Elarekibep (PRS-060/AZD1402), a new class of inhaled Anticalin medicine targeting IL-4Ra for type 2 endotype asthma.

BACKGROUND: Type 2 endotype asthma is driven by IL-4 and IL-13 signaling via IL-4Ra, which is highly expressed on airway epithelium, airway smooth muscle, and immunocytes in the respiratory mucosa, suggesting potential advantages of an inhalable antagonist. Lipocalin 1 (Lcn1), a 16 kDa protein abundant in human periciliary fluid, has a robust drug-like structure well suited to protein engineering, but it has never been used to make an inhaled Anticalin protein therapeutic. OBJECTIVES: We sought to reengineer Lcn1 into an inhalable IL-4Ra antagonist and assess its pharmacodynamic/kinetic profile. METHODS: Lcn1 was systematically modified by directed protein mutagenesis yielding a high-affinity, slowly dissociating, long-acting full antagonist of IL-4Ra designated PRS-060 with properties analogous to dupilumab, competitively antagonizing IL-4Ra-dependent cell proliferation, mucus induction, and eotaxin expression in vitro. Because PRS-060 displayed exquisite specificity for human IL-4Ra, with no cross-reactivity to rodents or higher primates, we created a new triple-humanized mouse model substituting human IL-4Ra, IL-4, and IL-13 at their correct syntenic murine loci to model clinical dosing. RESULTS: Inhaled PRS-060 strongly suppressed acute allergic inflammation indexes in triple-humanized mice with a duration of action longer than its bulk clearance, suggesting that it may act locally in the lung. CONCLUSION: Lcn1 can be reengineered into the Anticalin antagonist PRS-060 (elarekibep), exemplifying a new class of inhaled topical, long-acting therapeutic drugs with the potential to treat type 2 endotype asthma.

Characterization of High and Low IFNG-Expressing Subgroups in Atopic Dermatitis.

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases, with an increasing number of targeted therapies available. While biologics to treat AD exclusively target the key cytokines of type 2 immunity, Janus kinase inhibitors target a broad variety of cytokines, including IFN-γ. To better stratify patients for optimal treatment outcomes, the identification and characterization of subgroups, especially with regard to their IFNG expression, is of great relevance, as the role of IFNG in AD has not yet been fully clarified. This study aims to define AD subgroups based on their lesional IFNG expression and to characterize them based on their gene expression, T cell secretome and clinical attributes. RNA from the lesional and non-lesional biopsies of 48 AD patients was analyzed by RNA sequencing. Based on IFNG gene expression and the release of IFN-γ by lesional T cells, this cohort was categorized into three IFNG groups (high, medium, and low) using unsupervised clustering. The low IFNG group showed features of extrinsic AD with a higher prevalence of atopic comorbidities and impaired epidermal lipid synthesis. In contrast, patients in the high IFNG group had a higher average age and an activation of additional pro-inflammatory pathways. On the cellular level, higher amounts of M1 macrophages and natural killer cell signaling were detected in the high IFNG group compared to the low IFNG group by a deconvolution algorithm. However, both groups shared a common dupilumab response gene signature, indicating that type 2 immunity is the dominant immune shift in both subgroups. In summary, high and low IFNG subgroups correspond to intrinsic and extrinsic AD classifications and might be considered in the future for evaluating therapeutic efficacy or non-responders.

Roles of Periostin in Asthma.

Periostin is a matricellular protein that is deeply involved in type-2/eosinophilic airway inflammation and remodeling in asthma. While its expression in airway epithelial cells is correlated with the thickness of airway basement membrane, more importantly, periostin can be detected stably in blood with little variability, reflecting airway type-2 inflammation and remodeling. As for a result, serum periostin can serve as a valuable marker to identify patients with type-2 severe asthma who are insensitive to inhaled corticosteroids, and consequently have the excess decline of pulmonary function with asthma exacerbations. Serum periostin may significantly help to improve management of patients with severe asthma.

Chronic Pruritus: A Review of Neurophysiology and Associated Immune Neuromodulatory Treatments.

Chronic pruritus remains a difficult condition to treat with many non-specific therapeutic options. Recent scientific discoveries have elucidated the physiology associated with pruritus. Combined with clinical and experimental trials with immune-modulatory agents, chronic pruritus now has novel treatment options with known mechanisms of action. This review goes over recent scientific progress in understanding the molecular mechanisms governing pruritus, the cross-talk between the immune and nervous systems that regulate itch, and central nervous pathways and projections affected by itch. In light of these recent discoveries, we briefly discuss a growing body of data from relevant clinical trials investigating immunomodulatory drugs targeting specific interleukin receptors (IL-4/13/31) and intracellular signaling (e.g., Janus kinase) pathways. We focus on the physiological processes that control this complex physical and emotional experience, as well as the role of newer drugs used to treat chronic itch.

Identification of potential biomarkers for post-traumatic complications released after trauma-hemorrhage from murine Kupffer cells and its investigation in lung and liver.

CONTEXT: Early diagnosis of complications after severe trauma by specific biomarkers remains difficult. OBJECTIVE: Identify potential new biomarkers for early diagnosis of post-traumatic complications. MATERIAL AND METHODS: Mice underwent pressure-controlled hemorrhage or sham procedure. Four hours later, genome-wide expression of isolated Kupffer cells was compared with controls using Affymetrix-Genechip-Expression-Analysis and real-time-PCR. RESULTS: Expression analysis and real-time-PCR revealed a significant increase of gene expression of Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5. CONCLUSION: Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5 might represent new biomarkers for early diagnosis of post-traumatic complications, if they are linked to the development of post-traumatic complications.

Atopic Dermatitis Mediates the Association Between an IL4RA Variant and Food Allergy in School-Aged Children.

BACKGROUND: Atopic dermatitis (AD) and food allergy (FA) may share genetic risk factors. It is unknown whether genetic factors directly cause FA or are mediated through AD, as the dual-allergen hypothesis suggests. OBJECTIVE: To test the hypothesis that AD mediates the relationship between an IL-4 receptor alpha chain gene (IL4RA) variant, the human IL-4 receptor alpha chain protein-R576 polymorphism, and FA. METHODS: A total of 433 children with asthma enrolled in the School Inner-City Asthma Study underwent genotyping for the IL4RA576 allele. Surveys were administered to determine FA, AD, and associated allergic responses. Mediation analysis was performed adjusting for race and ethnicity, age, sex, and household income. Multivariate models were used to determine the association between genotype and FA severity. RESULTS: AD was reported in 193 (45%) and FA in 80 children (19%). Each risk allele increased odds of AD 1.39-fold ([1.03-1.87], P = .03), and AD increased odds of FA 3.67-fold ([2.05- 6.57], P < .01). There was an indirect effect of genotype, mediated by AD, predicting FA; each risk allele increased the odds of FA by 1.13 (odds ratio [95% CI], Q/R = 1.13 [1.02-1.24], R/R = 1.28 [1.04-1.51]; P < .01). Each risk allele increased the odds of severe FA symptoms 2.68-fold ([1.26-5.71], P = .01). CONCLUSIONS: In a cohort of children with asthma, AD is part of the causal pathway between an IL4RA variant and FA. This variant is associated with increased risk of severe FA reactions. Addressing AD in children with an IL4RA polymorphism may modulate the risk of FA.

IL-4-Responsive B Cells Are Detrimental During Chronic Tuberculosis Infection in Mice.

In tuberculosis, T cell-mediated immunity is extensively studied whilst B cells received limited attention in human and mice. Of interest, Mycobacterium tuberculosis (Mtb) does increase IL-4 Receptor-alpha (IL4Rα) expression in murine B cells. To better understand the role of IL4Rα signalling in B cells, we compared wild type mice with B cell-specific IL4Rα deficient mice (mb1creIL-4Rα-/lox mice). Chronic Mtb aerosol infection in mb1creIL-4Rα-/lox mice reduced lung and spleen bacterial burdens, compared to littermate (IL-4Rα-/lox) control animals. Consequently, lung pathology, inflammation and inducible nitric oxide synthase (iNOS) expression were reduced in the lungs of mb1creIL-4Rα-/lox mice, which was also accompanied by increased lung IgA and decreased IgG1 levels. Furthermore, intratracheal adoptive transfer of wild-type B cells into B cell-specific IL4Rα deficient mice reversed the protective phenotype. Moreover, constitutively mCherry expressing Mtb showed decreased association with B cells from mb1creIL-4Rα-/lox mice ex vivo. In addition, supernatants from Mtb-exposed B cells of mb1creIL-4Rα-/lox mice also increased the ability of macrophages to produce nitric oxide, IL-1ß, IL-6 and TNF. Together, this demonstrates that IL-4-responsive B cells are detrimental during the chronic phase of tuberculosis in mice with perturbed antibody profiles, inflammatory cytokines and tnf and stat1 levels in the lungs.

IL4RA gene expression in relation to I50V, Q551R and C-3223T polymorphisms.

BACKGROUND: Interleukin 4 (IL-4) and its receptor play important roles in the pathologies of asthma and atopy. The alpha subunit of the IL-4 receptor (IL-4RA) is included in 2 types of receptors which have different modulatory effects on immune responses. This distinct pattern reflects involvement in the immunopathology of both asthma and atopy. A number of studies have proven the association between IL4RA gene polymorphisms and asthma and atopy, but it is still an open question whether these variants are functional. OBJECTIVES: To analyze the data from IL4RA gene expression in PBMC in relation to specific polymorphisms - the most frequently studied I50V and Q551R and the less known C-3223T. MATERIAL AND METHODS: The analysis was performed for 36 subjects, both atopic and non-atopic. Real-time polymerase chain reaction (PCR) was used with specific primers for the quantification and genotyping. Delta Ct (ΔCT) and delta-delta Ct (ΔΔCT) values were used for the relative quantification of IL4RA expression in PBMC. RESULTS: We observed no significant differences in the IL4RA expression profile between the 3 genotypes. A trend toward higher relative expression was observed for homozygous minor I50V and C-3223T genotypes. CONCLUSIONS: We did not find a statistically significant relationship between the genetic polymorphisms and the relative expression of IL4RA. The effect of genetic polymorphism on IL4RA mRNA expression could interfere with other factors, such as environmental stimuli, and should be evaluated in future studies.

The establishment of humanized IL-4/IL-4RA mouse model by gene editing and efficacy evaluation.

Asthma is a common respiratory immune disease in children and adults, and interleukin-4 (IL-4) is one of the key factors for the onset of asthma. Therefore, targeting human IL-4 and IL-4 receptor alpha (IL-4RA) has become one of the strategies for targeted therapy of cytokines. Herein, we established an animal model of asthmatic airway inflammation using double humanized IL-4/IL-4RA (hIL-4/hIL-4RA) mice, where human IL-4 and IL-4RA replaced their murine counterparts, respectively. We successfully identified the phenotype by Southern blotting, ELISA, and flow cytometry. The hIL-4/hIL-4RA mice induced by ovalbumin (OVA) exhibited several important features of asthma, such as inflammatory cell infiltration, IgE release, goblet cell hyperplasia, and Th2 cytokine secretion. Furthermore, treatment of these humanized mice with anti-human IL-4RA antibodies significantly inhibited level of these pathological indicators. Thus, hIL-4/hIL-4RA mice provide a validated preclinical mouse model to interrogate new therapeutic agents targeting this specific cytokine pathway in asthma.

Macrophage phenotypic switch orchestrates the inflammation and repair/regeneration following acute pancreatitis injury.

BACKGROUND: Impaired or hyperactive pancreas regeneration after injury would cause exocrine insufficiency or recurrent / chronic pancreatitis and potentially carcinogenesis. Macrophages are the most abundant immune cells in the regenerative pancreas, however their phenotype and role remain poorly defined. METHOD: Using caerulein-induced acute pancreatitis (AP) model, we examined the dynamic landscape of pancreatic macrophages throughout the acute inflammation to regeneration phases by flow cytometric and RNA-seq analyses. Liposome depletion of macrophages, Il4ra-/- mice as well as inhibitors were used to elucidate the role and regulatory mechanism of macrophages during pancreatic regeneration. FINDINGS: We found that M1 macrophages dominated in the pro-inflammatory phase of AP, while M2-like macrophages dominated during pancreas repair/regeneration. Depletion of macrophages at early or late regenerative stage dramatically blocked the acinar-ductal metaplasia (ADM) or delayed inflammation resolution, respectively. Moreover, alternative activation of macrophages was partially dependent on IL-4RA signaling, and ECM/AKT activation in pancreatic macrophages facilitated inflammation resolution during tissue regeneration. INTERPRETATION: Our findings illustrate a dynamic phenotype and function of macrophages during AP repair/regeneration, helping us better understand the mechanism of pancreatic regeneration and providing clues for novel therapeutic strategy.

CCL2/MCP-1 signaling drives extracellular matrix turnover by diverse macrophage subsets.

Macrophage plasticity, cellular origin, and phenotypic heterogeneity are perpetual challenges for studies addressing the biology of this pivotal immune cell in development, homeostasis, and tissue remodeling/repair. Consequently, a myriad of macrophage subtypes has been described in these contexts. To facilitate the identification of functional macrophage subtypes in vivo, here we used a flow cytometry-based assay that allows for detailed phenotyping of macrophages engaged in extracellular matrix (ECM) degradation. Of the five macrophage subtypes identified in the remodeling dermis by using this assay, collagen degradation was primarily executed by Ly6C - CCR2 + and Ly6C - CCR2 low macrophages via mannose receptor-dependent collagen endocytosis, while Ly6C + CCR2 + macrophages were the dominant fibrin-endocytosing cells. Unexpectedly, the CCL2/MCP1-CCR2 signaling axis was critical for both collagen and fibrin degradation, while collagen degradation was independent of IL-4Ra signaling. Furthermore, the cytokine GM-CSF selectively enhanced collagen degradation by Ly6C + CCR2 + macrophages. This study reveals distinct subsets of macrophages engaged in ECM turnover and identifies novel wound healing-associated functions for CCL2 and GM-CSF inflammatory cytokines.

IL-4RA gene expression in PBMC with regard to place of living and atopy status.

BACKGROUND: IL-4 and IL-4RA are key factors in allergic inflammation. IL-4 stimulates both IgE production and Th2 lymphocyte differentiation. Increased levels of IL-4 and IL-4RA have been shown in allergic patients. Genetic analyses have confirmed that polymorphisms within the IL-4RA gene influence the risk of allergy and can change the expression of the protein. Due to gene-environment interactions, this process is also likely to be modified by environmental exposure. OBJECTIVES: The aim of the study was to evaluate the IL-4RA gene expression in peripheral blood mononuclear cells (PBMC) from atopic and non-atopic subjects with regard to place of living (urban vs rural). MATERIAL AND METHODS: We enrolled 38 subjects into the study, 18 of whom were atopic. Atopy was estimated according to the results of a skin prick test. PBMC were isolated from whole blood, total RNA was extracted and reverse transcribed into cDNA. We performed real-time PCR to measure gene expression, the ACTB gene was chosen as a reference and the delta-delta Ct (ΔΔCT) method was applied for relative quantification. The Mann-Whitney U test was used for statistics. RESULTS: We did not observe any statistically significant differences in the gene expression profile between atopic and non-atopic subjects regardless of their place of living. However, a trend was observed for atopic rural inhabitants to have lower levels of IL-4RA gene expression than atopic subjects living in the town. CONCLUSIONS: The regulation of IL-4RA gene expression is complex and probably influenced by both genetic and environmental factors, such as farming exposures, which could provide the counterbalance to atopy.

Interleukin-4 Receptor Alpha Expressing B Cells Are Essential to Down-Modulate Host Granulomatous Inflammation During Schistosomasis.

Schistosomiasis (bilharzia) is a parasitic helminth disease that can cause severe inflammatory pathology leading to organ damage in humans. Failure of the host to regulate egg-driven granulomatous inflammation causes host morbidity during chronic infection with Schistosoma mansoni. Although the importance of B cells in regulating pathology during chronic infection has been well defined, the specific contribution of IL-4Rα-expressing B cells is still unknown. To address this, we examined B cell-specific IL-4Rα-deficient (mb1creIL-4Rα-/lox) mice in three experimental models of schistosomiasis: high-dose (100 cercariae), low dose (30 cercariae), and a synchronous egg challenge. In the high dose model, we found that mice deficient in IL-4Rα-expressing B cells were more susceptible to acute schistosomiasis than B cell-deficient (µMT) mice, succumbing to infection at the acute stage whereas µMT mice survived until the chronic stage. An S. mansoni egg challenge model demonstrated that deleting IL-4Rα expression specifically on B cells resulted in increased lung granulomatous pathology, suggesting a role for this B cell subset in controlling granulomatous pathology. In agreement with this, a low dose model of schistosomiasis-which mimics the course of clinical chronic disease-demonstrated that depleting IL-4Rα-expressing B cells in mb1creIL-4Rα-/lox mice considerably impaired the host ability to down-modulate granulomatous inflammation in the liver and gut during chronic schistosomiasis. Taken together, our findings indicate that within the B cell compartment, IL-4Rα-expressing B cells in particular down-modulate the deleterious egg-driven tissue granulomatous inflammation to enable host survival during schistosomiasis in mice.

Polymorphic variants of interleukin-13 R130Q, interleukin-4 T589C, interleukin-4RA I50V, and interleukin-4RA Q576R in allergic rhinitis: A pilot study.

The development of allergic rhinitis is considered to be caused by the complex interactions between genetic predisposition and environmental factors. Polymorphisms in the interleukin (IL)-13/4/4RA pathway have previously been shown to be associated with atopic diseases. The purpose of this study was to determine the association between IL-13 R130Q, IL-4 T589C, IL4 receptor alpha (IL-4RA) I50V, or IL-4RA Q576R polymorphisms and risk of allergic rhinitis in a hospital-based Malaysian population. A case-control pilot study was undertaken and genotyping of these polymorphisms was performed using polymerase chain reaction-restriction fragment length polymorphism on 54 allergic rhinitis patients and 45 healthy individuals. Polymorphism of IL-13 R130Q showed significant difference in genotype (p = 0.048) and allele (p = 0.002) frequencies in allergic rhinitis when compared with healthy controls. Individuals who were GA heterozygotes (adjusted odds ratio [OR(adj)] = 3.567; 95% CI, 1.211-10.509), and carriers of A allele genotype (OR(adj) = 3.686; 95% CI, 1.300-10.451) and A allele (OR(adj) = 3.071; 95% CI, 1.514-6.232) had an elevated risk of developing allergic rhinitis. The genotype and allele frequencies of IL-4 T589C, IL-4RA I50V, and IL-4RA Q576R polymorphisms were not significantly different between the allergic rhinitis patients and normal healthy individuals and did not show an associated risk with allergic rhinitis. Our findings indicate that polymorphic variant of IL-13 R130Q appears to be associated with increased risk for development of allergic rhinitis in a hospital-based Malaysian population but not IL-4 T589C, IL-4RA I50V, and IL-4RA Q576 polymorphisms. Additional studies using larger sample size are required to confirm our findings and its exact role in allergic rhinitis.

Interference of IL-4RA transfection to express level of STAT6 in airways / 中国免疫学杂志

Objective:To study the effect of airway IL-4RA gene transfer on asthma-associated expression of STAT6 in the mice of allergic asthma.Methods:Forty BALB/c mice were randomly divided into four groups:blank control,asthma model,asthma model administered by pLNC-laz,and asthma model administered by glucocorticoid and pLNC-IL-4RA.The ten mice in each groups were sensitized by i.p.injection of 10 ?g of OVA emulsified in 40 mg of aluminum hydroxide in a total volume of 200 ?l on the days of 1st,7th and 14th.On the 15th day,the mice were challenged via airways with OVA for 7 days,20 min a day,blank control were sensitized by i.p.injection of 200 ?l pH=7.4 PBS on days 7 and 14.Asthma model was administered by pLNC-laz or pLNC-IL-4RA for 3 days beginning at the 12th day.The mice of the group with glucocorticoid was administered with glucocorticoid on the 15th day for 7 days.24 hours after the last challenge,the mice were sacrificed by bloodletting.Serum OVA-specific IgE and blood Eosinophils were measured.The lungs were fixed by immersion in 10% formalin.Expression of STAT6 was identified by immunohistochemistry.Results:It was shown that pLNC-IL-4RA was integrated to the genomic DNA of pneumonic tissues.Retrovirus-mediated delivery of IL-4RA to airways of the asthma mice weakened airway eosinophilia triggered by either IL-13 or IL-4.Furthermore,IL-4RA delivered by retrovirus was expressed in airways of the mice with allergen sensitization,resulting in significiant reduction of expressing level of asthma-associated STAT6 in the experimental mice of allergic asthma.Conclusion:Retrovirus mediated delivery of IL-4RA to airways reduces expression of STAT6 in pneumonic tissues of asthmatic mice.Thus,the gene theraphy can be a potential therapeutic option to treat and control chronic airway inflammation and asthmatic symptoms.