BACKGROUND

Generalized pustular psoriasis is a life-threatening disease of unknown cause. It is characterized by sudden, repeated episodes of high-grade fever, generalized rash, and disseminated pustules, with hyperleukocytosis and elevated serum levels of C-reactive protein, which may be associated with plaque-type psoriasis.

METHODS

We performed homozygosity mapping and direct sequencing in nine Tunisian multiplex families with autosomal recessive generalized pustular psoriasis. We assessed the effect of mutations on protein expression and conformation, stability, and function.

RESULTS

We identified significant linkage to an interval of 1.2 megabases on chromosome 2q13-q14.1 and a homozygous missense mutation in IL36RN, encoding an interleukin-36-receptor antagonist (interleukin-36Ra), an antiinflammatory cytokine. This mutation predicts the substitution of a proline residue for leucine at amino acid position 27 (L27P). Homology-based structural modeling of human interleukin-36Ra suggests that the proline at position 27 affects both the stability of interleukin-36Ra and its interaction with its receptor, interleukin-1 receptor-like 2 (interleukin-1 receptor-related protein 2). Biochemical analyses showed that the L27P variant was poorly expressed and less potent than the nonvariant interleukin-36Ra in inhibiting a cytokine-induced response in an interleukin-8 reporter assay, leading to enhanced production of inflammatory cytokines (interleukin-8 in particular) by keratinocytes from the patients.

CONCLUSIONS

Aberrant interleukin-36Ra structure and function lead to unregulated secretion of inflammatory cytokines and generalized pustular psoriasis. (Funded by Agence Nationale de la Recherche and Société Française de Dermatologie.).

Psoriasis is a chronic inflammatory disorder of the skin affecting approximately 2% of the world's population. Accumulating evidence has revealed that the IL-23/IL-17/IL-22 pathway is key for development of skin immunopathology. However, the role of keratinocytes and their crosstalk with immune cells at the onset of disease remains poorly understood. Here, we show that IL-36R-deficient (Il36r-/-) mice were protected from imiquimod-induced expansion of dermal IL-17-producing γδ T cells and psoriasiform dermatitis. Furthermore, IL-36R antagonist-deficient (Il36rn-/-) mice showed exacerbated pathology. TLR7 ligation on DCs induced IL-36-mediated crosstalk with keratinocytes and dermal mesenchymal cells that was crucial for control of the pathological IL-23/IL-17/IL-22 axis and disease development. Notably, mice lacking IL-23, IL-17, or IL-22 were less well protected from disease compared with Il36r-/- mice, indicating an additional distinct activity of IL-36 beyond induction of the pathological IL-23 axis. Moreover, while the absence of IL-1R1 prevented neutrophil infiltration, it did not protect from acanthosis and hyperkeratosis, demonstrating that neutrophils are dispensable for disease manifestation. These results highlight a central and unique IL-1-independent role for IL-36 in control of the IL-23/IL-17/IL-22 pathway and development of psoriasiform dermatitis.

Generalized pustular psoriasis (GPP) is a rare and yet potentially lethal clinical variant of psoriasis, characterized by the formation of sterile cutaneous pustules, neutrophilia, fever and features of systemic inflammation. We sequenced the exomes of five unrelated individuals diagnosed with GPP. Nonsynonymous, splice-site, insertion, and deletion variants with an estimated population frequency of <0.01 were considered as candidate pathogenic mutations. A homozygous c.338C>T (p.Ser113Leu) missense substitution of IL36RN was identified in two individuals, with a third subject found to be a compound heterozygote for c.338C>T (p.Ser113Leu) and a c.142C>T (p.Arg48Trp) missense substitution. IL36RN (previously known as IL1F5) encodes an IL-1 family receptor antagonist, which opposes the activity of the IL-36A and IL-36G innate cytokines. Homology searches revealed that GPP mutations alter evolutionarily conserved residues. Homozygosity for the c.338C>T (p.Ser113Leu) variant is associated with an elevated proinflammatory response following ex vivo stimulation with IL36A. These findings suggest loss of function of IL36RN as the genetic basis of GPP and implicate innate immune dysregulation in this severe episodic inflammatory disease, thereby highlighting IL-1 signaling as a potential target for therapeutic intervention.

Psoriasis vulgaris is a common, chronic inflammatory skin disease with a complex etiology involving genetic risk factors and environmental triggers. Here we describe the many known genetic predispositions of psoriasis with respect to immune genes and their encoded pathways in psoriasis susceptibility. These genes span an array of functions that involve antigen presentation (HLA-Cw6, ERAP1, ERAP2, MICA), the IL-23 axis (IL12Bp40, IL23Ap19, IL23R, JAK2, TYK2), T-cell development and T-cells polarization (RUNX1, RUNX3, STAT3, TAGAP, IL4, IL13), innate immunity (CARD14, c-REL, TRAF3IP2, DDX58, IFIH1), and negative regulators of immune responses (TNIP1, TNFAIP3, NFKBIA, ZC3H12C, IL36RN, SOCS1). The contribution of some of these gene products to psoriatic disease has also been revealed in recent years through targeting of key immune components, such as the Th17/IL-23 axis which has been highly successful in disease treatment. However, many of the genetic findings involve immune genes with less clear roles in psoriasis pathogenesis. This is particularly the case for those genes involved in innate immunity and negative regulation of immune specific pathways. It is possible that risk alleles of these genes decrease the threshold for the initial activation of the innate immune response. This could then lead to the onslaught of the pathogenic adaptive immune response known to be active in psoriatic skin. However, precisely how these various genes affect immunobiology need to be determined and some are speculated upon in this review. These novel genetic findings also open opportunities to explore novel therapeutic targets and potentially the development of personalized medicine, as well as discover new biology of human skin disease.

Generalized pustular psoriasis (GPP) is a rare inflammatory skin disease that can be life-threatening. Recently, it has been reported that familial GPP is caused by homozygous or compound heterozygous mutations of IL36RN. However, the majority of GPP cases are sporadic and it is controversial whether IL36RN mutations are a causative/predisposing factor for sporadic GPP. We searched for IL36RN mutations in two groups of GPP patients in the Japanese population in this study: GPP without psoriasis vulgaris (PV), and GPP with PV. Eleven cases of GPP without PV (GPP alone) and 20 cases of GPP accompanied by PV (GPP with PV) were analyzed. Surprisingly, 9 out of 11 cases of GPP alone had homozygous or compound heterozygous mutations in IL36RN. In contrast, only 2 of 20 cases of GPP with PV had compound heterozygous mutations in IL36RN. The two cases of GPP with PV who had compound heterozygous mutations in IL36RN are siblings, and both cases had PV-susceptible HLA-A*0206. We determined that GPP alone is a distinct subtype of GPP and is etiologically distinguished from GPP with PV, and that the majority of GPP alone is caused by deficiency of the interleukin-36 receptor antagonist due to IL36RN mutations.

Generalized pustular psoriasis (GPP) is a rare, potentially life threatening, and aggressive form of psoriasis, which is characterized by sudden onset with repeated episodic skin inflammation leading to pustule formation. Familial GPP is known to be caused by recessively inherited mutations in the IL36RN gene, which encodes interleukin 36 receptor antagonist (IL-36Ra). In this article, we performed mutation analysis of the IL36RN gene in 14 Japanese patients with GPP, and identified mutations in two of these patients analyzed. One patient was compound heterozygous for mutations c.115+6T>C and c.368C>G (p.Thr123Arg), whereas the other carried compound heterozygous mutations c.28C>T (p.Arg10*) and c.115+6T>C in the IL36RN gene. Expression studies using total RNA from the patients' skin revealed that the mutation c.115+6T>C resulted in skipping of exon 3, leading to a frameshift and a premature termination codon (p.Arg10Argfs*1). The protein structure analysis suggested that the missense mutation p.Thr123Arg caused misfolding and instability of IL-36Ra protein. In vitro studies in cultured cells showed impaired expression of the p.Thr123Arg mutant IL-36Ra protein, which failed to antagonize the IL-36 signaling pathway. Our data further underscore the critical role of IL36RN in pathogenesis of GPP.

YM is the first son of Tunisian consanguineous parents who developed, at 2 weeks of life, an erythematous and scaly eruption, with subsequent rapid evolution toward generalized pustular psoriasis. Afterward, cutaneous flares of diffuse erythematous rash and pustules involving the whole body appeared, with a once weekly periodicity. Intense irritability was present during flares without fever. Moreover, since 1 month of age the infant presented with diarrhea, dysphagia, and reduced feeding rate, with failure to thrive. Laboratory tests during acute flares showed marked leukocytosis, thrombocytosis, and anemia without C-reactive protein elevation. Skin biopsy and clinical presentation were consistent with pustular psoriasis; nevertheless, the patient did not respond to high-potency topical corticosteroids and retinoid acid. As the patient presented with repeated skin flares early after birth, as well as serious constitutional distress with failure to thrive, an autoinflammatory syndrome like interleukine-1-receptor antagonist deficiency or interleukin-36-receptor antagonist deficiency (DITRA) was considered. The hypothesis was reinforced by parental consanguinity, and absence of skin lesion improvement under standard topical treatment. Genetic analyses showed a homozygous mutation in the IL36RN gene (L27P), which represents the same mutation recently described in DITRA patients. At age 6 months we started treatment with the recombinant interleukin-1 receptor antagonist anakinra with efficacy both on constitutional symptoms and skin involvement. DITRA is a recently described autoinflammatory disease characterized by repeated flares of generalized pustular psoriasis, high fever, asthenia, and systemic inflammation. We report herein the first exhaustive clinical description of an infant with DITRA who was successfully treated with anakinra.

BACKGROUND

In present study we performed whole transcriptome analysis in plaque psoriasis patients and compared lesional skin with non-lesional skin and with the skin from healthy controls. We sequenced total RNA from 12 lesional (LP), 12 non-lesional (NLP) and from 12 normal (C) skin biopsies.

RESULTS

Compared with previous gene expression profiling studies we had three groups under analysis - LP, NLP and C. Using NLP samples allows to see the transcriptome of visually normal skin from psoriasis patient. In LP skin S100A12, S100A7A, LCE3E, DEFB4A, IL19 were found up regulated. In addition to already these well-described genes, we also found several other genes related to psoriasis. Namely, KLK9, OAS2, OAS3, PLA2G, IL36G, IL36RN were found to be significantly and consistently related to the psoriatic lesions and this finding is supported also by previous studies. The genes up-regulated in the LP samples were related to the innate immunity, IL17 and IL10 networks. In NLP samples innate immunity and IL17 network were activated, but activation of IL10 network was not evident. The transcriptional changes characteristic in the NLP samples can be considered as a molecular signature of "dormant psoriasis".

CONCLUSIONS

Taken together, our study described the transcriptome profile characteristic for LP and NLP psoriatic skin. RNA profile of the NLP skin is in between the lesional and healthy skin, with its own specific pattern. We found that both LP and NLP have up-regulated IL17 network, whereas LP skin has up regulated IL10 related cytokines (IL19, IL20, IL24). Moreover, IL36G and IL36RN were identified as strong regulators of skin pathology in both LP and NLP skin samples, with stronger influence in LP samples.

BACKGROUND

The term pustular psoriasis indicates a group of severe skin disorders characterized by eruptions of neutrophil-filled pustules. The disease, which often manifests with concurrent psoriasis vulgaris, can have an acute systemic (generalized pustular psoriasis [GPP]) or chronic localized (palmoplantar pustulosis [PPP] and acrodermatitis continua of Hallopeau [ACH]) presentation. Although mutations have been uncovered in IL36RN and AP1S3, the rarity of the disease has hindered the study of genotype-phenotype correlations.

OBJECTIVE

We sought to characterize the clinical and genetic features of pustular psoriasis through the analysis of an extended patient cohort.

METHODS

We ascertained a data set of unprecedented size, including 863 unrelated patients (251 with GPP, 560 with PPP, 28 with ACH, and 24 with multiple diagnoses). We undertook mutation screening in 473 cases.

RESULTS

Psoriasis vulgaris concurrence was lowest in PPP (15.8% vs 54.4% in GPP and 46.2% in ACH, P < .0005 for both), whereas the mean age of onset was earliest in GPP (31.0 vs 43.7 years in PPP and 51.8 years in ACH, P < .0001 for both). The percentage of female patients was greater in PPP (77.0%) than in GPP (62.5%; P = 5.8 × 10-5). The same applied to the prevalence of smokers (79.8% vs 28.3%, P < 10-15). Although AP1S3 alleles had similar frequency (0.03-0.05) across disease subtypes, IL36RN mutations were less common in patients with PPP (0.03) than in those with GPP (0.19) and ACH (0.16; P = 1.9 × 10-14 and .002, respectively). Importantly, IL36RN disease alleles had a dose-dependent effect on age of onset in all forms of pustular psoriasis (P = .003).

CONCLUSIONS

The analysis of an unparalleled resource revealed key clinical and genetic differences between patients with PPP and those with GPP.

Generalized pustular psoriasis is a distinct type of psoriasis characterized by recurrent febrile attacks with disseminated subcorneal pustules on generalized skin rashes. Recently, homozygous and compound heterozygous mutations of the IL36RN gene, which encodes the anti-inflammatory cytokine interleukin (IL)-36 receptor antagonist, were identified in familial and sporadic cases of various ethnicities with generalized pustular psoriasis. Here we report a 39-year-old Japanese male patient who had suffered from repeated attacks of generalized pustular psoriasis since infancy with intervals of several years. At presentation, erythematous lesions with a few pustules were found only on some parts of the body and controlled with topical corticosteroids. An analysis of the IL36RN gene revealed compound heterozygous mutations of c.28C>T and c.368C>T. While the former mutation causing the premature termination p.Arg10X is recurrent in Japanese cases, the latter missense mutation causing p.Thr123Met substitution is novel, but another mutation in the same position has been reported in one Japanese case. Our report further supports the presence of the Japanese-specific hot spots in the IL36RN gene, 28C and 368C, and suggests the functional significance of Thr123. This special type of generalized pustular psoriasis caused by IL36RN mutations has been designated as deficiency for IL-36 receptor antagonist, a new hereditary autoinflammatory disease, and its phenotypes have emerged to include other related pustular disorders, palmoplantar pustulosis, acrodermatitis continua of Hallopeau, and acute generalized exanthematous pustulosis. The genetic analysis of the cases with these diseases would be important for establishment and application of the specific treatments targeting the IL-36 signaling.

IL-36 cytokines have recently emerged as mediators of inflammation in autoimmune conditions including psoriasis vulgaris (PsV) and generalized pustular psoriasis (GPP). This study used RNA-seq to profile the transcriptome of primary epidermal keratinocytes (KCs) treated with IL-1B, IL-36A, IL-36B, or IL-36G. We identified some early IL-1B-specific responses (8 h posttreatment), but nearly all late IL-1B responses were replicated by IL-36 cytokines (24 h posttreatment). Type I and II interferon genes exhibited time-dependent response patterns, with early induction (8 h) followed by no response or repression (24 h). Altogether, we identified 225 differentially expressed genes (DEGs) with shared responses to all 4 cytokines at both time points (8 and 24 h). These involved upregulation of ligands (IL1A, IL1B, and IL36G) and activating proteases (CTSS) but also upregulation of inhibitors such as IL1RN and IL36RN. Shared IL-1B/IL-36 DEGs overlapped significantly with genes altered in PsV and GPP skin lesions, as well as genes near GWAS loci linked to autoimmune and autoinflammatory diseases (e.g., PsV, psoriatic arthritis, inflammatory bowel disease, and primary biliary cholangitis). Inactivation of MyD88 adapter protein using CRISPR/Cas9 completely abolished expression responses of such DEGs to IL-1B and IL-36G stimulation. These results provide a global view of IL-1B and IL-36 expression responses in epidermal KCs with fine-scale characterization of time-dependent and cytokine-specific response patterns. Our findings support an important role for IL-1B and IL-36 in autoimmune or autoinflammatory conditions and show that MyD88 adaptor protein mediates shared IL-1B/IL-36 responses.

Generalized pustular psoriasis (GPP) is often present in patients with existing or prior psoriasis vulgaris (PV; "GPP with PV"). However, cases of GPP have been known to arise without a history of PV ("GPP alone"). There has long been debate over whether GPP alone and GPP with PV are distinct subtypes that are etiologically different from each other. We recently reported that the majority of GPP alone cases is caused by recessive mutations of IL36RN. In contrast, only a few exceptional cases of GPP with PV were found to have recessive IL36RN mutations. Very recently, we also reported that CARD14 p.Asp176His, a gain-of-function variant, is a predisposing factor for GPP with PV; in contrast, the variant is not associated with GPP alone in the Japanese population. These results suggest that GPP alone is genetically different from GPP with PV. IL36RN mutations are also found in some patients with severe acute generalized exanthematous pustulosis, palmar-plantar pustulosis, and acrodermatitis continua of hallopeau. CARD14 mutations and variants are causal or disease susceptibility factors of PV, GPP, or pityriasis rubra pilaris, depending on the mutation or variant position of CARD14. It is clinically important to analyze IL36RN mutations in patients with sterile pustulosis. For example, identifying recessive IL36RN mutations leads to early diagnosis of GPP, even at the first episode of pustulosis. In addition, individuals with IL36RN mutations are very susceptible to GPP or GPP-related generalized pustulosis induced by drugs (e.g., amoxicillin), infections, pregnancy, or menstruation.

Given that entheses are sites of high mechanical stress that concentrate the forces of large contracting muscles down onto a small footprint of bone contact, it was recognized nearly 60 decades ago that stress and injury at such sites may play a role in the pathogenesis of mechanically related enthesopathy. In recent years, the role of mechanical stress and its related consequences on inflammatory enthesitis have also been recognized. Clinical imaging studies and experimental animal models of spondyloarthropathy including tumor necrosis factor (TNF) transgenic models and interleukin (IL)-23 overexpression systems are associated with a primary enthesitis with disease subsequently spreading to adjacent joint structures including the synovium and bone. Joint mechanical stress, without discernible microdamage or injury, leads to spondyloarthritis (SpA) in a TNF transgenic model. Normal-aged human entheses often demonstrate microdamage, but it is unclear whether an abnormal response to mechanical stress alone or the need for stress-induced microdamage is involved in human disease initiation. Clinically, the contribution of mechanical stress to SpA including psoriatic arthritis (PsA) helps conceptualize the disease in a new way and provides obvious mechanistic links to skin and nail Koebner responses. It also offers novel epidemiological explanations for why PsA develops in subjects with high body mass indices most typically in the fourth and fifth decades. Molecularly, the monogenic forms of SpA including caspase recruitment domain-containing protein 14 (CARD14) and IL36RN mutations have site-specific expression of mutated proteins in the skin, thus offering a direct molecular link between local inflammation-related pathway dysregulation and local stress or injury in disease causation. Given that many of the pathways that govern both immunity and mechanical stress including extracellular-signal-regulated kinase (ERK) and mitogen-activated protein kinase (MAPK) are shared, it may be difficult to develop strategies that selectively target mechanical stress-related pathways. However, occupational- and obesity-related factors may be potentially modifiable in susceptible individuals to prevent or ameliorate disease.

BACKGROUND

Rare variants in the genes IL36RN, CARD14 and AP1S3 have been identified to cause or contribute to pustular skin diseases, primarily generalized pustular psoriasis (GPP).

OBJECTIVE

To better understand the disease relevance of these genes, we screened our cohorts of patients with pustular skin diseases [primarily GPP and palmoplantar pustular psoriasis (PPP)] for coding changes in these three genes. Carriers of single heterozygous IL36RN mutations were screened for a second mutation in IL36RN.

METHODS

Coding exons of IL36RN, CARD14 and AP1S3 were sequenced in 67 patients - 61 with GPP, two with acute generalized exanthematous pustulosis and four with acrodermatitis continua of Hallopeau. We screened IL36RN and AP1S3 for intragenic copy-number variants and 258 patients with PPP for coding changes in AP1S3. Eleven heterozygous IL36RN mutations carriers were analysed for a second noncoding IL36RN mutation. Genotype-phenotype correlations in carriers/noncarriers of IL36RN mutations were assessed within the GPP cohort.

RESULTS

The majority of patients (GPP, 64%) did not carry rare variants in any of the three genes. Biallelic and monoallelic IL36RN mutations were identified in 15 and five patients with GPP, respectively. Noncoding rare IL36RN variants were not identified in heterozygous carriers. The only significant genotype-phenotype correlation observed for IL36RN mutation carriers was early age at disease onset. Additional rare CARD14 or AP1S3 variants were identified in 15% of IL36RN mutation carriers.

CONCLUSIONS

The identification of IL36RN mutation carriers harbouring additional rare variants in CARD14 or AP1S3 indicates a more complex mode of inheritance of pustular psoriasis. Our results suggest that, in heterozygous IL36RN mutation carriers, there are additional disease-causing genetic factors outside IL36RN.

Homozygous or compound heterozygous IL36RN gene mutations underlie the pathogenesis of psoriasis-related pustular eruptions including generalized pustular psoriasis, palmoplantar pustular psoriasis, acrodermatitis continua of Hallopeau, and acute generalized exanthematous pustular eruption. We identified two unreported IL36RN homozygous mutations (c.41C>A/p.Ser14X and c.420_426del/p.Gly141MetfsX29) in patients with familial generalized pustular psoriasis. We analyzed the impact of a spectrum of IL36RN mutations on IL-36 receptor antagonist protein by using site-directed mutagenesis and expression in HEK293T cells. This enabled us to differentiate null mutations with complete absence of IL-36 receptor antagonist (the two previously unreported mutations, c.80T>C/p.Leu27Pro, c.28C>T/p.Arg10X, c.280G>T/p.Glu94X, c.368C>G/p.Thr123Arg, c.368C>T/p.Thr123Met, and c.227C>T/p.Pro76Leu) from mutations with decreased (c.95A>G/p.His32Arg, c.142C>T/p.Arg48Trp, and c.308C>T/p.Ser113Leu) or unchanged (c.304C>T/p.Arg102Trp and c.104A>G/p.Lys35Arg) protein expression. Functional assays measuring the impact of mutations on the capacity to repress IL-36-dependent activation of the NF-κB pathway showed complete functional impairment for null mutations, whereas partial or no impairment was observed for other mutations considered as hypomorphic. Finally, null mutations were associated with severe clinical phenotypes (generalized pustular psoriasis, acute generalized exanthematous pustular eruption), whereas hypomorphic mutations were identified in both localized (palmoplantar pustular psoriasis, acrodermatitis continua of Hallopeau) and generalized variants. These results provide a preliminary basis for genotype-phenotype correlation in patients with deficiency of the IL-36Ra (DITRA), and suggest the involvement of other factors in the modulation of clinical expression.

As IL36RN mutations are a cause of generalized pustular psoriasis (GPP), three recent investigations attempted to correlate the IL36RN genotype with GPP clinical presentations. These studies found that IL36RN mutations account for only a fraction of GPP cases presenting with concomitant psoriasis vulgaris (PV; common or typical psoriasis). Pathogenic alleles were also found in control populations, indicating that environmental triggers and/or modifier genes may contribute to the disease.

Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, β and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.