The

n e w e ng l a n d j o u r na l

of

m e dic i n e

original article

Interleukin-36Receptor Antagonist Deficiency and Generalized Pustular Psoriasis

Slaheddine Marrakchi, M.D., Ph.D., Philippe Guigue, Ph.D., Blair R. Renshaw, B.S., Anne Puel, Ph.D., Xue-Yuan Pei, Ph.D., Sylvie Fraitag, M.D., Jihen Zribi, M.D., Elodie Bal, Ph.D., Cline Cluzeau, Ph.D., Maya Chrabieh, M.Sc., Jennifer E. Towne, Ph.D., Jason Douangpanya, B.A., Christian Pons, M.Sc., Sourour Mansour, M.Sc., Valrie Serre, Ph.D., Hafedh Makni, M.D., Nadia Mahfoudh, M.D., Faiza Fakhfakh, Ph.D., Christine Bodemer, M.D., Ph.D., Josu Feingold, Ph.D., Smail Hadj-Rabia, M.D., Ph.D., Michel Favre, Ph.D., Emmanuelle Genin, Ph.D., Mourad Sahbatou, Ph.D., Arnold Munnich, M.D., Ph.D., Jean-Laurent Casanova, M.D., Ph.D., John E. Sims, Ph.D., Hamida Turki, M.D., Ph.D., Herv Bachelez, M.D., Ph.D., and Asma Smahi, Ph.D.

A bs t r ac t
From the Department of Dermatology and the Laboratory of Immunology, Hedi Chaker Hospital, Sfax University, Sfax, Tunisia (S. Marrakchi, J.Z., H.M., N.M., F.F., H.T.); the Laboratory of Genetics, INSERM Unit 781 (S. Marrakchi, P.G., S.F., E.B., C.C., S. Mansour, V.S., C.B., J.F., S.H.-R., A.M., A.S.) and the Laboratory of Human Genetics of Infectious Diseases (A.P., M.C., J.-L.C.), INSERM Unit 980, Sorbonne Paris Cit Universit ParisDescartes, Hpital NeckerEnfants Malades; the Department of Pathology, Hpital NeckerEnfants Malades (S.F.); Institut Pasteur, Unit de Gntique, Papillomavirus et Cancer Humain (C.P., M.F.); INSERM UMRS 946, Institut Universitaire dHmatologie (E.G.), and INSERM Unit 976, Assistance Publique Hpitaux de Paris Hpital Saint-Louis (H.B.), Sorbonne Paris Cit Universit ParisDiderot; and Fondation Jean Dausset, Centre dEtude du Polymorphisme Humain (M.S.) all in Paris; the Department of Inflammation Research, Amgen, Seattle (B.R.R., J.E.T., J.D., J.E.S.); the Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (X.-Y.P.); and St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller University, New York (J.-L.C.). Address reprint requests to Dr. Smahi at Hpital Necker, INSERM U781, 149, Rue de Svres, 75015 Paris, France, or at asma.smahi@inserm.fr. Drs. Marrakchi and Guigue contributed equally to this article. N Engl J Med 2011;365:620-8.
Copyright 2011 Massachusetts Medical Society.

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-36receptor 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 receptorlike 2 (interleukin-1 receptorrelated 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 Socit Franaise de Dermatologie.)
n engl j med 365;7 nejm.org august 18, 2011

620

The New England Journal of Medicine Downloaded from nejm.org on November 6, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.

Interleukin-36Receptor Antagonist and Psoriasis

soriasis is a chronic inflammatory skin disease affecting 2 to 3% of persons of European descent.1 Psoriasis vulgaris, the most common form of the disease, accounts for 80% of cases and has a strong, albeit complex, genetic component.2 Numerous chromosomal loci have been implicated in genomewide association studies, but analyses of these loci have yielded only a few candidate genes, which mediate inflammatory cytokine signaling and adaptive immune responses.3-5 The disease follows mendelian transmission in a small minority of families. Generalized pustular psoriasis is a life-threatening, multisystemic inflammatory disease involving repeated flare-ups of sudden onset, which are characterized by a diffuse, erythematous, pustular rash combined with high-grade fever, general malaise, and extracutaneous organ involvement.6-9 Age at onset is variable. Generalized pustular psoriasis has been included within the spectrum of psoriasis because it is often observed in conjunction with psoriasis vulgaris and because it involves the recruitment of T cells and neutrophils.6-8,10-12 The purpose of this study was to identify genetic abnormalities responsible for familial generalized pustular psoriasis.

means of GeneScan, version 3.7, and genotyping software. Parametric multipoint linkage analysis was performed with the use of Merlin software, version 1.1.2, assuming a full recessive model. The age of the most recent common ancestor of the patients who carried the mutation for generalized pustular psoriasis was estimated with the use of the ESTIAGE program.13 See the Supplementary Appendix, available with the full text of this article at NEJM.org., for details regarding sequencing, quantitative real-time PCR analysis, human interleukin-36receptor antagonist (interleukin36Ra) L27P modeling, construction of interleukin-36Ra plasmids and their expression of in human embryonic kidney [HEK] 293T cells and HaCat cells, keratinocyte culture and immortalization, measurement of interleukin-8 by enzyme-linked immunosorbent assay (ELISA) in the patients serum and supernatants of stimulated keratinocytes, generation of recombinant human interleukin-36 proteins and an interleukin-36responsive Jurkat reporter cell line, and immunohistochemical analyses.

R e sult s
Clinical Characteristics of the Patients

Me thods
Affected Families

Members of nine Tunisian multiplex families with autosomal recessive generalized pustular psoriasis participated in the study, which was approved by the ethics committee at Hpital Necker. All patients or their parents provided written informed consent to take part in the study.
Genetic Analysis

We carried out a whole-genome scan with the use of an Affymetrix GeneChip Human Mapping 10K 2.0 Array (Atlas Biolabs). DNA was extracted from patients blood leukocytes with the use of the Illustra DNA extraction kit Nucleon BACC3 (GE Healthcare), according to the manufacturers instructions. Microsatellite DNA markers from chromosome region 2q12-q13 (UCSC Genome Browser) were amplified by means of a polymerase-chainreaction (PCR) assay, with primers labeled with fluorescent dye, and were analyzed by means of electrophoresis on a 310 Genetic Analyzer (Applied Biosystems); genotypes were determined by

The pedigrees of the nine families that agreed to participate in this study are shown in Figure 1A; the pattern of inheritance was consistent with autosomal recessive disease. Table S1 in the Supplementary Appendix provides details of the clinical features of the 16 affected family members. All families originated from southern Tunisia and reported varying degrees of consanguinity, with the exception of Family 5, in which the parents reported no consanguinity. All affected persons fulfilled the clinical and biologic criteria for generalized pustular psoriasis (Fig. 1B, and Table S1 in the Supplementary Appendix), defined by repeated flares of sudden onset, with the typical combination of signs: a diffuse, erythematous skin eruption characterized by rapid coverage with pustules, along with high-grade fever (40 to 42C), asthenia, marked leukocytosis, and elevated serum levels of C-reactive protein. In the aggregate, the families reported five deaths after septicemia. Disease developed in 12 of the affected persons during childhood (between 1 week and 11 years of age) and in 4 affected persons after they reached adulthood; 2 of these 4 had generalized pustular psoriasis dur-

n engl j med 365;7

nejm.org

august 18, 2011

621

The New England Journal of Medicine Downloaded from nejm.org on November 6, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.

The

n e w e ng l a n d j o u r na l

of

m e dic i n e

A
Family 1 I
1 2

Family 2 I
1 2 2 2 1 7 8 9 2 3 3 3 4 4 3 4 4

Family 3 I
1 2 1 2 3 2 1 2 4

II
1 2 2 1 3 3 2 3 4 5 6 4

II
1

II III
1 5 6 1 2 1

III
1 4

III IV

IV V
1 2

IV V

Family 4 I
1 2

Family 5 I
1 2 1

Family 6 I
1 2 3 2 3 2 1 2 3 4 34 34 5 6 4 4 5 5 6 5 6 7 8 9 7 10 6 7

II
1 2 3 2 4

II

II
1

III
1 2 1

III
1

IV V
1 2 3

IV V
1 2

Family 7 I
1 2

Family 8 I
1 2

Family 9 I
1 2 2 1 3 2 1 2 3 4 4

II
1 2 1 3 2 4

II
1 2 1 3 2 4

II
1

III IV
1 2 3 4

III IV
1 2 3 4

III IV

B a b c d e f

622

n engl j med 365;7

nejm.org

august 18, 2011

The New England Journal of Medicine Downloaded from nejm.org on November 6, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.

Interleukin-36Receptor Antagonist and Psoriasis

Figure 1 (facing page). Pedigrees of Nine Families Affected with Generalized Pustular Psoriasis and Clinical Characteristics of Affected Family Members. The pedigrees shown in Panel A suggest autosomal recessive segregation in the nine families affected with generalized pustular psoriasis. Solid symbols indicate affected family members, open symbols unaffected members, squares male family members, circles female family members, slashes deceased family members, and double lines consanguinity. Panel B shows representative clinical features in patients with generalized pustular psoriasis. Subpanels a, b, and c show diffuse erythematous and pustular eruptions on the trunk in patients from Families 1, 7, and 3, respectively; subpanel d shows a more localized, erythematosquamous plaque on the forearm of a member of Family 1, which persisted between attacks. Subpanel e shows the superficial desquamating stage in the legs, which follows the initial erythematous and pustular eruption, in a member of Family 6. Subpanels f and g show crusted erythematous and pustular lesions of the leg and scalp, respectively, in two patients (from Families 2 and 6, respectively) who also had psoriasis vulgaris. Subpanel h shows the erythrodermal form of generalized pustular psoriasis in a pregnant woman from Family 8 in whom impetigo herpetiformis was also diagnosed. Subpanels i and j show chronic flexural psoriatic lesions in two patients from Family 2. Subpanel k shows, in a member of Family 1, benign migratory glossitis mixed with the pustular lesions of generalized pustular psoriasis a mixture that was observed in most of the patients. Subpanel l shows, in a member of Family 5, acral pustular lesions of the digits with partial nail destruction, representative of the acrodermatitis continua form of generalized pustular psoriasis.

Figure 2. Molecular Modeling of Normal and Mutant Interleukin-36Receptor Antagonist (Interleukin-36Ra). Panel A shows a model of human wild-type interleukin-36Ra (white) superimposed on the L27P variant (blue). Panel B shows a close-up view of the mutated region with L27P and H22 in stick presentation. Panels C, D, and E show the surface presentation of the distribution of the electric charge of mouse interleukin-36Ra, human wild-type interleukin-36Ra, and the human L27P variant, respectively. Arrows point to the regions in which the charge distribution is altered.

ing pregnancy and received a diagnosis of impetigo herpetiformis. The frequency of flares was highly variable from one person to another, with some having chronic involvement, manifested as erythematous chronic plaques without pustules. Flares were associated with viral or bacterial infections in 12 patients, withdrawal of retinoid therapy in 7, menstruation in 6, and pregnancy in 2. Histopathological studies performed in 8 patients showed typical spongiform pustules, acanthosis with elongation of rete ridges, and parakeratosis in the stratum corneum (Fig. S1 in the Supplementary Appendix). Immunostaining showed infiltration of the skin by CD8 T cells, CD3 T cells, and macrophages (Fig. S1 in the Supplementary Appendix).
Linkage Analysis and Screening of Candidate Genes

We genotyped chromosomal markers in the members of Family 1 and identified an 11-megabase

(Mb) region of homozygosity on chromosome 2q13-q14 (Fig. S2 in the Supplementary Appendix). Subsequently, we observed cosegregation of the disease and a common haplotype of 1.2 Mb (contained within the 11-Mb region) in the other eight families, suggesting a founder effect (Zmax = 13 at = 0) (Fig. S2 and Table S2 in the Supplementary Appendix). Nine genes encoding members of the interleukin-1 family reside at this locus. Because these genes encode proteins known to have a role in the innate immune response in the skin,14,15 we sequenced their exons and exonintron boundaries. We identified a homozygous variant in IL36RN that was predicted to result in the substitution of proline for leucine at amino acid position 27 (L27P) of the interleukin-36Ra protein (Fig. S3 and Fig. S4 in the Supplementary Appendix) and was present in all affected persons whom we analyzed (Fig. 1A). We did not observe mutations in the other eight genes (see the Supplementary Appendix) on sequencing DNA and also complementary DNA obtained by reverse transcription of RNA (i.e., we analyzed both genes and their messenger RNA [mRNA] transcripts). On the basis of the size of the homozygous region in each of the families, we estimated that the most recent common ances-

n engl j med 365;7

nejm.org

august 18, 2011

623

The New England Journal of Medicine Downloaded from nejm.org on November 6, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.

The

n e w e ng l a n d j o u r na l

of

m e dic i n e

Figure 3 (facing page). Functional Effects of the L27P Mutation in the Interleukin-36Receptor Antagonist Gene. Panel A shows the expression of interleukin-36receptor antagonist (interleukin-36Ra) variants in HEK 293T cells as assessed by Western blot analysis. Myc-tagged ectodysplasin-A receptorassociated adapter protein (Edd) was used as an internal control and was cotransfected with Myc-tagged interleukin-36Ra. Two points are shown for each variant. The expression of the interleukin-36Ra L27P variant is severely impaired, as compared with the wild-type, L27R, and L27A variants. The same results were observed in HaCat cells. Panel B shows the inhibition of interleukin-36 by wild-type and mutant interleukin-36Ra. Truncated human interleukin-36 protein (5 ng per milliliter) was used to stimulate a cell line that overexpresses the interleukin-36 receptor (interleukin-1 receptorlike 2) and carries a reporter plasmid in which the luciferase gene is driven by the interleukin-8 promoter. Inhibitor on the x axis refers to the concentration of each form of interleukin-36Ra used. The y axis shows the light output from the luciferase encoded by the reporter plasmid, expressed in relative light units (RLU). Interleukin-36Ra (previously known as huIL-1F5) and interleukin-36RaL27P (previously known as huIL-1F5L27P) are the wild-type and mutant forms of human IL-1F5, respectively. The half-maximal inhibitory concentrations (IC50) were 0.074 and 0.64 g per milliliter for wild-type and mutant interleukin-36Ra, respectively. Panel C shows the thermal stability of interleukin-36Ra proteins. Wild-type interleukin-36Ra and mutant interleukin-36Ra (100 g per milliliter in phosphatebuffered saline) were heated for 15 minutes at the indicated temperatures, chilled on ice, and then added to a reporter assay similar to the one described for Panel B at their IC90 concentrations. The graph shows a loss of antagonistic activity with increasing temperature. These results are representative of two independent experiments in which two separate batches of interleukin-36Ra protein were used. The graph on the left in Panel D shows interleukin-8 production, which was assessed by means of an enzyme-linked immunosorbent assay (ELISA) with the use of immortalized keratinocytes from two patients (Patient V.2 from Family 1 and Patient III.4 from Family 2) and from four age-matched controls, which were stimulated with 50 ng of truncated human interleukin-36, interleukin-36, or interleukin-36 cytokines per milliliter for 24 hours. The graph on the right in Panel D represents interleukin-8 production after the stimulation of keratinocytes from patients and controls with 2.5 ng of polyinosinicpolycytidylic acid (poly[I:C]) per milliliter and 10 ng of interleukin-1 per milliliter for 24 hours. The mean (SD) values are shown from three independent experiments in which each stimulation was performed in triplicate. Panel E shows the production of interleukin-8, assessed by means of ELISA, in blood samples from five patients during inflammatory flares, as compared with four controls. Panel F shows the results of immunohistochemical studies in one of four patients with generalized pustular psoriasis who underwent such studies. There is strong and diffuse cytoplasmic staining of interleukin-36, specifically in epidermal keratinocytes (subpanel a). No staining is observed in the neutrophils filling the pustules (subpanel b). The expression of interleukin-36 is very low in a specimen of control epidermis (subpanel c), as well as in tissue samples of inflammatory skin disorders, lichen planus (subpanel d), and erythema multiforme (subpanel e). DE denotes dermis, and EP epidermis.

tor of family members carrying the L27P mutation lived 13 generations ago (95% confidence interval, 7 to 29).13 The mutation does not seem to affect the stability or rate of degradation of IL36RN mRNAs (data not shown). We observed the L27P mutation, in the heterozygous state, in 3 of 287 unaffected persons from Sfax, Tunisia, indicating a carrier prevalence of approximately 1% and an allele prevalence of 0.52% in this population. We did not detect this mutation in 500 unaffected Europeans, nor did we detect it in available databases (dbSNP, Ensembl Genome Browser).
Effect of L27P Substitution

The region of interleukin-36Ra protein surrounding amino acid position 27 is highly conserved across species, and leucine at position 27 is absolutely conserved across mammalian species (Fig. S4), suggesting that it is critical to biologic fitness. We created a three-dimensional model of human interleukin-36Ra based on the crystal structure of mouse interleukin-36Ra16 and suggest that the L27P mutation reduces both the stability of the in624

terleukin-36Ra protein and its affinity for the interleukin-1Rrp2 receptor (Fig. 2, and the Supplementary Appendix). Consistent with this hypothesis is the observation that very little mutant (L27P) interleukin-36R is expressed, as compared with nonmutant interleukin-36Ra, in HEK 293T and HaCat cells on transfection of these cells with the relevant construct; for example, transfection with a construct encoding an arginine or an alanine at position 27 resulted in levels of interleukin-36Ra that were similar to those generated by the nonmutant gene (Fig. 3A). To better evaluate the effect of the mutation on protein stability and antagonist function, we generated recombinant mutant and nonmutant interleukin-36Ra proteins and tested their abilities to inhibit signaling by interleukin-1 receptorlike 2 (interleukin-1RL2, also known as interleukin-1 receptorrelated protein 2), on stimulation with interleukin-36. The L27P mutant protein was substantively less able to inhibit interleukin-1RL2 signaling than was nonmutant interleukin-36Ra (Fig. 3B). We subjected the L27P mutant protein to brief incubation at increasing temperatures and

n engl j med 365;7

nejm.org

august 18, 2011

The New England Journal of Medicine Downloaded from nejm.org on November 6, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.

Interleukin-36Receptor Antagonist and Psoriasis

A
Ra Ra Ra n36 In te W rleu ild ki Ty n-3 p e 6R a Ed d L2 + In 7P t e rle uk in -3 6 leu ki leu ki Ed d W + In ild te Ty rle pe uk
Interleukin-36Ra

Myc-Tagged Edd Myc-Tagged Interleukin-36Ra

B
Interleukin-8 Promoter Activity (RLU; millions)
40 30 Interleukin-36RaL27P 20 10 0 104 Interleukin-36Ra

C
Inhibition by Interleukin 36Ra (%)
100 80 60 40 20 0 20 Interleukin-36RaL27P

Inhibitor (g/ml)

nt

D
Controls (N=4) Patient 1 Patient 2 50,000 45,000

Co

Interleukin-8 Level in Keratinocytes (pg/ml)

12,000 10,000 8,000 6,000 4,000 2,000 0

Interleukin-8 (pg/ml)

40,000 35,000 30,000 25,000 20,000 15,000 10,000 5,000 0

36

y( I:C )

leu

rle

rle

te

te

te r

In

F a
EP

In

b
EP

In

c EP
DE

In

te

rle

d
EP

e
EP

DE

DE

DE

ie n (N ts =5 Co ) nt ro (N ls =4 )

-3

-3

uk in

uk in

uk in

n-

-1

Pa t

Po l

ki

30 l . 35 0 . 41 4 . 46 7 . 50 8 . 55 0 . 61 5 . 66 8 .8 70 . 75 0 . 81 6 . 86 9 .9 90 .0

ro

103

102

101

100

101

102

Ed d L2 + In 7A t e r

Ed d L2 + In 7R t e r

Temperature (C)

E
600

500

400

300

200

100

in -3 6

n36

Ra

DE

n engl j med 365;7

nejm.org

august 18, 2011

625

The New England Journal of Medicine Downloaded from nejm.org on November 6, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.

The

n e w e ng l a n d j o u r na l

of

m e dic i n e

found that it was more readily denatured by heating than was the nonmutant protein (Fig. 3C). Keratinocytes from two patients who were homozygous for the L27P mutation produced much higher levels of interleukin-8 than keratinocytes from controls in response to the proinflammatory cytokines (interleukin-36, interleukin-36, and interleukin-36) as well as to interleukin-1 and polyinosinicpolycytidylic acid (poly[I:C]), a synthetic ligand specific for toll-like receptor 3 that is involved in responses to viral infections (Fig. 3D). Accordingly, we found that interleukin-36 is overproduced after the stimulation of keratinocytes with interleukin-1 and poly(I:C) (Fig. S5 in the Supplementary Appendix). The overproduction of interleukin-8 probably results from the impaired inhibitory activity of interleukin-36Ra that is caused by the mutation, because an antibody against interleukin-1RL2 blocked interleukin-8 production by normal keratinocytes, on exposure to interleukin-36 cytokines (Fig. S6 in the Supplementary Appendix). Consistent with this interpretation are the results of plasma cytokine measurements with the use of multiplex ELISA in six of the affected persons at the time of an acute flare: interleukin-8 levels were substantively increased, as compared with the levels in unaffected persons (Fig. 3E). Finally, to gauge the extent to which the interleukin-36interleukin-36Ra signaling pathway was stimulated in affected persons, we performed immunohistochemical analyses of skin lesions from four affected persons who were homozygous for the L27P mutation. The results suggest that all three proinflammatory cytokines interleukin36, interleukin-36, and in particular, interleukin36 were overexpressed in the skin lesions from persons who were homozygous for the mutation, as compared with the expression levels in skin samples from unaffected controls and in skin lesions from patients with inflammatory skin diseases other than psoriasis, such as lichen planus and erythema multiforme (Fig. 3F).

Discussion
Our study shows that generalized pustular psoriasis is an autoinflammatory disease resulting from excessive expression of interleukin-1 family proteins in the skin and disinhibition of the signaling pathway that these proteins activate (Fig. 4).14,15,17 We identified a homozygous mutation in IL36RN, the gene encoding interleukin-36Ra (also
626
n engl j med 365;7

known as interleukin-1F5), an antagonist of three cytokines belonging to the interleukin-1 family interleukin-36, interleukin-36, and interleukin-36 (also known as interleukin-1F6, interleukin-1F8, and interleukin-1F9, respectively).18-21 These cytokines activate several proinflammatory signaling pathways, such as the nuclear factor-B and mitogen-activated protein kinase pathways.22,23 The interleukin-36Ra antagonist and the three agonists interleukin-36, interleukin-36, and interleukin-36, as well as interleukin-1RL2, are highly expressed in epithelial tissues such as the skin, trachea, and esophagus.20 Moreover, we have shown that the expression of interleukin-36 agonist can be strongly induced in keratinocytes after treatment with immunostimulating agents such as interleukin-1 and poly(I:C). These data suggest that this pathway may play a role in the innate immune response to pathogens. The interleukin-1 family consists of 11 members (interleukin-1, interleukin-1, interleukin-1receptor antagonist [interleukin-1Ra], interleukin-18, interleukin-33, interleukin-36, interleukin-36, interleukin-36, interleukin-36Ra, interleukin-37, and interleukin1F10). They are encoded by highly conserved genes and probably arose from the duplication of a common ancestor gene.24 Interleukin-36Ra shares 44% homology with interleukin-1Ra, which negatively regulates both interleukin-1 and interleukin-1. The crucial role of innate immune pathways in tissue inflammation and protective immunity is supported by the identification of several genetically inherited defects involving either impaired or enhanced interleukin-1 immunity and inflammation.25-27 However, mutations in genes that encode the cytokines, their receptors, or their inhibitors have been ascribed to only two pathologic conditions. The first is a form of X-linked mental retardation associated with mutation of IL1RAPL1, which encodes the interleukin-1 receptor accessory protein like protein.28 Deficiency of interleukin-1receptor antagonist (DIRA), the second condition, is associated with excessive activity of interleukin-1.29,30 DIRA is characterized by pustular eruptions and osteoarticular, bone, and central nervous system defects. The third condition, described in this article, is associated with a mutation in a gene encoding another member of the interleukin-1 family, interleukin-36Ra. The mutation results in a reduction of interleukin-36Ra activity; we propose that the resulting disease be called DITRA (deficiency
august 18, 2011

nejm.org

The New England Journal of Medicine Downloaded from nejm.org on November 6, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.

Interleukin-36Receptor Antagonist and Psoriasis

Figure 4. Disinhibition of the Signaling Pathway Activated by Interleukin-1 Family Proteins in Generalized Pustular Psoriasis. Interleukin-36, interleukin-36, and interleukin-36 exert their actions by binding to the interleukin-36 receptor, interleukin-1 receptorlike 2. Ligand binding to this receptor enables the recruitment of the interleukin-1receptor accessory protein, leading to signal transduction involving activation of nuclear factor-B (NF-B) and mitogenactivated protein (MAP) kinases. The interleukin-36receptor antagonist (interleukin-36Ra) also binds to the interleukin-36 receptor, which blocks binding by the agonist ligands but fails to recruit accessory protein and therefore does not lead to biologic activity. Interleukin-36Ra antagonizes their action and inhibits downstream inflammatory signaling (NF-B and MAP kinases), avoiding exacerbated inflammatory responses. DITRA denotes deficiency of interleukin-36receptor antagonist.

of interleukin thirty-sixreceptor antagonist). Fewer organ systems seem to be affected in DITRA (which primarily involves the skin) than in DIRA. But persons with DITRA, in contrast to those with DIRA, have a very high-grade fever and general malaise during an attack. We found that keratinocytes from patients with a deficiency of interleukin36Ra (i.e., those with DITRA) have overproduction of interleukin-8 not only in response to interleukin-36, interleukin-36, and interleukin-36 but also after poly(I:C) stimulation possibly explaining the role of common infections in triggering pustular flares in all our patients. The mouse interleukin-36interleukin-36Ra pathway has been implicated in skin inflammation. Indeed, skinspecific overexpression of interleukin-36 in mice results in a psoriasis-like phenotype with pustu-

lar lesions,19,31 which can be exacerbated by a concomitant deficiency in interleukin-36Ra.19 Moreover, the psoriatic phenotype apparent in human skin lesions transplanted onto immunodeficient mice is normalized by treatment with an antihuman interleukin-1RL2 antibody.19 The interfamilial variation in the age at disease onset in our study may be related to modifying genes, epigenetic factors (including environmental factors), or both. During the course of generalized pustular psoriasis, patients may present with various subtypes of psoriasis,2,12 suggesting common pathophysiological mechanisms. For instance, impetigo herpetiformis is a rare pustular dermatosis of unknown cause that typically occurs in pregnant women. It is considered by most authors to be a generalized pustular psoriasis of
627

n engl j med 365;7

nejm.org

august 18, 2011

The New England Journal of Medicine Downloaded from nejm.org on November 6, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.

Interleukin-36Receptor Antagonist and Psoriasis

pregnancy.32 Our study supports this conclusion, dysregulation of the interleukin-36interleukin-36Ra since generalized pustular psoriasis during preg- signaling pathway may confer a predisposition to nancy (initially diagnosed in two patients as im- common forms of psoriasis. petigo herpetiformis) occurred in the same paSupported by grants from the Agence Nationale de la Rechertients in whom generalized pustular psoriasis was che and the Socit Franaise de Dermatologie. also triggered by other, classic factors. ApproxiDisclosure forms provided by the authors are available with mately 30% of patients with generalized pustular the full text of this article at NEJM.org. We thank all the study families for their participation and psoriasis present with the lesions of psoriasis Maxime Battistella and Manuelle Viguier for their critical read33 supporting our hypothesis that putative ing of the manuscript. vulgaris,
References
1. Lowes MA, Bowcock AM, Krueger JG.

Pathogenesis and therapy of psoriasis. Nature 2007;445:866-73. 2. Griffiths CE, Barker JN. Pathogenesis and clinical features of psoriasis. Lancet 2007;370:263-71. 3. Liu Y, Helms C, Liao W, et al. A genome-wide association study of psoriasis and psoriatic arthritis identifies new disease loci. PLoS Genet 2008;4(3):e1000041. 4. Nair RP, Duffin KC, Helms C, et al. Genome-wide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways. Nat Genet 2009;41:199-204. 5. Strange A, Capon F, Spencer CC, et al. A genome-wide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1. Nat Genet 2010;42:985-90. 6. Zelickson BD, Muller SA. Generalized pustular psoriasis: a review of 63 cases. Arch Dermatol 1991;127:1339-45. 7. Takematsu H, Rokugo M, Takahashi K, Tagami H. Juvenile generalized pustular psoriasis in a pair of monozygotic twins presenting strikingly similar clinical courses. Acta Derm Venereol 1992;72:443-4. 8. Muto M, Ohmura A, Hamamoto Y, et al. Generalized pustular psoriasis: strategy for identification of psoriasis susceptibility gene. Arch Dermatol Res 2003;295: Suppl 1:S60-S62. 9. Augey F, Renaudier P, Nicolas JF. Generalized pustular psoriasis (Zumbusch): a French epidemiological survey. Eur J Dermatol 2006;16:669-73. 10. Iizuka H, Takahashi H, Ishida-Yamamoto A. Pathophysiology of generalized pustular psoriasis. Arch Dermatol Res 2003;295:Suppl 1:S55-S59. 11. Hubler WR Jr. Familial juvenile generalized pustular psoriasis. Arch Dermatol 1984;120:1174-8. 12. Naldi L, Gambini D. The clinical spectrum of psoriasis. Clin Dermatol 2007; 25:510-8. 13. Genin E, Tullio-Pelet A, Begeot F,

Lyonnet S, Abel L. Estimating the age of rare disease mutations: the example of Triple-A syndrome. J Med Genet 2004;41: 445-9. 14. Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol 2009;27:519-50. 15. Sims JE, Smith DE. The IL-1 family: regulators of immunity. Nat Rev Immunol 2010;10:89-102. 16. Dunn EF, Gay NJ, Bristow AF, Gearing DP, ONeill LA, Pei XY. High-resolution structure of murine interleukin 1 homologue IL-1F5 reveals unique loop conformations for receptor binding specificity. Biochemistry 2003;42:10938-44. 17. Dinarello CA. Interleukin-1 and the autoinflammatory diseases. N Engl J Med 2009;360:2467-70. 18. Dinarello C, Arend W, Sims J, et al. IL-1 family nomenclature. Nat Immunol 2010; 11:973. 19. Blumberg H, Dinh H, Dean C Jr, et al. IL-1RL2 and its ligands contribute to the cytokine network in psoriasis. J Immunol 2010;185:4354-62. 20. Kumar S, McDonnell PC, Lehr R, et al. Identification and initial characterization of four novel members of the interleukin-1 family. J Biol Chem 2000;275:10308-14. 21. Smith DE, Renshaw BR, Ketchem RR, Kubin M, Garka KE, Sims JE. Four new members expand the interleukin-1 superfamily. J Biol Chem 2000;275:1169-75. 22. Debets R, Timans JC, Homey B, et al. Two novel IL-1 family members, IL-1 delta and IL-1 epsilon, function as an antagonist and agonist of NF-kappa B activation through the orphan IL-1 receptor-related protein 2. J Immunol 2001;167:1440-6. 23. Towne JE, Garka KE, Renshaw BR, Virca GD, Sims JE. Interleukin (IL)-1F6, IL-1F8, and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP to activate the pathway leading to NF-kappaB and MAPKs. J Biol Chem 2004;279:13677-88. 24. Taylor SL, Renshaw BR, Garka KE,

Smith DE, Sims JE. Genomic organization of the interleukin-1 locus. Genomics 2002; 79:726-33. 25. Henderson C, Goldbach-Mansky R. Monogenic IL-1 mediated autoinflammatory and immunodeficiency syndromes: finding the right balance in response to danger signals. Clin Immunol 2010;135: 210-22. 26. Puel A, Picard C, Ku CL, Smahi A, Casanova JL. Inherited disorders of NFkappaB-mediated immunity in man. Curr Opin Immunol 2004;16:34-41. 27. Casanova JL, Abel L, Quintana-Murci L. Human TLRs and IL-1Rs in host defense: natural insights from evolutionary, epidemiological, and clinical genetics. Annu Rev Immunol 2011;29:447-91. 28. Carri A, Jun L, Bienvenu T, et al. A new member of the IL-1 receptor family highly expressed in hippocampus and involved in X-linked mental retardation. Nat Genet 1999;23:25-31. 29. Aksentijevich I, Masters SL, Ferguson PJ, et al. An autoinflammatory disease with deficiency of the interleukin-1receptor antagonist. N Engl J Med 2009;360:242637. 30. Reddy S, Jia S, Geoffrey R, et al. An autoinflammatory disease due to homozygous deletion of the IL1RN locus. N Engl J Med 2009;360:2438-44. 31. Blumberg H, Dinh H, Trueblood ES, et al. Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation. J Exp Med 2007; 204:2603-14. 32. Chang SE, Kim HH, Choi JH, Sung KJ, Moon KC, Koh JK. Impetigo herpetiformis followed by generalized pustular psoriasis: more evidence of same disease entity. Int J Dermatol 2003;42:754-5. 33. Baker H, Ryan TJ. Generalized pustular psoriasis: a clinical and epidemiological study of 104 cases. Br J Dermatol 1968; 80:771-93.
Copyright 2011 Massachusetts Medical Society.

628

n engl j med 365;7

nejm.org

august 18, 2011

The New England Journal of Medicine Downloaded from nejm.org on November 6, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.