Progesterone amplifies allergic inflammation and airway pathology in association with higher lung ILC2 responses.

Perimenstrual worsening of asthma occurs in up to 40% of women with asthma, leading to increased acute exacerbations requiring clinical care. The role of sex hormones during these times remains unclear. In the current study, we used a translational approach to determine whether progesterone exacerbates allergic inflammation in the traditional chicken egg ovalbumin (OVA) model in BALB/c mice. Simultaneously, we used peripheral blood mononuclear cells (PBMC) from healthy human donors to assess the effects of progesterone on circulating group 2 innate lymphoid cells (ILC2). Briefly, lungs of ovariectomized (OVX) or sham-operated female (F-Sham) controls were implanted with a progesterone (P4, 25 mg) (OVX-P4) or placebo pellet (OVX-Placebo), followed by sensitization and challenge with ovalbumin (OVA). Progesterone increased total inflammatory histologic scores, increased hyper-responsiveness to methacholine (MCh), increased select chemokines in the bronchoalveolar lavage (BAL) and serum, and increased ILC2 and neutrophil numbers, along the airways compared with F-Sham-OVA and OVX-Placebo-OVA animals. Lung ILC2 were sorted from F-Sham-OVA, OVX-Placebo-OVA and OVX-P4-OVA treated animals and stimulated with IL-33. OVX-P4-OVA lung ILC2 were more responsive to interleukin 33 (IL-33) compared with F-Sham-OVA treated, producing more IL-13 and chemokines following IL-33 stimulation. We confirmed the expression of the progesterone receptor (PR) on human ILC2, and showed that P4 + IL-33 stimulation also increased IL-13 and chemokine production from human ILC2. We establish that murine ILC2 are capable of responding to P4 and thereby contribute to allergic inflammation in the lung. We confirmed that human ILC2 are also hyper-responsive to P4 and IL-33 and likely contribute to airway exacerbations following allergen exposures in asthmatic women with increased symptoms around the time of menstruation.NEW & NOTEWORTHY There is a strong association between female biological sex and severe asthma. We investigated the allergic immune response, lung pathology, and airway mechanics in the well-described chicken egg ovalbumin (OVA) model with steady levels of progesterone delivered throughout the treatment period. We found that progesterone enhances the activation of mouse group 2 innate lymphoid cells (ILC2). Human ILC2 are also hyper-responsive to progesterone and interleukin 33 (IL-33), and likely contribute to airway exacerbations following allergen exposures in women with asthma.

Vascular smooth muscle cells in response to cholesterol crystals modulates inflammatory cytokines release and promotes neutrophil extracellular trap formation.

BACKGROUND: The formation and accumulation of cholesterol crystals (CC) at the lesion site is a hallmark of atherosclerosis. Although studies have shown the importance of vascular smooth muscle cells (VSMCs) in the disease atherosclerosis, little is known about the molecular mechanism behind the uptake of CC in VSMCs and their role in modulating immune response. METHODS: Human aortic smooth muscle cells were cultured and treated with CC. CC uptake and CC mediated signaling pathway and protein induction were studied using flow cytometry, confocal microscopy, western blot and Olink proteomics. Conditioned medium from CC treated VSMCs was used to study neutrophil adhesion, ROS production and phagocytosis. Neutrophil extracellular traps (NETs) formations were visualized using confocal microscopy. RESULTS: VSMCs and macrophages were found around CC clefts in human carotid plaques. CC uptake in VSMCs are largely through micropinocytosis and phagocytosis via PI3K-AkT dependent pathway. The uptake of CC in VSMCs induce the release inflammatory proteins, including IL-33, an alarming cytokine. Conditioned medium from CC treated VSMCs can induce neutrophil adhesion, neutrophil reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. IL-33 neutralization in conditioned medium from CC treated VSMCs inhibited neutrophil ROS production and NETs formation. CONCLUSION: We demonstrate that VSMCs due to its vicinity to CC clefts in human atherosclerotic lesion can modulate local immune response and we further reveal that the interaction between CC and VSMCs impart an inflammatory milieu in the atherosclerotic microenvironment by promoting IL-33 dependent neutrophil influx and NETs formation.

Interleukin-33/ST2 axis involvement in atrial remodeling and arrhythmogenesis.

Interleukin (IL)-33, a cytokine involved in immune responses, can activate its receptor, suppression of tumorigenicity 2 (ST2), is elevated during atrial fibrillation (AF). However, the role of IL-33/ST2 signaling in atrial arrhythmia is unclear. This study explored the pathological effects of the IL-33/ST2 axis on atrial remodeling and arrhythmogenesis. Patch clamping, confocal microscopy, and Western blotting were used to analyze the electrical characteristics of and protein activity in atrial myocytes (HL-1) treated with recombinant IL-33 protein and/or ST2-neutralizing antibodies for 48 hrs. Telemetric electrocardiographic recordings, Masson's trichrome staining, and immunohistochemistry staining of the atrium were performed in mice receiving tail vein injections with nonspecific immunoglobulin (control), IL-33, and IL-33 combined with anti-ST2 antibody for 2 weeks. IL-33-treated HL-1 cells had a reduced action potential duration, lower L-type Ca2+ current, greater sarcoplasmic reticulum (SR) Ca2+ content, increased Na+/Ca2+ exchanger (NCX) current, elevation of K+ currents, and increased intracellular calcium transient. IL-33-treated HL-1 myocytes had greater activation of the calcium-calmodulin-dependent protein kinase II (CaMKII)/ryanodine receptor 2 (RyR2) axis and nuclear factor kappa B (NF-κB) / NLR family pyrin domain containing 3 (NLRP3) signaling than did control cells. IL-33 treated cells also had greater expression of Nav1.5, Kv1.5, NCX, and NLRP3 than did control cells. Pretreatment with neutralizing anti-ST2 antibody attenuated IL-33-mediated activation of CaMKII/RyR2 and NF-κB/NLRP3 signaling. IL-33-injected mice had more atrial ectopic beats and increased AF episodes, greater atrial fibrosis, and elevation of NF-κB/NLRP3 signaling than did controls or mice treated with IL-33 combined with anti-ST2 antibody. Thus, IL-33 recombinant protein treatment promotes atrial remodeling through ST2 signaling. Blocking the IL-33/ST2 axis might be an innovative therapeutic approach for patients with atrial arrhythmia and elevated serum IL-33.

Discovery of highly immunogenic spleen-resident FCGR3+CD103+ cDC1s differentiated by IL-33-primed ST2+ basophils.

Recombinant interleukin-33 (IL-33) inhibits tumor growth, but the detailed immunological mechanism is still unknown. IL-33-mediated tumor suppression did not occur in Batf3-/- mice, indicating that conventional type 1 dendritic cells (cDC1s) play a key role in IL-33-mediated antitumor immunity. A population of CD103+ cDC1s, which were barely detectable in the spleens of normal mice, increased significantly in the spleens of IL-33-treated mice. The newly emerged splenic CD103+ cDC1s were distinct from conventional splenic cDC1s based on their spleen residency, robust effector T-cell priming ability, and surface expression of FCGR3. DCs and DC precursors did not express Suppressor of Tumorigenicity 2 (ST2). However, recombinant IL-33 induced spleen-resident FCGR3+CD103+ cDC1s, which were found to be differentiated from DC precursors by bystander ST2+ immune cells. Through immune cell fractionation and depletion assays, we found that IL-33-primed ST2+ basophils play a crucial role in the development of FCGR3+CD103+ cDC1s by secreting IL-33-driven extrinsic factors. Recombinant GM-CSF also induced the population of CD103+ cDC1s, but the population neither expressed FCGR3 nor induced any discernable antitumor immunity. The population of FCGR3+CD103+ cDC1s was also generated in vitro culture of Flt3L-mediated bone marrow-derived DCs (FL-BMDCs) when IL-33 was added in a pre-DC stage of culture. FL-BMDCs generated in the presence of IL-33 (FL-33-DCs) offered more potent tumor immunotherapy than control Flt3L-BMDCs (FL-DCs). Human monocyte-derived DCs were also more immunogenic when exposed to IL-33-induced factors. Our findings suggest that recombinant IL-33 or an IL-33-mediated DC vaccine could be an attractive protocol for better tumor immunotherapy.

Soluble suppression of tumorigenicity-2 in pregnancy with a small-for-gestational-age fetus and with preeclampsia.

OBJECTIVE: Preeclampsia and fetal growth disorders are pregnancy-specific conditions that share common pathophysiological mechanisms. Yet, why some patients develop preeclampsia while others experience fetal growth restriction, or a combination of both clinical presentations, is unknown. We propose that the difference in severity of the maternal inflammatory response can contribute to the clinical phenotypes of preeclampsia vs. small for gestational age (SGA). To assess this hypothesis, we measured maternal plasma concentrations of the soluble isoform of suppression of tumorigenicity-2 (sST2), a member of the interleukin-1 receptor family that buffers proinflammatory responses. Previous reports showed that serum sST2 concentrations rise in the presence of intravascular inflammation and Th1-type immune responses and are significantly higher in patients with preeclampsia compared to those with normal pregnancy. The behavior of sST2 in pregnancies complicated by SGA has not been reported. This study was conducted to compare sST2 plasma concentrations in normal pregnancies, in those with preeclampsia, and in those with an SGA fetus. METHODS: This retrospective cross-sectional study included women with an SGA fetus (n = 52), women with preeclampsia (n = 106), and those with normal pregnancy (n = 131). Maternal plasma concentrations of sST2 were determined by enzyme-linked immunosorbent assay. Doppler velocimetry of the uterine and umbilical arteries was available in a subset of patients with SGA (42 patients and 43 patients, respectively). RESULTS: (1) Women with an SGA fetus had a significantly higher median plasma concentration of sST2 than normal pregnant women (p = .008); (2) women with preeclampsia had a significantly higher median plasma concentration of sST2 than those with normal pregnancy (p < .001) and those with an SGA fetus (p < .001); (3) patients with SGA and abnormal uterine artery Doppler velocimetry had a higher median plasma concentration of sST2 than controls (p < .01) and those with SGA and normal uterine artery Doppler velocimetry (p = .02); (4) there was no significant difference in the median plasma sST2 concentration between patients with SGA who had normal uterine artery Doppler velocimetry and controls (p = .4); (5) among patients with SGA, those with abnormal and those with normal umbilical artery Doppler velocimetry had higher median plasma sST2 concentrations than controls (p = .001 and p = .02, respectively); and (6) there was no significant difference in the median plasma sST2 concentrations between patients with SGA who did and those who did not have abnormal umbilical artery Doppler velocimetry (p = .06). CONCLUSIONS: Preeclampsia and disorders of fetal growth are conditions characterized by intravascular inflammation, as reflected by maternal plasma concentrations of sST2. The severity of intravascular inflammation is highest in patients with preeclampsia.

Soluble ST2 as a Potential Biomarker for Abdominal Aortic Aneurysms-A Single-Center Retrospective Cohort Study.

The maximal aortic diameter is the only clinically applied predictor of abdominal aortic aneurysm (AAA) progression and indicator for surgical repair. Circulating biomarkers resulting from AAA pathogenesis are attractive candidates for the diagnosis and prognosis of aneurysmal disease. Due to the reported role of interleukin 33 in AAA development, we investigated the corresponding circulating receptor molecules of soluble suppression of tumorigenesis 2 (sST2) in AAA patients regarding their marker potential in diagnosis and prognosis. We conducted a single-center retrospective cohort study in a diagnostic setting, measuring the circulating serum sST2 protein levels of 47 AAA patients under surveillance, matched with 25 peripheral artery disease (PAD) patients and 25 healthy controls. In a prognostic setting, we analyzed the longitudinal monitoring data of 50 monitored AAA patients. Slow versus fast AAA progression was defined as a <2 or ≥2 mm increase in AAA diameter over 6 months and a <4 or ≥4 mm increase over 12 months. Additionally, the association of circulating serum sST2 and AAA growth was investigated using a specifically tailored log-linear mixed model. Serum sST2 concentrations were significantly increased in AAA patients compared with healthy individuals: the median of AAA patient cohort was 112.72 ng/mL (p = 0.025) and that of AAA patient cohort 2 was 14.32 ng/mL (p = 0.039) versus healthy controls (8.82 ng/mL). Likewise, PAD patients showed significantly elevated sST2 protein levels compared with healthy controls (the median was 12.10 ng/mL; p = 0.048) but similar concentrations to AAA patients. Additionally, sST2 protein levels were found to be unsuited to identifying fast AAA progression over short-term periods of 6 or 12 months, which was confirmed by a log-linear mixed model. In conclusion, the significantly elevated protein levels of sST2 detected in patients with vascular disease may be useful in the early diagnosis of AAA but cannot distinguish between AAA and PAD or predict AAA progression.

IL-33 Enhances ACE2 Expression on Epidermal Keratinocytes in Atopic Dermatitis: A Plausible Issue for SARS-CoV-2 Transmission in Inflamed Atopic Skin.

BACKGROUND: Interleukin-33 (IL-33) is an important cytokine in the pathophysiology of atopic dermatitis (AD) and in the progression of COVID-19. Angiotensin converting enzyme 2 (ACE2), the entry receptor for SARS-CoV-2, is expressed in epidermal keratinocytes. Whether IL-33 could regulate the expression of ACE2 mechanistically in keratinocytes warrants investigation. OBJECTIVE: We questioned whether the ACE2 expression is increased in AD skin. We also questioned whether ACE2 is expressed in keratinocytes; if so, would its expression be enhanced mechanistically by IL-33. METHODS: We measured and compared the expression of ACE2 in skin from patients with AD, patients with psoriasis, and healthy controls using immunohistochemistry. Flow cytometry, immunofluorescent exam, and quantitative RT-PCR were used for measuring the ACE2 expression in cultured keratinocytes treated with IL-33 and IL-17. Blocking antibodies were utilized to study the intracellular signaling pathways governing the ACE2 expression using cytokines. RESULTS: The results showed that the ACE2 expression is increased in AD compared with that in healthy skin and psoriasis. In primary epidermal keratinocytes, ACE2 is constitutively expressed. IL-33 induces a time-dependent increase in ACE2 expression in cultured keratinocytes through quantitative PCR, flow cytometry, and immunofluorescent examinations. Furthermore, pretreatment of an ERK inhibitor, but not a STAT3 inhibitor, eliminated the increases in ACE2 by IL-33 in keratinocytes, indicating that IL-33 enhances ACE2 expression through ERK on epidermal keratinocytes. CONCLUSION: This is the first study to reveal that IL-33 enhances ACE2 expression on keratinocytes via ERK. Although further mechanistic studies are required, the increased ACE2 expression in IL-33 might have a biological implication on the transmission of SARS-CoV-2 in patients with AD.

Expression and clinical significance of IL-33 and its receptor ST2 in children with obstructive sleep apnea syndrome.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is characterized by a majority population of respiratory sleep disorders, which consists of simple snoring as well as increased upper airway resistance syndrome. Adenoid hypertrophy has been suggested as the main cause of OSAS in children. The role of interleukin-33 (IL-33) and its receptor suppressor of tumorigenicity 2 (ST2) in a variety of pediatric allergic diseases has been confirmed. We hypothesized that IL-33/ST2 path way might play a pivotal role in the pathogenesis of adenoid hypertrophy-associated OSAS in children. METHODS: A total of 40 children undergoing adenoidectomy due to OSAS in the Otolaryngology of Tianjin Children's Hospital were selected as the study participants. The quantity of IL-33 and ST2 positive cells in adenoids was detected by immunohistochemical (IHC) streptavidin-peroxidase conjugate (SP) method. RESULTS: The IL-33 positive cells were mainly distributed in the submucosa epithelium and vascular endothelium, and expressed in the nucleus and cytoplasm. Meanwhile, ST2 positive cells were primarily observed in the mucosa and expressed in the nucleus and cytoplasm, with a little expression of intercellular substance. There was a positive correlation between the proportion of adenoids in the posterior nostril diameter and the number of IL-33 positive cells. The expression of IL-33 in adenoids was positively correlated with the level of ST2 (r=0.809, P=0.000). The expression of IL-33 in adenoids was positively correlated with the level of eosinophil granulocyte (r=0.859, P=0.000). Moreover, the expression of ST2 in adenoids was positively correlated with the level of eosinophil granulocyte (r=0.814, P=0.000). The number of IL-33 positive cells was significantly higher in the moderate hypoxemia group than that in the mild hypoxemia group (P<0.05). There was no significant difference in the number of ST2 positive cells between the moderate hypoxemia group and mild hypoxemia group (P>0.05). CONCLUSIONS: Both IL-33 and its receptor ST2 were expressed in adenoids of OSAS children. The severity of airway obstruction caused by adenoid hypertrophy was positively correlated with the expression of IL-33.

Effect of Xiao Qinglongtang on IL-33/ST2 Signaling Pathway in Mice with Allergic Rhinitis / 中国实验方剂学杂志

ObjectiveTo investigate the therapeutic effect of Xiao Qinglongtang （XQLT） on ovalbumin （OVA）-induced allergic rhinitis （AR） in mice and its effect on the interleukin-33 （IL-33）/suppression of tumorigenicity 2 （ST2） signaling pathway. MethodSeventy-two female BALB/c mice of SPF grade were randomly divided into a control group， a model group， a positive control group （loratadine， 2.05 mg·kg-1）， and low-， medium-， and high-dose （5.005，10.01，20.02 g·kg-1） XQLT groups. All mice except for those in the control group were sensitized by intraperitoneal injection of OVA solution， and the AR model was induced by intranasal drops of OVA solution. Thirty minutes before local intranasal drops， drugs were administered once， and mice in the control group and the model group received phosphate buffered saline （PBS） at 20 mL·kg-1 for 7 days. After the last intranasal drop of OVA solution， the times of sneezing and nasal rubbing of mice within 10 min was recorded. After drug administration for 7 days， blood samples were collected， and nasal bones of mice were decalcified for the preparation of pathological sections. The content of OVA-specific immunoglobulin E （OVA-sIgE）， interleukin-4 （IL-4）， interleukin-5 （IL-5）， and interleukin-13 （IL-13） was detected by enzyme-linked immunosorbent assay （ELISA） kits. Hematoxylin-eosin （HE） staining， periodic acid-Schiff （PAS） staining， and Giemsa staining were used to observe the pathological changes， goblet cell hyperplasia， and eosinophil infiltration of nasal mucosa， respectively. Western blot was used to detect the expression levels of IL-33， ST2， and IL-1 receptor accessory protein （IL-1RAP） in nasal mucosa. ResultCompared with the control group， the model group showed increased times of sneezing and nasal rubbing （P<0.01）， edema and thickening of nasal mucosa， goblet cell hyperplasia and eosinophil infiltration， increased serum levels of OVA-sIgE， IL-4， IL-5 and IL-13 （P<0.01）， and increased protein expression of IL-33， ST2， and IL-1RAP in nasal mucosa （P<0.05，P<0.01）. After drug administration， compared with the model group， the high-dose XQLT group showed reduced times of sneezing and nasal rubbing （P<0.01）， improved pathological conditions of nasal mucosa， reduced serum levels of OVA-sIgE， IL-4， IL-5， and IL-13 （P<0.01）， and declining protein expression of IL-33， ST2， and IL-1RAP in nasal mucosa （P<0.05，P<0.01）. ConclusionXQLT has a therapeutic effect on OVA-sensitized AR mice， and the mechanism may be related to the regulation of the IL-33/ST2 signaling pathway and Th2 inflammatory cytokine to reduce Th2 inflammatory response and alleviate nasal mucosal injury.

The Concentration of Selected Inflammatory Cytokines (IL-6, IL-8, CXCL5, IL-33) and Damage-Associated Molecular Patterns (HMGB-1, HSP-70) Released in an Early Response to Distal Forearm Fracture and the Performed Closed Reduction With Kirschner Wire Fixation in Children.

The evaluation of trauma after surgery through objective analysis of biochemical markers can help in selecting the most appropriate therapy. Thus the aim of the study was the evaluation of the concentration of selected inflammatory cytokines (IL-6, IL-8, CXCL5, IL-33), C-reactive protein (CRP), and damaged-associated molecular patterns (DAMPs): HMGB-1, HSP-70 in the plasma of children in response to bone fracture and 12-14 hours after subsequent surgery performed by closed reduction with percutaneous Kirschner wire fixation (CRKF). The study will answer the question if the CRFK procedure leads to excessive production of inflammatory and damage markers. Blood samples from 29 children with distal forearm fractures were collected 30 min. before CRKF procedure and 12-14 hours after performance of the procedure. The control group was composed of 17 healthy children. IL-6 and CRP concentrations were analyzed using routinely performed in vitro diagnostics tests; the remaining proteins were analyzed with the use of the ELISA method. Increased values of IL-6, CRP, and HSP-70 represented an early inflammatory response to distal forearm fractures classified as SH-II type according to the Salter-Harris classification system. However, the median CRP concentration was within the reference values not indicative of inflammation. The CRKF procedure may be a good solution for the treatment of bone fractures, as damaged associated molecular patterns - HMGB-1 and HSP-70 - did not significantly differ 12-14 hours after the approach was applied as compared to the control group. Moreover, the increase in IL-6 concentration after the CRKF procedure was 1.5-fold to the level before CRKF, while the increase of this marker in response to the distal forearm fracture was 4.3-fold compared to the control group. Based on this data, it appears reasonable to suggest that the CRKF approach caused less damage and inflammatory response in comparison to the response to the fracture itself.

Transgenic Overexpression of Galectin-3 in Pancreatic ß Cells Attenuates Hyperglycemia in Mice: Synergistic Antidiabetic Effect With Exogenous IL-33.

Galectin-3 (Gal-3) has diverse roles in inflammatory and autoimmune diseases. There is evidence that Gal-3 plays a role in both type 1 and type 2 diabetes. While the role of Gal-3 expression in immune cells invading the pancreatic islets in the experimental model of type 1 diabetes mellitus has been already studied, the importance of the overexpression of Gal-3 in the target ß cells is not defined. Therefore, we used multiple low doses of streptozotocin (MLD-STZ)-induced diabetes in C57Bl/6 mice to analyze the effect of transgenic (TG) overexpression of Gal-3 in ß cells. Our results demonstrated that the overexpression of Gal-3 protected ß cells from apoptosis and attenuated MLD-STZ-induced hyperglycemia, glycosuria, and ketonuria. The cellular analysis of pancreata and draining lymph nodes showed that Gal-3 overexpression significantly decreased the number of pro-inflammatory cells without affecting the presence of T-regulatory cells. As the application of exogenous interleukin 33 (IL-33) given from the beginning of MLD-STZ diabetes induction attenuates the development of disease, by increasing the presence of regulatory FoxP3+ ST2+ cells, we evaluated the potential synergistic effect of the exogenous IL-33 and TG overexpression of Gal-3 in ß cells at the later stage of diabetogenesis. The addition of IL-33 potentiated the survival of ß cells and attenuated diabetes even when administered later, after the onset of hyperglycemia (12-18 days), suggesting that protection from apoptosis and immunoregulation by IL-33 may attenuate type 1 diabetes.

IL-33/ST2 Axis Plays a Protective Effect in Streptococcus pyogenes Infection through Strengthening of the Innate Immunity.

Group A Streptococcus (GAS) causes invasive human diseases with the cytokine storm. Interleukin-33 (IL-33)/suppression of tumorigenicity 2 (ST2) axis is known to drive TH2 response, while its effect on GAS infection is unclear. We used an air pouch model to examine the effect of the IL-33/ST2 axis on GAS-induced necrotizing fasciitis. GAS infection induced IL-33 expression in wild-type (WT) C57BL/6 mice, whereas the IL-33- and ST2-knockout mice had higher mortality rates, more severe skin lesions and higher bacterial loads in the air pouches than those of WT mice after infection. Surveys of infiltrating cells in the air pouch of GAS-infected mice at the early stage found that the number and cell viability of infiltrating cells in both gene knockout mice were lower than those of WT mice. The predominant effector cells in GAS-infected air pouches were neutrophils. Absence of the IL-33/ST2 axis enhanced the expression of inflammatory cytokines, but not TH1 or TH2 cytokines, in the air pouch after infection. Using in vitro assays, we found that the IL-33/ST2 axis not only enhanced neutrophil migration but also strengthened the bactericidal activity of both sera and neutrophils. These results suggest that the IL-33/ST2 axis provided the protective effect on GAS infection through enhancing the innate immunity.

A comprehensive in Silico analysis of the functional and structural impact of single nucleotide polymorphisms (SNPs) in the human IL-33 gene.

Interleukin 33 (IL-33) is the latest member of the IL-1 cytokine family, which plays both pro - and anti-inflammatory functions. Numerous Single-nucleotide polymorphisms (SNPs) in the IL-33 gene have been recognized to be associated with a vast variety of inflammatory disorders. SNPs associated studies have become a crucial approach in uncovering the genetic background of human diseases. However, distinguishing the functional SNPs in a disease-related gene from a pool of both functional and neutral SNPs is a major challenge and needs multiple experiments of hundreds or thousands of SNPs in candidate genes. This study aimed to identify the possible deleterious SNPs in the IL-33 gene using bioinformatics predictive tools. The nonsynonymous SNPs (nsSNPs) were analyzed by SIFT, PolyPhen, PROVEAN, SNP&GO, MutPred, SNAP, PhD SNP, and I-Mutant tools. The Non-coding SNPs (ncSNPs) were also analyzed by SNPinfo and RegulomeDB tools. In conclusion, our in-silico analysis predicted 5 nsSNPs and 22 ncSNPs as potential candidates in the IL-33 gene for future genetic association studies.

Neutralization of IL-33 modifies the type 2 and type 3 inflammatory signature of viral induced asthma exacerbation.

BACKGROUND: Respiratory viral infections are one of the leading causes of need for emergency care and hospitalizations in asthmatic individuals, and airway-secreted cytokines are released within hours of viral infection to initiate these exacerbations. IL-33, specifically, contributes to these allergic exacerbations by amplifying type 2 inflammation. We hypothesized that blocking IL-33 in RSV-induced exacerbation would significantly reduce allergic inflammation. METHODS: Sensitized BALB/c mice were challenged with aerosolized ovalbumin (OVA) to establish allergic inflammation, followed by RSV-A2 infection to yield four treatment groups: saline only (Saline), RSV-infected alone (RSV), OVA alone (OVA), and OVA-treated with RSV infection (OVA-RSV). Lung outcomes included lung mRNA and protein markers of allergic inflammation, histology for mucus cell metaplasia and lung immune cell influx by cytospin and flow cytometry. RESULTS: While thymic stromal lymphopoietin (TSLP) and IL-33 were detected 6 h after RSV infection in the OVA-RSV mice, IL-23 protein was uniquely upregulated in RSV-infected mice alone. OVA-RSV animals varied from RSV- or OVA-treated mice as they had increased lung eosinophils, neutrophils, group 2 innate lymphoid cells (ILC2) and group 3 innate lymphoid cells (ILC3) detectable as early as 6 h after RSV infection. Neutralized IL-33 significantly reduced ILC2 and eosinophils, and the prototypical allergic proteins, IL-5, IL-13, CCL17 and CCL22 in OVA-RSV mice. Numbers of neutrophils and ILC3 were also reduced with anti-IL-33 treatment in both RSV and OVA-RSV treated animals as well. CONCLUSIONS: Taken together, our findings indicate a broad reduction in allergic-proinflammatory events mediated by IL-33 neutralization in RSV-induced asthma exacerbation.

WFDC2 Promotes Spasmolytic Polypeptide-Expressing Metaplasia Through the Up-Regulation of IL33 in Response to Injury.

BACKGROUND & AIMS: WAP 4-disulfide core domain protein 2 (WFDC2), also known as human epididymis protein 4, is a small secretory protein that is highly expressed in fibrosis and human cancers, particularly in the ovaries, lungs, and stomach. However, the role of WFDC2 in carcinogenesis is not fully understood. The present study aimed to investigate the role of WFDC2 in gastric carcinogenesis with the use of preneoplastic metaplasia models. METHODS: Three spasmolytic polypeptide-expressing metaplasia (SPEM) models were established in both wild-type and Wfdc2-knockout mice with DMP-777, L635, and high-dose tamoxifen, respectively. To reveal the functional role of WFDC2, we performed transcriptomic analysis with DMP-777-treated gastric corpus specimens. RESULTS: Wfdc2-knockout mice exhibited remarkable resistance against oxyntic atrophy, SPEM emergence, and accumulation of M2-type macrophages in all 3 SPEM models. Transcriptomic analysis revealed that Wfdc2-knockout prevented the up-regulation of interleukin-33 (IL33) expression in the injured mucosal region of SPEM models. Notably, supplementation of recombinant WFDC2 induced IL33 production and M2 macrophage polarization, and ultimately promoted SPEM development. Moreover, long-term treatment with recombinant WFDC2 was able to induce SPEM development. CONCLUSIONS: WFDC2 expressed in response to gastric injury promotes SPEM through the up-regulation of IL33 expression. These findings provide novel insights into the role of WFDC2 in gastric carcinogenesis.

Interleukin-33 Promotes Cell Survival via p38 MAPK-Mediated Interleukin-6 Gene Expression and Release in Pediatric AML.

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of immature myeloid blasts in the bone marrow (BM). Cytokine provide signals for leukemia cells to improve their survival in the BM microenvironment. Previously, we identified interleukin-33 (IL-33) as a promoter of cell survival in a human AML cell line and primary mouse leukemia cells. In this study, we report that the cell surface expression of IL-33-specific receptor, Interleukin 1 Receptor Like 1 (IL1RL1), is elevated in BM cells from AML patients at diagnosis, and the serum level of IL-33 in AML patients is higher than that of healthy donor controls. Moreover, IL-33 levels are found to be positively associated with IL-6 levels in pediatric patients with AML. In vitro, IL-33 treatment increased IL-6 mRNA expression and protein level in BM and peripheral blood (PB) cells from AML patients. Evidence was also provided that IL-33 inhibits cell apoptosis by activating p38 mitogen-activated protein kinase (MAPK) pathway using human AML cell line and AML patient samples. Finally, we confirmed that IL-33 activated IL-6 expression in a manner that required p38 MAPK pathway using clinical AML samples. Taken together, we identified a potential mechanism of IL-33-mediated survival involving p38 MAPK in pediatric AML patients that would facilitate future drug development.

Cancer stem cells and their niche in the progression of squamous cell carcinoma.

Most cancers harbor a small population of highly tumorigenic cells known as cancer stem cells (CSCs). Because of their stem cell-like properties and resistance to conventional therapies, CSCs are considered to be a rational target for curable cancer treatment. However, despite recent advances in CSC research, CSC-targeted therapies are not as successful as was initially hoped. The proliferative, invasive, and drug-resistant properties of CSCs are regulated by the tumor microenvironment associated with them, the so-called CSC niche. Thus, targeting tumor-promoting cellular crosstalk between CSCs and their niches is an attractive avenue for developing durable therapies. Using mouse models of squamous cell carcinoma (SCC), we have demonstrated that tumor cells responding to transforming growth factor ß (TGF-ß) function as drug-resistant CSCs. The gene expression signature of TGF-ß-responding tumor cells has accelerated the identification of novel pathways that drive invasive tumor progression. Moreover, by focusing on the cytokine milieu and macrophages in the proximity of TGF-ß-responding tumor cells, we recently uncovered the molecular basis of a CSC-niche interaction that emerges during early tumor development. This review article summarizes the specialized tumor microenvironment associated with CSCs and discusses mechanisms by which malignant properties of CSCs are maintained and promoted.

IL33 and Mast Cells-The Key Regulators of Immune Responses in Gastrointestinal Cancers?

The Interleukin (IL-)1 family IL33 is best known for eliciting type 2 immune responses by stimulating mast cells (MCs), regulatory T-cells (Tregs), innate lymphoid cells (ILCs) and other immune cells. MCs and IL33 provide critical control of immunological and epithelial homeostasis in the gastrointestinal (GI) tract. Meanwhile, the role of MCs in solid malignancies appears tissue-specific with both pro and anti-tumorigenic activities. Likewise, IL33 signaling significantly shapes immune responses in the tumor microenvironment, but these effects remain often dichotomous when assessed in experimental models of cancer. Thus, the balance between tumor suppressing and tumor promoting activities of IL33 are highly context dependent, and most likely dictated by the mixture of cell types responding to IL33. Adding to this complexity is the promiscuous nature by which MCs respond to cytokines other than IL33 and release chemotactic factors that recruit immune cells into the tumor microenvironment. In this review, we integrate the outcomes of recent studies on the role of MCs and IL33 in cancer with our own observations in the GI tract. We propose a working model where the most abundant IL33 responsive immune cell type is likely to dictate an overall tumor-supporting or tumor suppressing outcome in vivo. We discuss how these opposing responses affect the therapeutic potential of targeting MC and IL33, and highlight the caveats and challenges facing our ability to effectively harness MCs and IL33 biology for anti-cancer immunotherapy.

A critical role for expression of atypical chemokine receptor 2 in multiple sclerosis: A preliminary project.

BACKGROUND: ACKR2 is an atypical chemokine receptor that promotes acute inflammation by acting as a scavenger receptor for inflammatory chemokines in experimental models of some inflammatory disorders, but its function in multiple sclerosis (MS) is unclear. Therefore we aimed to evaluate the mRNA expression of ACKR2 as a scavenger receptor in patients with MS and also the correlation of this expression with certain cytokines that are important for MS pathogenesis. METHODS: The ACKR2 mRNA expression was examined on peripheral blood mononuclear cells (PBMCs) of 40 patients with relapsing-remitting MS (RRMS) and 35 healthy individuals. ACKR2 mRNA expressions were measured using real-time quantitative polymerase chain reaction (qPCR). In addition, circulating cytokine levels (TNF-α, IL-6, IL-33) in all patients and controls were evaluated using enzyme-linked immunosorbent assay. RESULTS: mRNA expression of ACKR2 was decreased on PBMCs compared to healthy subjects (p < 0.001). ACKR2 expression in peripheral blood leucocytes could be regulated by circulating cytokines but there are no correlations with these cytokines (p > 0.05). In addition, the patients' plasma levels of IL-33 significantly increased (p = 0.039) and no significant difference was found between other cytokine levels in the patients (p > 0.05). CONCLUSION: Our data clearly show a decreased ACKR2 mRNA expression on PBMCs and increased plasma IL-33 levels of patients with MS. There was no significant relationship between ACKR2 and other cytokine levels. Within our knowledge, this is the first study that evaluates the ACKR2 mRNA expression in the PBMCs of MS patients.

Dual-specificity phosphatase 5 suppresses ovarian cancer progression by inhibiting IL-33 signaling.

Ovarian cancer (OC) is the leading cause of death from gynecological malignancy. Dual-specificity phosphatases (DUSPs) are proteins that are reported involved in carcinogenesis, but their roles in OC have not be extensively studied. Here, we found that DUSP5 is markedly down-regulated in OC tissues. We reanalyzed DUSP5 expression in OC using published microarray data from the Gene Expression Omnibus (GEO) database and found that patients with low DUSP5 expression have significantly shorter overall survival than those with high expression (P < 0.001). Down-regulation of DUSP5 in OC tissues was immunohistochemically confirmed in tissue microarrays containing 15 normal ovary tissue samples and 60 OC specimens. Functional studies suggest that DUSP5 silence facilitates cell proliferation, migration, and invasion of OC cells in vitro. DUSP5 over-expression inhibits cell proliferation but has no effect on OC cell migration or invasion. Mechanistically, silencing DUSP5 transcriptionally activates interleukin 33 (IL-33) expression and secretion. Blockage of IL-33 with a neutralizing anti-IL33 antibody attenuates the effect of DUSP5 silencing to promote cell proliferation, migration, and invasion. Moreover, recombinant IL-33 protein treatment dramatically promotes OC cell proliferation, migration, and invasion with DUSP5 over-expression. Our study provides proof of principle that DUSP5 down-regulation promotes proliferation, migration, and invasion of OC cells via activation of IL-33 signaling.