SIGIRR has been described as a negative regulator of several IL-1R/TLR family members and has been implicated in several inflammatory disease conditions. However, it is unknown whether it can suppress IL-36 family cytokines, which are members of the broader IL-1 superfamily that have emerged as critical orchestrators of psoriatic inflammation in both humans and mice. In this study, we demonstrate that SIGIRR is downregulated in psoriatic lesions in humans and mice, and this correlates with increased expression of IL-36 family cytokines. Using Sigirr−/− mice, we identify, for the first time (to our knowledge), SIGIRR as a negative regulator of IL-36 responses in the skin. Mechanistically, we identify dendritic cells and keratinocytes as the primary cell subsets in which IL-36 proinflammatory responses are regulated by SIGIRR. Both cell types displayed elevated IL-36 responsiveness in absence of SIGIRR activity, characterized by enhanced expression of neutrophil chemoattractants, leading to increased neutrophil infiltration to the inflamed skin. Blockade of IL-36R signaling ameliorated exacerbated psoriasiform inflammation in Sigirr−/− mice and inhibited neutrophil infiltration. These data identify SIGIRR activity as an important regulatory node in suppressing IL-36–dependent psoriatic inflammation in humans and mice.

Cytokines of the IL-36 family have emerged as major mediators of inflammation, including skin inflammation. The family consists of the agonistic ligands IL-36α, IL-36β and IL-36γ and the antagonist IL-36Ra, all of which act through the IL-36 receptor (IL-36R/IL-1RL2) (1). Several studies in psoriasis patients and murine models of psoriasis have recognized a major role of IL-36 cytokines in dermal inflammation (25). IL-36 cytokines were further established as orchestrators of psoriatic inflammation when it was discovered that loss of function mutations of IL36RN led to an autoinflammatory disease known as generalized pustular psoriasis, a severe form of psoriasis (6, 7). Indeed, neutralizing IL-36R Abs have been developed as a treatment of psoriasis and are currently under clinical investigation (8, 9). IL-36 signaling induces proinflammatory responses on different cell types involved in psoriatic skin inflammation, including keratinocytes, dendritic cells (DC), macrophages, and T cell subsets (1, 10). However, it was recently shown by our group that IL-36R expression on keratinocytes is required for psoriasiform inflammation (11).

SIGIRR (also known as TIR8 or IL-1R8) is a member of the IL-1 receptor superfamily, which is a negative regulator of TLR and IL-1R responses (12). It was thought to be an orphan receptor, but more recently has been shown to act as a coreceptor, with IL-1R5/IL-18Rα, for IL-37 (13). Its structure is unique from other IL-1Rs, as it contains only one extracellular Ig like domain instead of three, its intracellular Toll/IL-1 receptor (TIR) domain contains two amino acid substitutions (Ser447 and Tyr536 replaced by Cys222 and Leu305), indicating distinct signal transduction capabilities, and it also encompasses an unusually long cytoplasmic tail (12, 14). SIGIRR has been shown to modulate ligand-induced activation of IL-1R1, IL-1R5/IL-18Rα, IL-1R4/ST2, TLR4, TLR7, TLR9, TLR3, and TLR1/2 (12, 1519). Its regulatory mechanism is not precisely known, but it is thought to involve both interference with receptor dimerization through its extracellular Ig domain, and binding of adaptor molecules such as MyD88 and IL-1 receptor–associated kinase through its TIR domain, thus blocking downstream signaling and NF-κB, MAPKs, or JNK activation (16, 17, 19). Because of its crucial role in regulating proinflammatory IL-1R/TLR responses, SIGIRR has been implicated as being involved in several inflammatory and autoimmune conditions, including rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease (18, 2023). We previously demonstrated that SIGIRR deficiency exacerbated psoriatic inflammation through increased infiltration of IL-17A–expressing γδ T cells (24). SIGIRR has been found to be downregulated in the peripheral blood of psoriatic arthritis patients (25); however, it is not currently known what is the status of SIGIRR expression in psoriatic skin. Another important gap in existing knowledge about SIGIRR as a modulator of inflammatory responses, especially in the context of skin inflammation, is whether SIGIRR can regulate IL-36R responses, which are established as central orchestrators of skin inflammation particularly in the context of psoriasis.

In this study, we demonstrate that SIGIRR can negatively regulate IL-36R responses in the skin. Alongside elevated IL-36 family levels, SIGIRR gene expression was downregulated in the skin of psoriasis patients. These observations were mirrored in an imiquimod (IMQ) induced model of psoriatic inflammation in mice. Moreover, mice deficient in SIGIRR were more susceptible to IL-36–dependent psoriatic skin inflammation, identifying SIGIRR activity as a constitutive regulator of IL-36R–driven inflammatory dermal responses. Mechanistically, SIGIRR suppressed IL-36–driven neutrophil infiltration through restricting the induced expression of the neutrophil chemokines CXCL1 and CXCL2 by keratinocytes and DC subsets.

Gene Expression Omnibus (GEO) database GDS4602 (reference series GSE13355) was analyzed for expression of IL36A, IL36B, IL36G, IL36RN, IL1RL2, SIGIRR, CXCL1, CXCL2, and CXL8. Database contains microarray gene expression analysis of skin punch biopsies from lesional and nonlesional skin from psoriasis patients and healthy control skin, analyzed by Affymetrix Human Genome U133 Plus 2.0 Array (26). Databases GDS4600 and GDS2518, containing paired lesional and nonlesional psoriatic skin samples, were also analyzed for SIGIRR expression (27, 28).

All mice used were of C57BL/6 background, 6–18 wk old, male and female, bred in-house in specific pathogen–free conditions in Trinity Translational Medicine Institute, St. James’ Hospital, Dublin, Ireland. Sigirr−/− mice on a C57BL/6 background were provided by C. Garlanda (Humanitas University). Il17eGFP mice (29) were crossbred with Sigirr−/− mice in-house to produce Sigirr−/− Il17eGFP mice. All animal experiments were performed with ethical approval by Trinity College Dublin Animal Research Ethics Committee and under license by the Irish Health Products Regulatory Authority (previously Irish Medicines Board) (project authorization numbers: B100/4272; AE19136/P036).

Aldara cream (5% IMQ, MEDA Pharmaceuticals) or control cream (Vaseline) was applied on different ears of wild-type (wt) or Sigirr−/− mice daily, for 2–7 d, depending on experiment. For experiments with IL-36R neutralization, mice were injected i.p. with 150 μg anti-mouse IL-36R Ab (M616; Amgen) or rat IgG2a isotype control (2A3; BioXCell) daily, starting from day –1 before IMQ application (day 0) and continued for all the course of the treatment. Ears were collected and either fixed in 10% neutral buffer formalin (Medical Supply) for subsequent histological and immunohistochemical analysis, or stored in RNA later (Sigma-Aldrich) for RNA isolation.

wt and Sigirr−/− mice were anesthetized and injected intradermally on their ears with either 200 ng active IL-36α (aa 8–160) or PBS in a 20 μl volume every day for 5 d using a 30-gauge needle. Ear thickness was measured using a thickness gauge (Hitec) before the initiation of the experiment on day 0 and every day thereafter until day 5.

RNA from ear tissue was isolated using ISOLATE II RNA Mini Kit (Bioline) according to the manufacturer’s instructions. Ears stored in RNA later were transferred in Lysis Buffer RLY and homogenized using BeadBug-prefilled tubes with 1.5-mm zirconium beads (Sigma-Aldrich) in a FastPrep-24 5G system (MP Biomedicals), before continuing with RNA isolation. RNA was reverse transcribed to cDNA using the High-Capacity cDNA kit with RNase Inhibitor (Applied Biosystems). Real time PCR was performed with the cDNA produced, in triplicate duplex reactions with 18S (VIC) as control and target TaqMan Gene Expression Assay (FAM; Table I), using TaqMan Fast Universal PCR Master Mix in a QuantStudio 3 System (Applied Biosystems). Analysis was performed using the ΔΔCt method, with 18S rRNA used for normalization.

Table I.

TaqMan Gene Expression Assays

GeneTaqMan Assay ID
Il17c Mm00521397_m1 
S100a8 Mm00496696_g1 
S100a9 Mm00656925_m1 
Il17a Mm00439618_m1 
Il23a Mm01160011_g1 
Cxcl1 Mm04207460_m1 
Cxcl2 Mm00436450_m1 
Il1f6 (Il36a) Mm00457645_m1 
Il1f8 (Il36b) Mm01337546_g1 
Il1f9 (Il36g) Mm00463327_m1 
Il1f5 (Il36rn) Mm01333586_m1 
Il1rl2 Mm00519245_m1 
Sigirr Mm00491700_m1 
18S Hs99999901_s1 
GeneTaqMan Assay ID
Il17c Mm00521397_m1 
S100a8 Mm00496696_g1 
S100a9 Mm00656925_m1 
Il17a Mm00439618_m1 
Il23a Mm01160011_g1 
Cxcl1 Mm04207460_m1 
Cxcl2 Mm00436450_m1 
Il1f6 (Il36a) Mm00457645_m1 
Il1f8 (Il36b) Mm01337546_g1 
Il1f9 (Il36g) Mm00463327_m1 
Il1f5 (Il36rn) Mm01333586_m1 
Il1rl2 Mm00519245_m1 
Sigirr Mm00491700_m1 
18S Hs99999901_s1 

Bone marrow cells were extracted from femur and tibia bones of wt and Sigirr−/− mice, then cultured in complete RPMI media (RPMI 1640 + 10% FCS + 1% penicillin-streptomycin) (Sigma-Aldrich) supplemented with 20 ng/ml recombinant mouse (rm) GM-CSF (PeproTech) for bone marrow–derived DC (BMDC) generation, or 20% L929 cell supernatants for bone marrow–derived macrophages (BMDM) generation. After sufficient days in culture to differentiate into the desired cell type, BMDC or BMDM were treated with 500 ng/ml rmIL-36α (aa 8–160) or 100 ng/ml LPS for 24 h, then supernatants were collected to be analyzed by ELISA.

Keratinocytes from wt and Sigirr−/− adult mouse tail skin were isolated using protocol described previously by Li et al. (30). Cultured cells were treated with 100 ng/ml rmIL-36α (aa 8–160) (R&D Systems) for 24 h, then total RNA was isolated and used for gene expression analysis by quantitative RT-PCR.

Cell supernatants, or peritoneal washes were used to measure levels of mouse CXCL1/KC and CXCL2/MIP-2 proteins using DuoSet ELISA Kits (R&D Systems) according to the manufacturer’s instructions. For innate IL-17A stimulation, splenocytes and inguinal lymph node cells were cultured together in complete media supplemented with desired cytokines for 24 h, then supernatants were collected and used to measure IL-17A protein levels by ELISA (Invitrogen eBioscience), according to the manufacturer’s instructions.

For peritoneal recruitment assay, wt and Sigirr−/− mice were injected i.p. with 100 ng rmIL-36α (aa 8–160), or PBS. Six hours later, peritoneal lavage was collected, and cells were analyzed by flow cytometry. Supernatants were kept at –20°C to be analyzed by ELISA.

For air pouch assay, wt and Sigirr−/− mice were injected s.c. in the back with sterile air under general anesthesia on day 1. Injections were repeated on days 2, 4, and 5 to establish air pouch creation. On day 6, 200 ng rmIL-36α (aa 8–160) or PBS were injected into the air pouch. Four hours later, air pouch was lavaged to collect cells, which were then analyzed by flow cytometry.

For flow cytometry analysis, cells were surface stained with fluorophore-conjugated primary Abs for mouse CD3e (145-2C11), CD19 (eBio1D3), CD11b (M1/70), F4/80 (BM8), Ly6G (1A8), CD11c (N418), γδ-TCR (eBioGL3) (eBioscience, Invitrogen) as required. For analysis of IL-17A expression, Il17eGFP mice were used instead. Anti-mouse CD16/CD32 (93, eBioscience) was added in all samples to block Fc receptors. Cells were then analyzed in BD LSRFortessa flow cytometer (BD Biosciences), using Live/Dead Aqua stain (Invitrogen) to gate on live cells. Representative gating strategy for in vivo recruitment assays shown in Supplemental Fig. 3. Data analysis was performed using FlowJo software (Treestar).

Ear tissue fixed in 10% formalin overnight was dehydrated and embedded into paraffin blocks. Sections of 5-μm thickness were cut, stained with H&E, and scored blindly for histopathological manifestations of psoriasis on a scale of 0–4 (0, no differences over control; 1, mild; 2, moderate; 3, marked; 4, severe) for acanthosis, desquamation, parakeratosis, and infiltration. Scores for each parameter were combined for an overall histological severity score.

For immunofluorescence staining of Ly6G+ cells, ear sections of 5-μm thickness were cleared, rehydrated and Ag was unmasked by boiling in microwave in 1× Citrate Buffer pH 6.0 (DiaPath). Free aldehydes group was blocked with 1 M glycine in PBS for 30 min, then 2.5% goat serum (Vector) was used to block unspecific binding. Sections were then incubated overnight with primary Ab rat anti-mouse Ly6G (1A8; BioXCell) diluted 1:4000. Sections were washed and incubated for 2 h with secondary Ab goat anti-rat IgG Alexa Fluor 594 (Invitrogen) diluted 1:500. After washing, slides were mounted with SlowFade Gold antifade reagent with DAPI (Invitrogen), sealed and allowed to dry. Slides were analyzed using LSM Confocal microscope (Zeiss). Ly6G+ cells were counted per Z stack, for three different stacks per sample, using ImageJ software.

For statistical data analysis Prism 6 software (GraphPad) was used. Data were first analyzed for normality and homoscedasticity, before choosing the appropriate test, either unpaired two-tailed Student t test or Mann–Whitney, or one-way ANOVA or Kruskal–Wallis test for multiple comparisons, as indicated in figure legends. For correlation analysis, Spearman correlation was used. Statistical significance was determined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Although SIGIRR expression has been reported to be downregulated in the peripheral blood of patients suffering from psoriatic arthritis, to our knowledge, there have been no reports describing whether its expression is altered in the skin of psoriasis patients (25). To address this question and determine whether SIGIRR activity may be implicated in disease pathogenesis, we analyzed publicly available microarray data from the GEO database (GDS4602), analyzing gene expression profiles of skin biopsies taken from psoriatic lesional and nonlesional skin, as well as normal controls (26). Consistent with previous reports, gene expression of all IL-36 family cytokines, IL36A, IL36B, IL36G, and IL36RN, were upregulated in lesional psoriatic skin, compared with nonlesional and control skin (Fig. 1A–D). IL36G and IL36RN expression were also found to be significantly elevated in nonlesional skin when compared with control biopsies, albeit to a much lesser extent (Fig. 1C, 1D). The IL-36R gene IL1RL2 was also upregulated in psoriatic lesional and nonlesional skin (Fig. 1E).

FIGURE 1.

IL-36 family and SIGIRR expression in psoriatic skin. Expression levels of IL36A (A), IL36B (B), IL36G (C), IL36RN (D), IL1RL2 (IL-36R) (E), and SIGIRR (F) in lesional (PP) or nonlesional (PN) skin of psoriatic patients (n = 58), or skin from normal controls (NN) (n = 64), as analyzed from public GEO microarray data (GDS4602). Kruskal–Wallis test with Dunn test for multiple comparisons for (A)–(D), ordinary one-way ANOVA with Tukey test for multiple comparisons for (E) and (F). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 1.

IL-36 family and SIGIRR expression in psoriatic skin. Expression levels of IL36A (A), IL36B (B), IL36G (C), IL36RN (D), IL1RL2 (IL-36R) (E), and SIGIRR (F) in lesional (PP) or nonlesional (PN) skin of psoriatic patients (n = 58), or skin from normal controls (NN) (n = 64), as analyzed from public GEO microarray data (GDS4602). Kruskal–Wallis test with Dunn test for multiple comparisons for (A)–(D), ordinary one-way ANOVA with Tukey test for multiple comparisons for (E) and (F). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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In contrast to the IL-36 family members, SIGIRR gene expression was lower in both psoriatic lesional and nonlesional skin compared with healthy skin (Fig. 1F). Further analysis of two independent datasets also demonstrated that SIGIRR gene expression was significantly decreased in inflamed lesional skin from psoriasis patients (Supplemental Fig. 1). These data demonstrate that gene expression levels of SIGIRR, a negative regulator of IL-1 family cytokines, are decreased in patients’ psoriatic skin and suggest that SIGIRR activity may act as an important regulator of dermal inflammation and disease pathogenesis.

As a next step to investigate the role of SIGIRR in regulating IL-36–driven inflammation in the skin, we analyzed Sigirr−/− mice. We have previously demonstrated that these mice exhibit an exacerbated disease phenotype across different models of psoriasiform inflammation (24). As previously reported, intradermal injection of rIL-36α in mouse ear skin, resulted in increased inflammation as measured by ear thickening in wt mice (31). The level of ear thickening observed was significantly increased in Sigirr−/− animals, indicating that SIGIRR expression can act to suppress IL-36–driven responses in the skin (Fig. 2A). To extend these observations, we next induced psoriasiform inflammation in both wt and Sigirr−/− mice through the topical application of 5% IMQ cream. As expected, Sigirr−/− mice displayed a more severe disease phenotype, with a higher combined histological score (a sum of acanthosis, parakeratosis, desquamation, and infiltration) when compared with wt mice (Fig. 2B, 2C). In agreement with previous reports, administration of an anti–IL-36R–blocking Ab significantly reduced IMQ-induced psoriasiform inflammation in wt mice (9). Interestingly, IL-36R blockade also resulted in a significant inhibition of inflammation in the skin of Sigirr−/− mice, offering similar levels of protection to those observed in their wt counterparts. Together these data confirm that SIGIRR can act as a negative regulator of IL-36–driven psoriasiform inflammation (Fig. 2B, 2C).

FIGURE 2.

SIGIRR negatively regulates IL-36–driven dermal inflammation. (A) The wt and Sigirr−/− mice were injected intradermally with 200 ng IL-36 or PBS for 5 d and ear thickness was measured daily. ANOVA with multiple comparisons. (B and C) The wt and Sigirr−/− mice (n = 5–6 per group) were treated with 150 μg anti–IL-36R–blocking Ab or isotype control i.p. starting 1 d before a 7-d application of 5% IMQ cream (Aldara). On day 7, ears were collected and stained with H&E for histopathological assessment. (B) Representative micrographs of stained tissue, control: Vaseline treated ear. (C) Combined histological score for acanthosis, desquamation, parakeratosis, and infiltration. Unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2.

SIGIRR negatively regulates IL-36–driven dermal inflammation. (A) The wt and Sigirr−/− mice were injected intradermally with 200 ng IL-36 or PBS for 5 d and ear thickness was measured daily. ANOVA with multiple comparisons. (B and C) The wt and Sigirr−/− mice (n = 5–6 per group) were treated with 150 μg anti–IL-36R–blocking Ab or isotype control i.p. starting 1 d before a 7-d application of 5% IMQ cream (Aldara). On day 7, ears were collected and stained with H&E for histopathological assessment. (B) Representative micrographs of stained tissue, control: Vaseline treated ear. (C) Combined histological score for acanthosis, desquamation, parakeratosis, and infiltration. Unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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As first step to investigate the mechanisms through which SIGIRR activity may regulate IL-36–dependent psoriatic inflammation, we analyzed which genes, known to be induced by IL-36 are affected differentially in wt and Sigirr−/− skin after IMQ treatment. Il17c, S100a8, and S100a9 are genes expressed by keratinocytes, that have previously been shown to be regulated by IL-36 (9, 32). Whereas IMQ-induced expression levels of Il17c were similar in both wt and Sigirr−/− skin, levels of S100a8 and S100a9 expression were significantly enhanced (Fig. 3A–C). Interestingly, both S100a8 and S100a9 are also expressed by neutrophils, and elevated expression of these genes may be reflective of enhanced neutrophil infiltration in the absence of SIGIRR. IMQ-induced upregulation of signature psoriasis proinflammatory cytokines Il17a and Il23 was also enhanced in Sigirr–/– mice (Fig. 3D, 3E). As increased neutrophil infiltration is a key pathogenic feature in this model, as well as in human psoriatic lesions, we also examined the relative expression of the neutrophil chemoattractants Cxcl1 and Cxcl2, both of which were found to be elevated in the skin of SIGIRR-deficient mice (Fig. 3F, 3G).

FIGURE 3.

SIGIRR regulation of IL-36–responsive gene expression in the IMQ model. The wt and Sigirr−/− mice (n = 4–6 per group) were treated with 5% IMQ or control cream for 2 d, then ears were collected for RNA isolation and gene expression analysis. Relative mRNA expression of Il17c (A), S100a8 (B), S100a9 (C), Il17a (D), Il23a (E), Cxcl1 (F), Cxcl2 (G), Il36a/Il1f6 (H), Il36b/Il1f8 (I), Il36g/Il1f9 (J), Il36rn/Il1f5 (K), Il1rl2 (IL-36R) (L), Sigirr (M). Mann–Whitney U test. *p < 0.05, **p < 0.01.

FIGURE 3.

SIGIRR regulation of IL-36–responsive gene expression in the IMQ model. The wt and Sigirr−/− mice (n = 4–6 per group) were treated with 5% IMQ or control cream for 2 d, then ears were collected for RNA isolation and gene expression analysis. Relative mRNA expression of Il17c (A), S100a8 (B), S100a9 (C), Il17a (D), Il23a (E), Cxcl1 (F), Cxcl2 (G), Il36a/Il1f6 (H), Il36b/Il1f8 (I), Il36g/Il1f9 (J), Il36rn/Il1f5 (K), Il1rl2 (IL-36R) (L), Sigirr (M). Mann–Whitney U test. *p < 0.05, **p < 0.01.

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We and others have previously demonstrated that IL-36 family cytokines themselves are elevated in IMQ-treated skin, and can act to induce their own expression (11, 32, 33). As such, we also examined whether their expression levels were altered in the absence of SIGIRR. As expected, and analogous to psoriasis patients’ lesional skin (Fig. 1), IMQ treatment caused an increase in Il36a, Il36b, Il36g, and Il36rn expression (Fig. 3H–K), although Il1rl2 expression was not affected (Fig. 3L). Significantly, elevated gene expression levels of IL-36 family ligands were unaffected by the absence of SIGIRR, demonstrating that its activity does not suppress the induction of IL-36 gene expression in inflamed skin. Expression of Il1rl2 was marginally lower in inflamed Sigirr−/− skin, possibly indicating that SIGIRR activity may play a role in regulating IL-36R expression (Fig. 3L).

Interestingly, and mirroring human psoriasis lesional skin (Fig. 1F), Sigirr gene expression was downregulated in inflamed murine skin (Fig. 3M). These data demonstrate that SIGIRR activity acts to restrict the expression of specific proinflammatory genes in the skin known to be downstream of the IL-36R, which are known to play important roles in driving psoriatic inflammation in the IMQ model of disease.

To determine on which cell subsets SIGIRR activity may be important in regulating IL-36–dependent responses, in the context of dermal inflammation, we next examined the responses of primary cells characterized as playing important roles in disease pathogenesis in this model. To address this question, we analyzed IL-36–dependent responses in DC, macrophages, keratinocytes, and γδ-T cells. Informed by our previous studies (24) and our gene expression data from inflamed skin (Fig. 3), we focused primarily on analyzing the induction of neutrophil chemokines and IL-17A expression as the key pathogenic pathways suppressed by SIGIRR in inflamed skin. Both BMDC and BMDM expressed the chemokines CXCL1/KC and CXCL2/MIP-2 in response to IL-36α stimulation to similar levels observed upon LPS stimulation (Fig 4A, 4B). The receptor for LPS, TLR4, has previously been shown to be negatively regulated by SIGIRR activity and serves as a positive control for innate stimulation in this context (19). Interestingly, loss of Sigirr expression resulted in enhanced CXCL1/KC and CXCL2/MIP-2 expression by BMDC upon stimulation with either IL-36α or LPS (Fig 4A). Levels of innate inflammatory cytokines IL-6 and IL-12p70 were also similarly elevated (Supplemental Fig. 2A). In contrast, no such differences were observed in BMDM (Fig. 4B). These data are consistent with previous observations which indicated that SIGIRR does not play a significant role in regulating macrophage TLR/IL-1R family responses (17, 18). Interestingly, SIGIRR activity also suppressed IL-36α induction of Il17c, S100a8, and S100a9, as well as Cxcl1 and Cxcl2, in primary keratinocytes. Together, these data indicate that SIGIRR can play an important regulatory role in limiting the responses of these key cellular targets of pathogenic IL-36 receptor signaling (Fig. 4C).

FIGURE 4.

SIGIRR is a negative regulator of IL-36 responses in DC and keratinocytes but not in macrophages or γδ T cells. (A and B) CXCL1/KC and CXCL2/MIP-2 levels in supernatants from BMDC (A) or BMDM (B) from wt or Sigirr−/− mice, stimulated with 500 ng/ml IL-36α or 100 ng/ml LPS or vehicle for 24 h, as measured by ELISA. (C) Il17c, S100a8, S100a9, Cxcl1, and Cxcl2 mRNA expression in keratinocytes from wt or Sigirr−/− mice, with or without treatment with 100 ng/ml IL-36α for 24 h, represented as fold change over untreated cells. (D) Splenocytes and inguinal lymph node cells from wt Il17eGFP mice were stimulated with 20 ng/ml each of IL-1β or IL-23 or IL-36α, or a combination of these for 24 h, then analyzed by flow cytometry. IL-17A+ cells were gated on CD3+ γδ TCR+ cells. (E) Splenocytes and inguinal lymph node cells from wt or Sigirr−/− Il17eGFP mice were stimulated with 20 ng/ml each of IL-1β or IL-23 or IL-36α, or a combination of these for 24 h, then IL-17A expression was measured in supernatants by ELISA. Data representative of at least two independent experiments with cells in triplicate wells. Unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4.

SIGIRR is a negative regulator of IL-36 responses in DC and keratinocytes but not in macrophages or γδ T cells. (A and B) CXCL1/KC and CXCL2/MIP-2 levels in supernatants from BMDC (A) or BMDM (B) from wt or Sigirr−/− mice, stimulated with 500 ng/ml IL-36α or 100 ng/ml LPS or vehicle for 24 h, as measured by ELISA. (C) Il17c, S100a8, S100a9, Cxcl1, and Cxcl2 mRNA expression in keratinocytes from wt or Sigirr−/− mice, with or without treatment with 100 ng/ml IL-36α for 24 h, represented as fold change over untreated cells. (D) Splenocytes and inguinal lymph node cells from wt Il17eGFP mice were stimulated with 20 ng/ml each of IL-1β or IL-23 or IL-36α, or a combination of these for 24 h, then analyzed by flow cytometry. IL-17A+ cells were gated on CD3+ γδ TCR+ cells. (E) Splenocytes and inguinal lymph node cells from wt or Sigirr−/− Il17eGFP mice were stimulated with 20 ng/ml each of IL-1β or IL-23 or IL-36α, or a combination of these for 24 h, then IL-17A expression was measured in supernatants by ELISA. Data representative of at least two independent experiments with cells in triplicate wells. Unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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In our previous study, we demonstrated that increased psoriasiform inflammation in Sigirr−/− mice was associated with elevated expression of IL-17A by γδ-T cells. Moreover, SIGIRR negatively regulated the innate induction of γδ-T cells IL-17A secretion in response to a combination of IL-23 and IL-1β in vitro (24) (Fig. 4E). Therefore, we next examined whether IL-36α could act in a similar fashion and provide an innate stimulus to drive γδ-T cell responses. In contrast to IL-1β, IL-36α treatment did not have any effect on the induction of IL-17A expression by γδ-T cells, either alone or in combination with IL-23, as determined by flow cytometry or by ELISA (Fig. 4D, 4E). In summary, SIGIRR acts to negatively regulate innate IL-36–driven responses in DC and keratinocytes without affecting macrophage responses. Importantly, IL-36 does not act in a similar fashion to IL-1β in providing an innate stimulus to directly enhance IL-17A expression by γδ-T cells. Interestingly, although less relevant for the IMQ-induced skin inflammation model, CD4+ Th1 cell polarization is also induced by IL-36 and this effect is enhanced in the absence of Sigirr (Supplemental Fig. 2B).

As our data demonstrated that SIGIRR can act to restrict IL-36–driven expression of CXCL1 and CXCL2 from different cell types, without significantly altering the innate expression of IL-17A, we next investigated whether neutrophil infiltration in response to IL-36 stimulation was altered in Sigirr−/− mice. To answer this question, we used two in vivo models to analyze innate cell recruitment to the peritoneum and dorsal air pouch in response to injection of IL-36α (Supplemental Fig. 3). In both of these models, IL-36α injection caused an influx of CD3CD19Ly6G+F4/80 neutrophils, which was enhanced in Sigirr−/− mice (Fig. 5A, 5D). This increase was evident both as percentage and total neutrophil cell number (Fig. 5A–C). Although the relative percentage of CD3CD19CD11b+F4/80+ macrophages recruited was decreased, no difference in macrophage cell numbers was evident (Fig. 5B, 5C). The recruitment of CD3+CD19 T cells, CD3CD19+ B cells and CD3CD19CD11c+F4/80 DCs were also not affected (data not shown). In agreement with our in vitro data, the levels of CXCL1/KC and CXCL2/MIP-2 chemokines were also elevated in the peritoneum of Sigirr−/− mice after IL-36α injection (Fig. 5E). These data confirm that SIGIRR activity negatively regulates IL-36–driven chemokine expression and neutrophil infiltration in vivo.

FIGURE 5.

SIGIRR negatively regulates IL-36–driven neutrophil infiltration in vivo. (AC) The wt and Sigirr−/− mice (n = 3 per group) were injected with 200 ng/ml IL-36α or PBS in dorsal generated air pouches, then recruited cells were collected 4 h later and analyzed by flow cytometry. (A) Representative FACS plots for Ly6G+F4/80 neutrophils, gated on CD3CD19 cells. (B) Percentages of Ly6G+F4/80 neutrophils and F4/80+CD11b+ macrophages, gated on CD3CD19 cells. (C) Absolute cell numbers for total cells in the air pouch, Ly6G+F4/80 neutrophils, and F4/80+CD11b+ macrophages gated on CD3CD19 cells. (D and E) The wt and Sigirr−/− mice (n = 3 per group) were injected with 100 ng/ml IL-36α or PBS i.p., then 6 h later peritoneal lavage was collected and analyzed by flow cytometry and ELISA. (D) Representative FACS plots for Ly6G+F4/80 neutrophils, gated on CD3CD19 cells. (E) CXCL1/KC and CXCL2/MIP-2 levels in lavage supernatants as measured by ELISA. Unpaired Student t test. *p < 0.05, **p < 0.01.

FIGURE 5.

SIGIRR negatively regulates IL-36–driven neutrophil infiltration in vivo. (AC) The wt and Sigirr−/− mice (n = 3 per group) were injected with 200 ng/ml IL-36α or PBS in dorsal generated air pouches, then recruited cells were collected 4 h later and analyzed by flow cytometry. (A) Representative FACS plots for Ly6G+F4/80 neutrophils, gated on CD3CD19 cells. (B) Percentages of Ly6G+F4/80 neutrophils and F4/80+CD11b+ macrophages, gated on CD3CD19 cells. (C) Absolute cell numbers for total cells in the air pouch, Ly6G+F4/80 neutrophils, and F4/80+CD11b+ macrophages gated on CD3CD19 cells. (D and E) The wt and Sigirr−/− mice (n = 3 per group) were injected with 100 ng/ml IL-36α or PBS i.p., then 6 h later peritoneal lavage was collected and analyzed by flow cytometry and ELISA. (D) Representative FACS plots for Ly6G+F4/80 neutrophils, gated on CD3CD19 cells. (E) CXCL1/KC and CXCL2/MIP-2 levels in lavage supernatants as measured by ELISA. Unpaired Student t test. *p < 0.05, **p < 0.01.

Close modal

The infiltration and activation of neutrophils is a key pathogenic driver of psoriatic inflammation. As our data demonstrated that SIGIRR can act to restrict IL-36–driven neutrophil infiltration, we next sought to determine whether the infiltration of neutrophils to the inflamed dermis was altered in association with protection from psoriasiform disease observed upon treatment with an anti–IL-36R blocking Ab. To address this, we treated wt and Sigirr−/− mice undergoing IMQ-induced psoriasiform inflammation with either a neutralizing anti–IL-36R Ab or an isotype control Ab, and subsequently analyzed Ly6G+ neutrophil infiltration through immunofluorescent staining. Sigirr−/− mice displayed enhanced infiltration of neutrophils in the ear dermis compared with wt mice, which was reduced to levels observed in wt mice after treatment with anti–IL-36R (Fig. 6A, 6B). In association with these observations, elevated gene, and protein expression of the neutrophil chemokine CXCL2 in the inflamed skin of Sigirr−/− mice was also significantly reduced upon treatment with anti–IL-36R (Fig 6C, 6D). Levels of CXCL1 expression were not significantly altered (data not shown). These data confirm that, in protecting from enhanced disease severity, blockade of IL-36R signaling, also, inhibits the enhanced infiltration of neutrophils to the inflamed skin observed in the absence of SIGIRR.

FIGURE 6.

Blockade of IL-36R inhibits neutrophil infiltration to the inflamed skin. The wt and Sigirr−/− mice (n = 5–6 per group) were treated with 150 μg of anti–IL-36R–blocking Ab or isotype control i.p. starting 1 d before a 7-d application of 5% IMQ cream. Ears were collected and stained for immunofluorescence with anti-Ly6G Ab (red) and DAPI nuclear stain (blue), before analysis in a confocal microscope. (A) Representative micrographs in which Ly6G+ cells can be seen in different groups, including control (Vaseline)-treated ears. Scale bar: 50 μm. (B) Average number of Ly6G+ cells per field (measured in three-dimensional stacks, three fields per sample). Mann–Whitney U test. (C and D) Levels of CXCL2/MIP-2 mRNA (C) measured by quantitative RT-PCR and protein (D) measured by ELISA and normalized for total protein levels, analyzed in the ears of anti–IL-36R or isotype control Ab treated (from day –1) wt and Sigirr−/− mice (n = 4–7 per group), after 3-d IMQ application. Ears of naive wt and Sigirr−/− mice were used as controls (ctrl). Unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6.

Blockade of IL-36R inhibits neutrophil infiltration to the inflamed skin. The wt and Sigirr−/− mice (n = 5–6 per group) were treated with 150 μg of anti–IL-36R–blocking Ab or isotype control i.p. starting 1 d before a 7-d application of 5% IMQ cream. Ears were collected and stained for immunofluorescence with anti-Ly6G Ab (red) and DAPI nuclear stain (blue), before analysis in a confocal microscope. (A) Representative micrographs in which Ly6G+ cells can be seen in different groups, including control (Vaseline)-treated ears. Scale bar: 50 μm. (B) Average number of Ly6G+ cells per field (measured in three-dimensional stacks, three fields per sample). Mann–Whitney U test. (C and D) Levels of CXCL2/MIP-2 mRNA (C) measured by quantitative RT-PCR and protein (D) measured by ELISA and normalized for total protein levels, analyzed in the ears of anti–IL-36R or isotype control Ab treated (from day –1) wt and Sigirr−/− mice (n = 4–7 per group), after 3-d IMQ application. Ears of naive wt and Sigirr−/− mice were used as controls (ctrl). Unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

To explore the translational significance of our mechanistic observations above we undertook a further analysis of the patient dataset shown in (Fig. 1. Interestingly, neutrophil chemoattractant chemokine genes CXCL1, CXCL2, and CXCL8 were all upregulated in lesional psoriatic skin (Supplemental Fig. 4A), and their elevated expression correlated with the expression of IL-36 family ligands IL36A and IL36G (Supplemental Fig. 4B, 4C), indicating that IL-36 signaling can drive neutrophil chemokine expression in the psoriatic skin of humans, as well as in mice. In addition, the expression of SIGIRR, which is downregulated in psoriasis patients, negatively correlated with expression levels of IL36A and IL36G, as well as IL36RN and IL1RL2, in lesional psoriatic skin (Supplemental Fig. 4D). These data suggest that SIGIRR is downregulated in parallel with an upregulation in IL-36 cytokines, leading to increased chemokine production and potentially resulting in enhanced neutrophil recruitment in inflamed skin. Together, these data identify SIGIRR gene expression and activity as a novel, to our knowledge, regulatory mechanism of suppression of pathogenic psoriatic inflammatory responses mediated by IL-36 family cytokines.

Although SIGIRR activity has emerged as an important negative regulator of IL-1R and TLR induced responses in various inflammatory conditions, much of the data surrounding its immunosuppressive function has stemmed from the analysis of preclinical models with relatively little known of its role in human disease. We previously demonstrated that SIGIRR activity suppressed psoriasiform inflammation in a murine model of disease (24). In agreement with these findings, we have now demonstrated, using publicly available psoriasis patient datasets, that SIGIRR expression is also downregulated in lesional skin of psoriasis patients. Moreover, this decreased expression correlated with the upregulation of IL-36 family cytokines, indicating that SIGIRR may act as negative regulator of IL-36–mediated skin pathology. The IL-36 family of cytokines are emerging as central orchestrators of dermal inflammation in psoriatic disease with early clinical studies indicating that targeting their activity may prove beneficial for some patients. As such, a deeper understanding of the mechanisms which regulate their activity are of particular importance. Significantly, we observed reduced SIGIRR expression in lesional psoriatic skin in three independent patient datasets, indicating that its activity may be reduced at dermal sites during active disease (Supplemental Fig. 1). Although the expression of SIGIRR has not previously been examined in human skin, it is relevant that expression has also previously been found to be lower in peripheral blood cells of psoriatic arthritis patients (25). It is also noteworthy that SIGIRR expression was significantly reduced in nonlesional psoriatic skin versus skin from healthy control subjects in the most comprehensive dataset identified (Fig. 1). These findings indicate that SIGIRR expression is reduced even in noninflamed skin from psoriasis patients and may represent a genetic/environmental marker for the onset of psoriatic inflammation. Along these lines, it would be of interest to determine whether any genetic variants at the SIGIRR gene locus are associated with susceptibility and/or pathogenesis of psoriatic disease as has recently been reported for IL1RL2 (9).

Although SIGIRR has been found to suppress proinflammatory signaling by related IL-1 family members, to our knowledge, this has not previously been reported for the IL-36 receptor. Our data identify SIGIRR as a negative regulator of IL-36 responses, particularly in DCs and keratinocytes. Similarly, SIGIRR has been shown to regulate TLR4 and 9 responses in DCs (18), TLR1/2, TLR3, TLR4, and TLR9 responses in mouse spleen monocytes (15), IL-1R, and TLR4 responses in splenocytes and mouse lung, kidney and colon tissue (17) and IL-33 responses in Th2 cells (16). Recently, SIGIRR has also been shown to act as a coreceptor for IL-37, an anti-inflammatory cytokine of the IL-1 family (13). Furthermore, IL-37, like SIGIRR, is downregulated in lesional psoriatic skin and restricts inflammation in experimental models of the disease (3436). Although such observations raise the possibility that the interaction between IL-37 and SIGIRR may be significant in human skin, our data demonstrates that SIGIRR can also act in an IL-37–independent fashion to suppress dermal inflammation as no murine homolog of the IL37 gene has been identified (37).

We demonstrate that SIGIRR activity, in restricting IL-36–driven proinflammatory responses, is evident in DCs and keratinocytes. It has previously been shown that DCs are major producers and responders of IL-36 cytokines in the skin, and their absence in IMQ model of psoriasis abrogates inflammation and neutrophil infiltration (4). In the same report, bone marrow chimera studies revealed that radio-resistant skin cells were required for IL-36–mediated psoriasiform inflammation (4), and we and others have identified keratinocytes as critical orchestrators of IL-36 signaling in this model of psoriasiform inflammation (11, 33). In the absence of keratinocyte IL-36R expression, levels of neutrophil infiltration and expression of the neutrophil chemoattractant CXCL1 were significantly reduced (11). In this study, we demonstrate for the first time, to our knowledge, a role for SIGIRR activity on keratinocytes, whereby it acts to restrict these IL-36R–mediated responses. In addition, SIGIRR inhibited the enhancement of CD4+ Th1 cell differentiation induced by IL-36 ligands (38) (Supplemental Fig. 2). Interestingly, SIGIRR has also previously been shown to suppress Th1 cell responses in a Pseudomonas aeruginosa–induced model of keratitis (39). Although Th1 cells are not considered as playing a prominent role in IMQ-induced psoriasiform inflammation in mice, they are an established feature of human psoriatic inflammation and may play an important role in this setting (40, 41). Although the specific pathogenic role of neutrophils in psoriatic inflammation remains to be fully determined, they are considered to play a prominent pathogenic role in certain types of psoriatic disease, such as generalized pustular psoriasis (GPP) (6, 7). It is also noteworthy that IL-36 cytokines have emerged as key orchestrators of pustular inflammation in GPP and the activation of proinflammatory neutrophil responses in the skin are an established pathogenic feature of IMQ-induced IL-36R–dependent inflammation in mice (4, 11). As such it will also be of interest to determine whether SIGIRR activity is altered in GPP patients.

The IL-17A/IL-23 axis is a central pathway in driving psoriatic skin inflammation (42, 43), and SIGIRR-deficient mice were found to have increased expression of these cytokines in inflamed skin. These data are consistent with previously published data from our group, demonstrating SIGIRR regulation of the IL-17A/IL-23 axis in the IMQ model of psoriasis, possibly through negative regulation of IL-1R signaling on γδ-T cells (24). As IL-36 cytokines have previously been reported to enhance γδ-T cell responses (44), we investigated whether, similar to IL-1β, IL-36 can also induce IL-17A secretion by γδ-T cells. However, IL-36 did not induce any changes in expression of IL-17A by γδ-T cells. Together these data indicate that negative regulation of IL-1R, and not IL-36R signaling, is the likely mechanism through which SIGIRR activity directly modulates γδ-T cell responses seen in the skin.

The observation that SIGIRR regulates IL-36 responses in keratinocytes agrees with previous studies indicating that both SIGIRR and IL-36 cytokines act at epithelial sites, raising the possibility of importance in epithelial barrier function and microbiome regulation. SIGIRR has previously been shown to maintain homeostasis by controlling immune tolerance to commensal bacteria in the intestine (23), whereas we and others have previously shown that IL-36 cytokines can act to regulate intestinal microbiome and barrier function (45, 46). Although it is unclear as to whether it plays a role in disease pathogenesis, skin microbiome dysbiosis is a feature of psoriatic disease (47, 48), and a potential role for IL-36R and SIGIRR in regulating the maintenance of the skin microbiome and barrier function warrants further investigation.

In summary, we have described a novel role for SIGIRR in regulating pathogenic IL-36 cytokine signaling in the inflamed skin in psoriasis. A deeper understanding of how this central orchestrating pathway is dysregulated in disease pathogenesis will facilitate efforts aimed at targeting activity more effectively in the clinic.

This work was supported by research funding awards to P.T.W. from the National Children’s Research Centre, Dublin, Ireland (K/17/1) and the Health Research Board, Ireland (HRA-POR-2015-1066).

The online version of this article contains supplemental material.

Abbreviations used in this article

BMDC

bone marrow–derived DC

BMDM

bone marrow--derived macrophage

DC

dendritic cell

GEO

Gene Expression Omnibus

GPP

generalized pustular psoriasis

IMQ

imiquimod

rm

recombinant mouse

wt

wild-type

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The authors have no financial conflicts of interest.

Supplementary data