AIM OF THE STUDY:Lebrikizumab monotherapy demonstrated efficacy for adults and adolescents with moderate-to-severe atopic dermatitis (AD). This study aims to evaluate the relationship between lebrikizumab serum levels and stable deep response after treatment cessation in Week 16-responder patients who maintained stable EASI 90 response post-induction up to Week 52. MATERIALS AND METHODS:Analysis was pooled from two identically designed, randomized, double-blind, placebo-controlled, phase 3 trials, ADvocate1 (NCT04146363; conducted from September 24, 2019, to May 3, 2022) and ADvocate2 (NCT04178967; conducted from October 29, 2019, to April 28, 2022). The analysis subgroup includes those patients who were Week-16 responders, have been re-randomized to receive placebo (withdrawal arm), and maintained a stable EASI 90 response for at least 80% of visits up to 52 weeks, without rescue medication. Serum lebrikizumab was measured at Weeks 4, 16, 32, and 52. RESULTS:During withdrawal, 28% of responders (17/60) maintained a stable EASI 90 response with no or minimal fluctuations, including at Week 52, without rescue medication. Mean serum lebrikizumab decreased by 92% and over 99% from Week 16 to Weeks 32 and 52, respectively (Week 16 = 92.4 μg/mL; Week 32 = 7.3 μg/mL; Week 52 = 0.15 μg/mL). CONCLUSION:Lebrikizumab demonstrated off-therapy maintenance of response in an AD patient subset for at least 38 weeks post-discontinuation.

Lebrikizumab is a novel monoclonal antibody that selectively binds to interleukin (IL)-13 with high affinity and a slow dissociation rate. We assayed serum from select patients enrolled in ADvocate1 and ADvocate2 to determine the impact of lebrikizumab on circulating biomarkers and pathways relevant to atopic dermatitis (AD) and to assess the correlation between key biomarkers and clinical measures of improvement. At baseline, IL-13, CC motif chemokine ligand (CCL)13, CCL17, CCL22, total immunoglobulin (Ig)E, IL-5, and periostin were elevated in patients with moderate-to-severe AD versus healthy controls (p < 0.001). Baseline Eczema and Area Severity Index (EASI) and Investigator’s Global Assessment (IGA) scores were significantly correlated with IL-13, IL-5, CCL13, CCL22, and CCL26. Lebrikizumab induced rapid and progressive reductions in CCL13, CCL17, CCL22, and periostin at weeks 4, 16, and 52 compared with baseline (p < 0.05). AD-associated pathways linked to cytokine signaling were significantly improved at weeks 4 and 16. Improvements in EASI, IGA, and the Pruritus Numeric Rating Scale were correlated with reductions in CCL13, CCL17, CCL22, CCL26, and periostin across all time points. After multiple testing correction and adjusting for sex and race as covariates, we identified the chemokine CCL26 as a pharmacodynamic marker for lebrikizumab response at weeks 4 and 16. Selective inhibition of IL-13 with lebrikizumab monotherapy induced progressive inhibition of systemic biomarkers and pathways of type 2 inflammation, which correlated with clinical measures of improvement in patients with moderate-to-severe AD. NCT04146363 and NCT04178967. Atopic dermatitis (AD, also called atopic eczema) can cause various debilitating symptoms including red, scaly, and itchy skin that can worsen a person’s quality of life. Imbalance of normal immune system molecules, such as cytokines (substances released by cells involved in inflammation), play a central role in the development of AD. The aim of this study was to assess the impact of lebrikizumab, a treatment for AD that targets a cytokine called interleukin (IL)-13, on normalizing the levels of immune system molecules. Patients with AD were treated with lebrikizumab in two phase 3, placebo-controlled studies for 52 weeks. The results of this analysis demonstrated that numerous cytokines and other immune system molecules, including IL-13, were elevated in patients with moderate-to-severe AD at the start of the study compared with healthy controls (people who did not have AD). However, after patients with AD received treatment with lebrikizumab, rapid and progressive reductions in molecules associated with AD were observed through 52 weeks. In addition, the pathways that signal the cytokines associated with AD were significantly improved 4 and 16 weeks after receiving treatment. These improvements correlated with improved disease activity measures. Overall, this analysis demonstrated that treatment with lebrikizumab normalized several molecular pathways in patients with moderate-to-severe AD as compared with the levels of these molecules in healthy controls. These results suggest that lebrikizumab works in inhibiting IL-13 levels in patients with AD and is sufficient to improve the signs and symptoms of the disease.

Abstract Introduction In ADvocate1 (NCT04146363) and ADvocate2 (NCT04178967), lebrikizumab demonstrated statistical superiority vs. placebo in patients with moderate-to-severe atopic dermatitis (AD). The objective of this analysis is to test for correlations between improvements in clinical outcomes and reductions in AD-relevant serum biomarkers during ADvocate1 and ADvocate2. Objectives To assess the correlation between clinical measures of atopic dermatitis (AD) and AD-specific biomarkers at baseline; and to assess the correlation between clinical measures of AD and changes in AD-specific biomarkers after treatment with lebrikizumab. Methods Full details of these studies were previously reported. Protein biomarkers were determined in available serum samples from patients receiving lebrikizumab 250 mg every 2 weeks (n=72) or placebo (n=36). Tested biomarkers included: IL-13 (baseline only), CCL2, CCL4, CCL11, CCL13, CCL17 (TARC), CCL22, CCL26 (eotaxin-3), CXCL10, total IgE, IL-4, IL-5, and periostin. Baseline biomarker levels and baseline clinical endpoints were compared using a Spearman correlation. A repeated measures correlation analysis was used to characterize paired measurement of within-patient biomarker levels and clinical endpoints at baseline, week 4, week 16, and week 52. Clinical measures of AD included Investigator’s Global Assessment (IGA), Eczema Area and Severity Index (EASI), and Pruritus Numeric Rating Scale (NRS). Results At baseline, EASI score was positively correlated with IL-13, periostin, IL-5, IgE, CCL13, CCL17, CCL22 and CCL26 (correlation coefficient>0.3, p<0.05), but not IL-4. During the studies, improvements in EASI, IGA, and Pruritus NRS were each correlated with reductions in periostin, CCL13, CCL17, CCL22, and CCL26 (correlation coefficient>0.3, p<0.05). Improvement in IGA was also correlated with a reduction in IgE (correlation coefficient>0.3, p<0.05). Conclusions Clinical measures of improvement in the signs and symptoms of AD are correlated with reductions in AD biomarkers in patients treated with lebrikizumab.

Lebrikizumab demonstrated robust efficacy as monotherapy for moderate-to-severe atopic dermatitis (AD) in two Phase 3, randomized, double-blind, placebo-controlled, 52-week trials, ADvocate1 (NCT04146363) and ADvocate2 (NCT04178967) (Silverberg, et al. N Engl J Med 2023;388:1080-91; Blauvelt, et al. Br J Dermatol 2023; 188:740–748). Among lebrikizumab responders at the end of the 16-Week induction period, EASI 75 was maintained in 82% of patients treated with continuous lebrikizumab every 4 weeks (Q4W) and by 66% of the patients in the lebrikizumab withdrawal arm at Week 52. To better understand the relationship between lebrikizumab serum concentration levels and sustained clinical response after treatment cessation in a subpopulation of lebrikizumab responders who discontinue treatment. In ADvocate1 and ADvocate2, patients received 500-mg loading doses at Week 0 and Week 2, followed by 250-mg doses every two weeks (Q2W) from Week 4 to Week 14 of the induction period. At Week 16, lebrikizumab responders were re-randomized 2:2:1 to receive lebrikizumab 250 mg Q2W, lebrikizumab 250 mg Q4W or placebo Q2W (lebrikizumab withdrawal). Patients in the current analysis included lebrikizumab responders who were withdrawn from treatment and maintained EASI 90 for 80% of the visits during the withdrawal period, achieved EASI 90 at the Week-52 visit, and did not use rescue medication with data pooled from ADvocate1 and ADvocate2. From Weeks 16 to 52, EASI was assessed every 4 weeks. Lebrikizumab serum concentration levels were measured at Weeks 16, 32, and 52. We evaluated the mean serum concentrations over time; the reduction in the lebrikizumab concentrations; and the number of half-lives during the withdrawal period, as calculated by a population pharmacokinetic (popPK) model-estimated half-life. 17 patients (28%) of the 60 lebrikizumab responders who were withdrawn from treatment maintained EASI 90 for 80% of the visits during the 38-week withdrawal period, achieved EASI 90 at Week 52, and did not use rescue medication. Withdrawal-period pharmacokinetic data were available from 16 of these 17 patients. At Week 16, the mean (SD) serum lebrikizumab concentration was 92.4 (29.9) μg/mL. The mean (SD) serum concentrations decreased to 7.3 (14.0) μg/mL at Week 32 and 0.15 (0.20) μg/mL at Week 52, representing 92% and >99% reductions, respectively. At Week 52, 12 of the 16 patients had serum concentrations below the lower level of quantification (LLOQ: 0.09 μg/mL) for the clinical assay. In the popPK analysis, the mean elimination half-life for lebrikizumab was approximately 24.5 days. Lebrikizumab, therefore, had undergone approximately 5 half-lives at Week 32 and 10.9 half-lives at Week 52; it should be noted 5 to 7 half-lives for a biologic are often considered for a washout period (Evans. J Exp Stroke Transl Med 2010; 9:8-18). In this analysis from ADvocate1 and ADvocate2, a subset of patients who were randomly withdrawn from lebrikizumab maintained a stable EASI 90 response up to Week 52 with negligible remaining lebrikizumab serum concentrations. This is the first analysis that could provide additional insights into lebrikizumab therapy-free remission. Further studies are needed to identify and characterize this subpopulation of AD patients and lebrikizumab’s potential disease-modifying properties.

AimLebrikizumab is an interleukin (IL)-13 inhibitor that specifically blocks IL-13 signaling. Here, we report the effects of lebrikizumab on asthma serum biomarkers in 2 phase 3 clinical studies. MethodsLAVOLTA I and LAVOLTA II are replicate, double-blind, placebo-controlled trials with 52-week placebo-controlled treatment periods that evaluated lebrikizumab 37.5- and 125-mg doses every 4 weeks. Patients were aged 18-75 years with uncontrolled asthma on stable background therapy. Biomarkers assessed included immunoglobulin E (IgE), periostin, CC motif chemokine ligand (CCL)13, and CCL17. Statistical significance was assessed for difference in fold-change for lebrikizumab versus placebo using a mixed-effects model for repeated measures. ResultsAt early time points in LAVOLTA I and II (weeks 1 and 4), decreases in periostin and CCL13 were statistically significant versus placebo (all p < 0.001) for both lebrikizumab doses. For the 125-mg lebrikizumab dose at week 1 in LAVOLTA I, the decrease in CCL17 was statistically significant (p = 0.001). Reductions in periostin, CCL13, and CCL17 were maintained throughout the trial duration. Significant decreases versus placebo (p <= 0.001) were seen in IgE by weeks 12 and 24 in LAVOLTA I and LAVOLTA II, respectively. ConclusionSignificant reductions in relevant inflammatory biomarkers were observed in the LAVOLTA I and LAVOLTA II studies.

Background Prurigo nodularis (PN) is a severely pruritic inflammatory skin condition characterized by symmetrically distributed hyperkeratotic nodules. Immune dysregulation is suggested to underly the mechanism of itch in PN with type 2 immune mediators found to be upregulated in the PN lesional skin (1). However, no study characterizing the systemic inflammatory signature of PN has yet been conducted.

Lebrikizumab (LEB) was studied for treating immune mediated disorders, including asthma and atopic dermatitis. LAVOLTA I (NCT01867125) and II (NCT01868061) (LI&LII) were duplicate randomized, placebo-controlled Phase 3 LEB trials in uncontrolled asthma that enrolled subjects irrespective of asthma exacerbation history, baseline blood eosinophilia, or FeNO. LI&II failed to show consistently significant results in asthma exacerbation rate reduction. Here, effects of LEB on inflammatory biomarkers were assessed in patients with asthma. Serum samples were tested using immunoassays for periostin (IL-13 activation marker), CCL13 (driver of chemotaxis in immune cells), CCL17 (driver of chemotaxis in Th2 cells), and IgE (hallmark of allergic inflammation). Mean changes from baseline in these markers are reported and change from baseline comparisons were evaluated using MMRM with different contrasts. Within-biomarker multiplicity controls were performed. In LI, for 125mg LEB Q4W (N=359) vs placebo Q4W (N=362) at week 1, reductions from baseline were -35.6 vs -1.7 pg/mL for CCL13; -59.3 vs -4.6 pg/mL for CCL17; and -5.3 vs -1.2 ng/mL for periostin; and week 12 reductions from baseline were -49.5 vs -8.8 IU/mL for IgE. Differences with 125mg LEB Q4W were significant (p <0.0001), differences with placebo were not. Similar reductions were seen for LEB 37.5mg Q4W and for LII. Reductions were maintained during the 52-week LI&LII treatment periods. LEB treatment decreased inflammatory biomarkers in asthma. Reductions in circulating inflammatory biomarkers were observed as early as 1 week following a single LEB dose. Improvements in inflammation were maintained during 52 weeks of LEB treatment.

IL-13 is the primary upregulated cytokine in atopic dermatitis (AD) skin and is the pathogenic mediator driving AD pathophysiology. Lebrikizumab, tralokinumab and cendakimab are therapeutic monoclonal antibodies (mAb) that target IL-13. We undertook studies to compare in vitro binding affinities and cell-based functional activities of lebrikizumab, tralokinumab and cendakimab. Lebrikizumab bound IL-13 with higher affinity (as determined using surface plasma resonance) and slower off-rate. It was more potent in neutralizing IL-13-induced effects in STAT6 reporter and primary dermal fibroblast periostin secretion assays than either tralokinumab or cendakimab. Live imaging confocal microscopy was employed to determine the mAb effects on IL-13 internalization into cells via the decoy receptor IL-13Rα2, using A375 and HaCaT cells. The results showed that only the IL-13/lebrikizumab complex was internalized and co-localized with lysosomes, whereas IL-13/tralokinumab or IL-13/cendakimab complexes did not internalize. Lebrikizumab is a potent, neutralizing high-affinity antibody with a slow disassociation rate from IL-13. Additionally, lebrikizumab does not interfere with IL-13 clearance. Lebrikizumab has a different mode of action to both tralokinumab and cendakimab, possibly contributing to the clinical efficacy observed by lebrikizumab in Ph2b/3 AD studies.

Background: Prurigo nodularis (PN) is an extremely pruritic, chronic inflammatory skin disease. Little is known about systemic inflammation in PN. Objective: To characterize plasma inflammatory biomarkers in patients with PN and investigate the presence of disease endotypes. Methods: In this cross-sectional study, Olink proteomic analysis was performed on plasma samples from patients with PN (n = 29) and healthy controls (n = 18). Results: Patients with PN had increased levels of 8 circulating biomarkers compared to controls, including tumor necrosis factor, C-X-C Motif Chemokine Ligand 9, interleukin-12B, and tumor necrosis factor receptor superfamily member 9 (P \.05). Two PN clusters were identified in cluster 1 (n = 13) and cluster 2 (n = 16). Cluster 2 had higher levels of 25 inflammatory markers than cluster 1. Cluster 1 had a greater percentage of patients with a history of myelopathy and spinal disc disease compared with cluster 2 (69% vs 25%, P = .03). Patients in cluster 2 were more likely to have a history of atopy (38% in cluster 2 vs 8% in cluster 1, P = .09). Limitations: Small sample size precludes robust subgroup analyses. Conclusion: This study provides evidence of neuroimmune-biased endotypes in PN and can aid clinicians in managing patients with PN that are nonresponsive to traditional therapies. ( J Am Acad Dermatol 2023;88:1101-9.)

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Importance:Prurigo nodularis (PN) is a chronic heterogeneous inflammatory skin disease.Objective:To elucidate which components of type 2 inflammation are dysregulated systemically in PN.Design:Whole blood was obtained from PN patients with uncontrolled disease and control patients without pruritus. Plasma was assayed for IL-4, IL-5, IL-13, IgE, and periostin. ANOVA was utilized to compare PN and control patients and multiple-hypothesis adjusted p-value was calculated with the significance threshold at 0.05. Clustering was performed using K-means clustering.Participants:PN patients (n = 29) and controls (n = 18) from Johns Hopkins Dermatology had similar age sex, and race distributions.Results:Single-plex assays of the biomarkers demonstrated elevated circulating plasma IL-13 (0.13 vs. 0.006 pg/mL, p = 0.0008) and periostin (80.3 vs. 60.2 ng/mL, p = 0.012) in PN compared to controls. IL-4 (0.11 vs. 0.02 pg/mL, p = 0.30) and IL-5 (0.75 vs. 0.40 pg/mL, p = 0.10) were not significantly elevated, while IgE approached significance (1202.0 vs. 432.7 ng/mL, p = 0.08). Clustering of PN and control patients together revealed two clusters. Cluster 1 (n = 36) consisted of 18 PN patients and 18 controls. Cluster 2 (n = 11) consisted entirely of PN patients (p < 0.01). Cluster 2 had higher levels of IL-13 (0.33 vs. 0.008 pg/mL, p = 0.0001) and IL-5 (1.22 vs. 0.43 pg/mL, p = 0.03) compared to cluster 1.Conclusion and relevance:This study demonstrates elevation of IL-13 and periostin in the blood of PN patients, with distinct clusters with varying degrees of type 2 inflammation. Given this heterogeneity, future precision medicine approaches should be explored in the management of PN.

Dear Editor, therapeutic intervention for atopic dermatitis (AD) focuses on neutralizing effector cytokines such as interleukin (IL)‐4 and IL‐13·1,2 IL‐33 is a driver of T helper (Th)2‐mediated inflammation and is upstream of IL‐4 and IL‐13. AD lesional skin includes more IL‐33‐expressing cells than nonlesional skin,3 while serum levels of IL‐33 are reported to be sevenfold higher in patients with AD than in healthy control participants and correlated with disease severity·4 This and proof of concept data led to the hypothesis that an anti‐IL‐33 antibody could be efficacious for the treatment of AD·5 This phase II, randomized, double‐blind, placebo‐controlled study (NCT03831191) aimed to evaluate the safety and efficacy of LY3375880, a human IgG4‐variant monoclonal antibody that binds and neutralizes soluble human IL‐33, in patients with moderate‐to‐severe AD. Investigators evaluated patients in 58 centres in nine countries in Asia, Europe, South America, and North America. Patients [Validated Investigator's Global Assessment ‐ Atopic Dermatitis (vIGA‐AD) score of ≥ 3 and an inadequate response to topical medications] were stratified according to disease severity (vIGA‐AD 3 vs. 4) and geographic region (Japan vs. non‐Japan) and randomized to receive subcutaneous injections of placebo or LY3375880 (50, 150 or 600 mg) (1: 1: 1: 1 ratio using an interactive web‐response system) every 4 weeks for 16 weeks. The primary outcome sought was the proportion of patients achieving vIGA‐AD of 0 or 1 with a ≥ 2‐point improvement at week 16. An internal assessment committee conducted an unblinded interim analysis of efficacy and safety data when one‐third of patients had completed 16 weeks of treatment or discontinued.

Background Interleukin-33 (IL-33) is an alarmin that is released following cellular damage, mechanical injury, or necrosis. It is a member of the IL-1 family and binds to a heterodimer receptor consisting of ST2 and IL-1RAP to induce the production of a wide range of cellular mediators, including the type 2 cytokines IL-4, IL-5 and IL-13. This relationship has led to the hypothesis that the IL-33/ST2 pathway is a driver of allergic disease and inhibition of the IL-33 and ST2 association could have therapeutic benefit. Methods In this paper, we describe the selection of a phage antibody through the ability to bind human IL-33 and block IL-33/ST2 interaction. This hit antibody was then affinity matured by site-directed mutagenesis of the antibody complementarity-determining regions (CDRs). Further characterization of a fully human monoclonal antibody (mAb), torudokimab (LY3375880) included demonstration of human IL-33 neutralization activity in vitro with an NFκB reporter assay and IL-33 induced mast cell cytokine secretion assay, followed by an in vivo IL-33-induced pharmacodynamic inhibition assay in mice that used IL-5 production as the endpoint. Results Torudokimab is highly specific to IL-33 and does not bind any of the other IL-1 family members. Furthermore, torudokimab binds human and cynomolgus monkey IL-33 with higher affinity than the binding affinity of IL-33 to ST2, but does not bind mouse, rat, or rabbit IL-33. Torudokimab’s half-life in cynomolgous monkey projects monthly dosing in the clinic. Conclusion Due to torudokimab’s high affinity, its ability to completely neutralize IL-33 activity in vitro and in vivo, and the observed cynomolgus monkey pharmacokinetic properties, this molecule was selected for clinical development.

3D multiplanar ultrasound with Doppler and high resolution probes studied cases of inflammatory disease over 20 years including 18 months of Covid-19 inflammation. The depth of epidermal and dermal disease was followed to guide treatment including biologics and adverse events were documented by imaging as they occurred. Toxins from heavy metals including uranium were detected and quantified as discrete focal high echoes on the 4D image reconstruction and inflammatory neovascularity was documented with the 3D Doppler histogram technology in cases of dermatomyositis, lupus, scleroderma, psoriasis and rosacea. In rosacea, the depth and density of the sebaceous glands correlated with the erythema and enthesitis of the digits and joint synovitis was associated with nail bed hyperemia in cases of psoriasis. In Covid-19 the vascular hyperemia and presence of microvessel thrombosis in the upper and lower extremities indicated worse clinical prognosis which was verified with point of care lung ultrasound observations and Doppler analysis of pleural based parenchymal pneumonitis. Breast erythematous changes included subdermal cystic components and 3 cases of inflammatory breast cancer were discovered by intradermal lymphedema after unsuccessful conventional therapies.

Background Many patients with migraines suffer from allergies and vice versa, suggesting a relationship between biological mechanisms of allergy and migraine. It was proposed many years ago that mast cells may be involved in the pathophysiology of migraines. We set out to investigate the relationship between mast cell activation and known neurogenic peptides related to migraine. Methods Cultured human mast cells were assayed for the presence of neuropeptides and their receptors at the RNA and protein level. Immunohistochemistry analyses were performed on tissue resident and cultured mast cells. Mast cell degranulation assays were performed and pituitary adenylate cyclase-activating polypeptide (PACAP) activity was measured with a bioassay. Results We found that cultured and tissue resident human mast cells contain PACAP in cytoplasmic granules. No other neurogenic peptide known to be involved in migraine was detected, nor did mast cells express the receptors for PACAP or other neurogenic peptides. Furthermore, mast cell degranulation through classic IgE-mediated allergic mechanisms led to the release of PACAP. The PACAP released from mast cells was biologically active, as demonstrated using PACAP receptor reporter cell lines. We confirmed existing literature that mast cell degranulation can also be induced by several neurogenic peptides, which also resulted in PACAP release. Conclusion Our data provides a potential biological explanation for the association between allergy and migraine by demonstrating the release of biologically active PACAP from mast cells.

Breaking tolerance is a key event leading to autoimmunity, but the exact mechanisms responsible for this remain uncertain. Here we show that the alarmin IL-33 is able to drive the generation of autoantibodies through induction of the B cell survival factor BAFF. A temporary, short-term increase in IL-33 results in a primary (IgM) response to self-antigens. This transient DNA-specific autoantibody response was dependent on the induction of BAFF. Notably, radiation resistant cells and not myeloid cells, such as neutrophils or dendritic cells were the major source of BAFF and were critical in driving the autoantibody response. Chronic exposure to IL-33 elicited dramatic increases in BAFF levels and resulted in elevated numbers of B and T follicular helper cells as well as germinal center formation. We also observed class-switching from an IgM to an IgG DNA-specific autoantibody response. Collectively, the results provide novel insights into a potential mechanism for breaking immune-tolerance via IL-33-mediated induction of BAFF.

Interleukin (IL)-33 is a member of the IL-1 family. IL-33 effects are mediated through its receptor, ST2 and IL-1RAcP, and its signaling induces the production of a number of pro-inflammatory mediators, including TNFα, IL-1β, IL-6, and IFN-γ. There are conflicting reports on the role of IL-33 in bone homeostasis, with some demonstrating a bone protective role for IL-33 whilst others show that IL-33 induces inflammatory arthritis with concurrent bone destruction. To better clarify the role IL-33 plays in bone biology in vivo, we studied IL-33 KO mice as well as mice in which the cytokine form of IL-33 was overexpressed. Mid-femur cortical bone mineral density (BMD) and bone strength were similar in the IL-33 KO mice compared to WT animals during the first 8 months of life. However, in the absence of IL-33, we observed higher BMD in lumbar vertebrae and distal femur in female mice. In contrast, overexpression of IL-33 resulted in a marked and rapid reduction of bone volume, mineral density and strength. Moreover, this was associated with a robust increase in inflammatory cytokines (including IL-6 and IFN-γ), suggesting the bone pathology could be a direct effect of IL-33 or an indirect effect due to the induction of other mediators. Furthermore, the detrimental bone effects were accompanied by increases in osteoclast number and the bone resorption marker of C-terminal telopeptide collagen-I (CTX-I). Together, these results demonstrate that absence of IL-33 has no negative consequences in normal bone homeostasis while high levels of circulating IL-33 contributes to pathological bone loss.

For over 80 years, human vaccines have used aluminium-based adjuvants (alum) despite incomplete understanding of how alum enhances the immune response. One proposed mechanism is that alum induces cellular necrosis and release of endogenous danger signals. These signals induce inflammation-associated cytokines that lead to humoral immunity. IL-33 is proposed to be released from necrotic cells and can elicit TH2-biased cytokines. Therefore, we investigated a role for IL-33 in the adjuvant activity of alum. We show that alum induces cellular necrosis and elevates IL-33 levels shortly after intraperitoneal administration. Alum or IL-33 injection induces similar significant increases in IL-5, IL-6, KC, MCP-1 and MIP-1β. We establish that many of the alum-induced cytokines are dependent on IL-33 using IL-33 knockout mice or an IL-33-neutralizing recombinant ST2 receptor. Interestingly, IL-33 itself can function as an adjuvant. While IL-33 only induces a marginal primary response, it facilitates a secondary response comparable to alum and correlates with delayed splenic germinal center formation. Alum-induced antibody responses in IL-33 knockout mice are similar to those in wild-type mice, demonstrating that IL-33 is not absolutely required for the alum-induced humoral response. Our results provide novel insights into the mechanism of action behind alum-induced cytokine responses and show that IL-33 is sufficient to provide a robust secondary antibody response independently of alum.

Human vaccines have used aluminium-based adjuvants (alum) for >80 years despite incomplete understanding of how alum enhances the immune response. Alum can induce the release of endogenous danger signals via cellular necrosis which elicits inflammation-associated cytokines resulting in humoral immunity. IL-33 is proposed to be one such danger signal that is released from necrotic cells. Therefore, we investigated whether there is a role for IL-33 in the adjuvant activity of alum. We show that alum-induced cellular necrosis results in elevated levels of IL-33 following injection in vivo. Alum and IL-33 induce similar increases in IL-5, KC, MCP-1, MIP-1α and MIP-1β; many of which are dependent on IL-33 as shown in IL-33 knockout mice or by using an IL-33-neutralizing recombinant ST2 receptor. Furthermore, IL-33 itself functions as an adjuvant that, while only inducing a marginal primary response, facilitates a robust secondary response comparable to that observed with alum. However, IL-33 is not absolutely required for alum-induced antibody responses since alum mediates similar humoral responses in IL-33 knockout and wild-type mice. Our results provide novel insights into the mechanism of action behind alum-induced cytokine responses and show that IL-33 is sufficient to provide a robust secondary antibody response independently of alum.