the MRL/lpr mouse model of systemic lupus led to reduced tolerance to skin self-antigens and exacerbated cutaneous manifestations, but did not lead to increased systemic manifestations, implicating LCs as having a pivotal role in maintenance of tolerance in skin-specific autoimmune responses [182, 183]. Depletion of LCs in a WT mouse model resulted in disruption of the skin barrier and delayed repair to acute skin injury [176].

Alterations in LC function enable photosensitive responses in lupus models. LCs have been shown to be necessary for immunosuppression following UV exposure in healthy skin, with loss of LCs leading to limited Treg induction normally characteristic of a healthy response to UV [184]. Loss of LCs has effects on the transcriptomic profiles of both keratinocytes and skin-resident dendritic epidermal T cells (DETCs), underlining a role in LC crosstalk in the maintenance of the skin immune system [185]. LCs have been further shown to directly mediate keratinocyte survival and skin monocyte recruitment in response to UV exposure through an EGFR-ligand dependent interaction, which is dysregulated in both lupus mouse models and in SLE patients [186]. Berthier et al confirmed this by identifying repression of EGFR signaling pathway genes in CLE skin compared with healthy control[89]. Together, these findings suggest that decreased tolerogenic functions of LCs may facilitate the development of cutaneous autoimmune manifestations and additionally contribute to photosensitivity following UV exposure.

4.2.4. Plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are a specialized subset of dendritic cells that express TLR7 and TLR9 [187]. pDCs can acquire autoantibody-nucleic acid immune complexes through FcγRII-mediated uptake, leading to TLR7/9 ligation, pDC activation, and type I IFN production[115, 188–190]. pDCs can also be triggered to produce type I IFNs in response to apoptotic cells[191, 192] and neutrophil NETs[193, 194], which have been identified in CLE lesions. As such, pDCs have classically been thought to contribute significantly to the robust type I IFN signature of lesional CLE[195]. Presence of pDCs has been identified in lesional CLE but not in healthy control skin[196, 197]. Following UV exposure, pDCs have been shown to traffic to irradiated skin in lupus-prone mice greater than wild-type control mice, possibly secondary to upregulation of the pDC chemoattractant chemerin[198], and sunscreen administration prior to photoprovocation led to diminished pDC recruitment to human lupus-prone skin[198, 199]. In human lupus skin, pDCs have been found to localize either to the dermis or to the DEJ[200]. While pDCs in the dermis co-localize with dermal dendritic cells and are thought to contribute to skewing toward Th1 cells, dermoepidermal pDCs intriguingly express granzyme B and co-localize with perforin-expressing CTLs[200, 201], suggesting that pDCs may play a more direct role in apoptosis in CLE skin.

Another proposed mechanism by which pDCs are stimulated to make IFNα is through interaction with exosomes. Exosomes are extracellular vesicles which carry molecules like proteins, nucleic acids, and lipids, and can be extruded by a variety of live cells[202], and exosomes from SLE patient plasma have been shown to induce IFNα production by PBMCs[203] and pDCs specifically[204]. Keratinocytes are among the cell types documented to release exosomes in response to various stimuli[205–207], and keratinocyte-derived exosomes have been shown to stimulate pDCs to make IFNs as well, suggesting that keratinocytes may be actively communicating with immune cells to drive CLE lesion development[204].

Depletion of pDCs has shown clinical benefit in reducing IFN-regulated gene expression and alleviating cutaneous manifestations of lupus in humans[208], suggesting the importance of pDCs in human CLE pathogenesis. However, while a body of evidence suggests pDCs as the major IFN producers in lesional CLE, recent paradigm-shifting work from Patel et al demonstrate a relative scarcity of type I IFN-producing pDCs in lesional CLE[110]. Instead, their work shows that the major producers of type I IFNs in CLE are myeloid lineage cells[110], which has been shown in murine models as well[156]. This is in line with data suggesting that pDCs adopt an exhausted phenotype late in lupus disease and become defective in producing type I IFNs – findings supported by mouse models and in human lupus[209–211]. Thus, while pDCs may play a role in development of cutaneous inflammation in lupus, the exact roles they play through disease progression should be interrogated.

4.2.5. Neutrophils

Molecular subtyping of CLE lesions suggests that a subset of lesions may express a neutrophilic gene signature[212], and lesional neutrophils display signatures suggestive of production of pro-inflammatory cytokines like IL-18 and type I IFNs[110]. Neutrophils, and particularly a subset called low-density granulocytes (LDGs), can partake in a process called NETosis wherein they release a mass of genetic material, chromatin, and proteolytic enzymes to trap invading pathogens[213]. In lupus, neutrophils can be stimulated to undergo NETosis by immune complexes following priming with type I IFNs[214] as well as exposure to UV light[215]. Netting neutrophils have been hypothesized to drive SLE pathogenesis[216], by inducing pDC production of type I IFNs[193, 194], triggering inflammasome activation[217], and potentially by providing a source of nuclear autoantigens[218]. Supporting this hypothesis, one study identified autoantibodies against NETs in 36% of SLE patients[219], and NETs themselves have been shown to trigger autoantibody production by B cells in lupus[220]. However, others have argued the opposite, suggesting that inhibiting NETosis in lupus-prone mice has little effect on disease pathogenesis[221–223]. In CLE specifically, NETs have been identified in CLE lesions, and the presence of cutaneous LDGs has been associated with skin lesions [193, 223]. NETs are present in varying quantities depending on CLE subset; DLE, ACLE, and tumid lupus lesions exhibit increased NETs compared with SCLE lesions[224]. Interestingly, NETs found in DLE lesional biopsies are decorated with IL-17A and tissue factor, which can trigger the fibrotic activity of fibroblasts and contribute to skin damage in DLE[225]. Furthermore, UV light exposure has been shown to induce NETosis[215], potentially providing a mechanism for initiation of UV-induced CLE flare.

Though some suggest neutrophil depletion has no impact on skin inflammation in murine lupus[226], other mouse models of cutaneous disease in lupus suggest that neutrophils infiltrate lesional skin more than in control mouse skin[118] and may represent early drivers of CLE pathogenesis. A recently-described murine CLE model with knockout of PD-1H demonstrated that neutrophils infiltrate skin prior to disease onset, suggesting that neutrophils may play a role in initiation of CLE lesions[227]. Another CLE-like mouse model has been described in which epidermal injury in lupus-prone NZB/NZW F1 mice induces chronic CLE-like lesions; in this model, there is persistent infiltration of netting neutrophils and activated pDCs, suggesting that NETs may contribute a source of activating TLR7 and TLR9 ligands that cause chronic inflammation[228]. Inhibition of NETosis in the MRL/lpr mouse model protected against formation of skin lesions, further evidencing the role for netting neutrophils in driving cutaneous disease[229]. Interestingly, UV irradiation of wild-type mice leads to kidney infiltration by skin-educated neutrophils, which mediate inflammation transient injury and proteinuria, and contribute to type I IFN signatures, suggesting that neutrophils may serve as a bridge between cutaneous inflammation and systemic disease flare in lupus[230]. Supporting the notion of a connection between skin inflammation and kidney disease, rare subtypes of neutrophilic SLE skin disease have been identified, including bullous SLE (BSLE); one study identified that 50% of patients with BSLE also have lupus nephritis[231].

4.2.6. Mast Cells and Basophils

Mast cells and basophils have also been implicated in CLE pathogenesis. CLE skin exhibits higher mast cell numbers than healthy control skin, and greater mast cell numbers have been identified in sun-exposed versus sun-protected CLE[232]. Mast cells are thought to be recruited to UV-exposed skin through chemotaxis to keratinocyte-derived IL-15 and CCL5[232]. IL-15 can also induce mast cell production of activated matrix metalloproteinases (MMPs)[232], which are associated with CLE lesions and which correlate with cutaneous disease activity[233]. The role of MMPs in CLE is poorly understood; while MMPs may contribute to tissue damage in CLE, MMPs have also been shown to degrade immune complexes[234], which may be protective against further immune activation. Consistent with the idea of mast cells as protective in CLE, one murine lupus model lacking mast cells was shown to have more rapid onset and more severe cutaneous inflammation than their mast cell-sufficient counterparts[235]. Further interrogation of the role of mast cells in CLE is needed to understand their contributions to disease progression.

Like mast cells, basophils have classically been associated with allergic disease, although evidence points to their potential contributions in SLE[236, 237]. In Lyn−/− mice which develop lupus-like illness, basophils have been shown to promote B cell production of anti-nuclear antibodies and subsequent nephritis[236], and circulating basophils from SLE patients have been shown to promote B cell antibody production and Th17 differentiation in vitro[237]. In contrast to healthy control skin, basophils have been shown to infiltrate lesional CLE skin, where they are likely recruited by skin-derived RANTES/CCL5 and MCP-1 production[238]. The precise mechanisms by which basophils contribute to skin disease in lupus is yet undetermined.

4.2.7. Natural Killer Cells

Like CD8+ cytotoxic T cells, natural killer (NK) cells are thought to contribute to keratinocyte apoptosis in CLE. NK cells are less numerous in the peripheral blood of lupus patients, which is thought to be secondary to NK cells trafficking to diseased tissues like the skin[239]. Immunohistochemical staining identifies NK cells at the DEJ in lesional skin, where they co-localize with pDCs and CD8+ T cells and are thought to promote granzyme B-mediated killing of keratinocytes[201]. Furthermore, NKG2D, a receptor that promotes NK cell cytotoxic activity, is present in CDLE lesions[240]. Though NK cells are relatively rare in lesional skin, they are highly activated and may also contribute to the documented IFN signature of CLE skin through production of type I IFNs[110]. Whether and how these cells contribute to lesional inflammation remains unknown and represents an important area for further investigation.

5. PHOTOSENSITIVITY

As noted above, exposure to UV light can trigger immune cell recruitment and skin lesions in murine models and SLE patients. This response, termed photosensitivity, refers to the aberrant inflammatory response to UV light exposure in the skin. Dissecting out the reasons for photosensitivity is a critical endeavor in prevention of CLE.

Components of UV light can be split according to wavelength, defined as UVA (320–400 nm), UVB (280–400 nm), and UVC (100–280 nm). UV exposure, especially in UVA and UVB wavelengths, can trigger both cutaneous and systemic manifestations in lupus [241]. This is in stark contrast to healthy skin, in which an immunosuppressive response to UV exposure is well established [242, 243]. UV-induced immunosuppression occurs in an antigen-specific manner through the limiting of memory and effector T cell responses and the generation of a tolerogenic regulatory T cell (Treg) response [243–245]. This Treg-mediated immunosuppression appears to function through IL-10 production and CTLA-4 signaling [246, 247]. Additional mechanisms of UV-induced immunosuppression include the increased skin infiltration of an IL-10-producing neutrophil population as well as increased activation of regulatory B cells in the skin-draining lymph nodes, which produce IL-10 and suppress dendritic cell activation of T cells [248, 249]. Interestingly, UV induces both a local and systemic type I interferon response, which appears to have a protective role in healthy skin [250]. In contrast, chronic IFN exposure, which is a hallmark of non-lesional SLE skin, have a central role in immune dysregulation and pathology in the context of lupus [251]. Besides IFNs, several additional factors are thought to lead to photosensitivity in lupus, namely dysfunctional DNA repair, increased keratinocyte death, defective clearance of cellular debris, increased autoantigen accessibility and expression, and increased production of cytokines and chemokines.

UV exposure is well-known to induce DNA damage in keratinocytes, which commonly results in the generation of cyclobutane pyrimidine dimers and (6–4) pyrimidine-pyrimidone photoproducts [252, 253]. Reactive oxygen species (ROS) are also generated as a result of UV exposure and cause damage to nucleic acids and proteins, leading to the induction of cell death pathways [254]. One marker of oxidative damage to DNA, 8-hydroxyguanosine (8-OHG), is elevated in CLE lesional skin and has been shown to be immunogenic through a STING-dependent mechanism [218]. Hindered repair of 8-OHG damage to DNA is thought to contribute to inflammation in cutaneous manifestations of lupus, with the 8-oxoguanine DNA glycolase (OGG) DNA repair enzyme found to be decreased in CLE lesions [255]. OGG knockout leads to increased severity of skin disease in the pristane-induced mouse model of lupus, suggesting that DNA damage and dysfunctional DNA repair mechanisms may contribute to lesion generation in photosensitive responses[256].

UV exposure increases autoantigen accessibility in lupus. Specifically, UVB was found to induce the expression of Ro/SSA and La/SSB on the surface of apoptotic keratinocytes, exposing these antigens and allowing for the binding of autoantibodies [51, 257]. Interestingly, circulating anti-Ro and anti-La autoantibodies, in addition to other antibodies against RNA-binding proteins, have associations with photosensitivity in multiple conditions [258–261].Together, these findings indicate autoantigen redistribution as a potential mechanistic player in the aberrant UV response in lupus.

An elevated and sustained induction of pro-inflammatory cytokines and chemokines is characteristic of photosensitive responses in lupus. Both basal expression and UV-induced secretion of type I interferons (IFNs) are increased in CLE skin and are thought to play a central role in enhanced keratinocyte cell death as well as amplification of cytokine induction [13, 20]. Indeed, keratinocytes overexpressing IFN-κ demonstrate increased rates of UVB-mediated apoptosis relative to wild-type cells, while IFN-κ knockout cells have decreased rates[251]. Further, IL-6, TNF-α, and IL-1β are secreted by keratinocytes in response to UV, and for IL-6, this is increased in SLE KCs in an IFN-κ dependent manner[129]. Enhanced production of chemokines such as CXCL9, CXCL10, CXCL11, CXCL12, and CCL27 is also noted in SLE after UVB exposure[14, 24]. These chemokines mediate recruitment of memory and effector T cells and plasmacytoid dendritic cells into the skin, leading to an amplification of inflammatory responses [24, 262]. Further, suppression of Tregs by type I IFNs also contributes to an increase in systemic T cell activation after UVB exposure[92]. Overall, increased DNA damage and cell death, increased autoantigen accessibility, and higher induction of cytokines and chemokines appear to work in conjunction in photosensitive skin to result in the overriding of the immunosuppressive pathways normally induced by UV exposure.

6.1. TOWARDS BETTER THERAPIES FOR CLE

There are no FDA-approved therapies specifically for CLE. Many drugs that are used for inflammatory conditions and SLE do result in some improvement in CLE lesions, especially anti-malarial therapy, which will induce a response in about 50% of patients at risk-acceptable doses[263]. Notably, the rapid accumulation of data in the pathogenic mechanisms of CLE has spurred a large interest in using CLE improvement as an outcome measure in therapeutic trials that involve use of drugs that target pathways and cells described above. Notable trials include TULIP I and TULIP II for anifrolumab, an FDA-approved monoclonal antibody that blocks type I IFN signaling, which exhibited robust improvements in CLE skin activity scores [264]. Other oral inhibitors of JAK/STAT signaling, which block type I IFNs and other cytokine pathways, have also shown promise in early phase clinical trials. Antibodies that target blood DC antigen 2 (BDCA2), which inhibits the capacity to produce IFNα in pDCs, show promise in improving CLE manifestations as well [265]. Targeting lymphocyte transcription factors Ikaros and Aiolos for degradation with a cereblon ligand, iberdomide, has also shown promise for improvement in CLE skin activity in patients with SLE[266]. Continued research into mechanisms of CLE is needed so that beyond drug discovery, we will understand how to use the right drugs in the right patients to ameliorate and prevent CLE.

7.1. CONCLUSIONS

In the last decade, there has been great progress in understanding the drivers of CLE. Studies of non-lesional skin support a pro-inflammatory phenotype that involves chronic hyperproduction of type I IFNs and ongoing innate inflammatory cell recruitment, which primes for aberrant responses to triggers such as UV light. Activation of adaptive immune pathways following these triggers then leads to more antigen-specific responses that can contribute to further tissue injury, fibrosis, and potentially to systemic immune activation. Further research will lead to better, less toxic therapies to prevent and treat CLE lesions.