Floxed Il1rl2 Locus with mCherry Reporter Element Reveals Distinct Expression Patterns of the IL-36 Receptor in Barrier Tissues.

IL-36 cytokines are emerging as beneficial in immunity against pathogens and cancers but can also be detrimental when dysregulated in autoimmune and autoinflammatory conditions. Interest in targeting IL-36 activity for therapeutic purposes is rapidly growing, yet many unknowns about the functions of these cytokines remain. Thus, the availability of robust research tools is essential for both fundamental basic science and pre-clinical studies to fully access outcomes of any manipulation of the system. For this purpose, a floxed Il1rl2, the gene encoding the IL-36 receptor, mouse strain was developed to facilitate the generation of conditional knockout mice. The targeted locus was engineered to contain an inverted mCherry reporter sequence that upon Cre-mediated recombination will be flipped and expressed under the control of the endogenous Il1rl2 promoter. This feature can be used to confirm knockout in individual cells but also as a reporter to determine which cells express the IL-36 receptor IL-1RL2. The locus was confirmed to function as intended and further used to demonstrate the expression of IL-1RL2 in barrier tissues. Il1rl2 expression was detected in leukocytes in all barrier tissues. Interestingly, strong expression was observed in epithelial cells at locations in direct contact with the environment such as the skin, oral mucosa, the esophagus, and the upper airways, but almost absent from epithelial cells at more inward facing sites, including lung alveoli, the small intestine, and the colon. These findings suggest specialized functions of IL-1RL2 in outward facing epithelial tissues and cells. The generated mouse model should prove valuable in defining such functions and may also facilitate basic and translational research.

IL-33-ST2 signaling in fibro-adipogenic progenitors alleviates immobilization-induced muscle atrophy in mice.

BACKGROUND: The regenerative and adaptive capacity of skeletal muscles reduces with age, leading to severe disability and frailty in the elderly. Therefore, development of effective therapeutic interventions for muscle wasting is important both medically and socioeconomically. In the present study, we aimed to elucidate the potential contribution of fibro-adipogenic progenitors (FAPs), which are mesenchymal stem cells in skeletal muscles, to immobilization-induced muscle atrophy. METHODS: Young (2-3 months), adult (12-14 months), and aged (20-22 months) mice were used for analysis. Muscle atrophy was induced by immobilizing the hind limbs with a steel wire. FAPs were isolated from the hind limbs on days 0, 3, and 14 after immobilization for transcriptome analysis. The expression of ST2 and IL-33 in FAPs was evaluated by flow cytometry and immunostaining, respectively. To examine the role of IL-33-ST2 signaling in vivo, we intraperitoneally administered recombinant IL-33 or soluble ST2 (sST2) twice a week throughout the 2-week immobilization period. After 2-week immobilization, the tibialis anterior muscles were harvested and the cross-sectional area of muscle fibers was evaluated. RESULTS: The number of FAPs increased with the progression of muscle atrophy after immobilization in all age-groups. Transcriptome analysis of FAPs collected before and after immobilization revealed that Il33 and Il1rl1 transcripts, which encode the IL-33 receptor ST2, were transiently induced in young mice and, to a lesser extent, in aged mice. The number of FAPs positive for ST2 increased after immobilization in young mice. The number of ST2-positive FAPs also increased after immobilization in aged mice, but the difference from the baseline was not statistically significant. Immunostaining for IL-33 in the muscle sections revealed a significant increase in the number of FAPs expressing IL-33 after immobilization. Administration of recombinant IL-33 suppressed immobilization-induced muscle atrophy in aged mice but not in young mice. CONCLUSIONS: Our data reveal a previously unknown protective role of IL-33-ST2 signaling against immobilization-induced muscle atrophy in FAPs and suggest that IL-33-ST2 signaling is a potential new therapeutic target for alleviating disuse muscle atrophy, particularly in older adults.

IL-33 Reduces Saturated Fatty Acid Accumulation in Mouse Atherosclerotic Foci.

The cellular and molecular mechanisms of atherosclerosis are still unclear. Type 2 innate lymphocytes (ILC2) exhibit anti-inflammatory properties and protect against atherosclerosis. This study aimed to elucidate the pathogenesis of atherosclerosis development using atherosclerosis model mice (ApoE KO mice) and mice deficient in IL-33 receptor ST2 (ApoEST2 DKO mice). Sixteen-week-old male ApoE KO and ApoEST2 DKO mice were subjected to an 8-week regimen of a high-fat, high-sucrose diet. Atherosclerotic foci were assessed histologically at the aortic valve ring. Chronic inflammation was assessed using flow cytometry and real-time polymerase chain reaction. In addition, saturated fatty acids (palmitic acid) and IL-33 were administered to human aortic endothelial cells (HAECs) to assess fatty acid metabolism. ApoEST2 DKO mice with attenuated ILC2 had significantly worse atherosclerosis than ApoE KO mice. The levels of saturated fatty acids, including palmitic acid, were significantly elevated in the arteries and serum of ApoEST2 DKO mice. Furthermore, on treating HAECs with saturated fatty acids with or without IL-33, the Oil Red O staining area significantly decreased in the IL-33-treated group compared to that in the non-treated group. IL-33 potentially prevented the accumulation of saturated fatty acids within atherosclerotic foci.

The IL-33/ST2 axis is protective against acute inflammation during the course of periodontitis.

Periodontitis, which is induced by repeated bacterial invasion and the ensuing immune reactions that follow, is the leading cause of tooth loss. Periodontal tissue is comprised of four different components, each with potential role in pathogenesis, however, most studies on immune responses focus on gingival tissue. Here, we present a modified ligature-induced periodontitis model in male mice to analyze the pathogenesis, which captures the complexity of periodontal tissue. We find that the inflammatory response in the peri-root tissues and the expression of IL-6 and RANKL by Thy-1.2- fibroblasts/stromal cells are prominent throughout the bone destruction phase, and present already at an early stage. The initiation phase is characterized by high levels of ST2 (encoded by Il1rl1) expression in the peri-root tissue, suggesting that the IL-33/ST2 axis is involved in the pathogenesis. Both Il1rl1- and Il33-deficient mice exhibit exacerbated bone loss in the acute phase of periodontitis, along with macrophage polarization towards a classically activated phenotype and increased neutrophil infiltration, indicating a protective role of the IL-33/ST2 axis in acute inflammation. Thus, our findings highlight the hidden role of the peri-root tissue and simultaneously advance our understanding of the etiology of periodontitis via implicating the IL-33/ST2 axis.

IL-33 Induces Cellular and Exosomal miR-146a Expression as a Feedback Inhibitor of Mast Cell Function.

IL-33 is an inflammatory cytokine that promotes allergic disease by activating group 2 innate lymphoid cells, Th2 cells, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. Homeostatic control of IL-33 signaling is poorly understood. Because the IL-33 receptor, ST2, acts via cascades used by the TLR family, similar feedback mechanisms may exist. MicroRNA (miR)-146a is induced by LPS-mediated TLR4 signaling and serves as a feedback inhibitor. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow-derived mast cells (BMMCs). BMMCs transfected with a miR-146a antagonist or derived from miR-146a knockout mice showed enhanced cytokine expression in response to IL-33, suggesting that miR-146a is a negative regulator of IL-33-ST2 signaling. In vivo, miR-146a expression in plasma exosomes was elevated after i.p. injection of IL-33 in wild-type but not mast cell-deficient KitW-sh/W-sh mice. Finally, KitW-sh/W-sh mice acutely reconstituted with miR-146a knockout BMMCs prior to IL-33 challenge had elevated plasma IL-6 levels compared with littermates receiving wild-type BMMCs. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.

Epithelial overexpression of IL-33 induces eosinophilic esophagitis dependent on IL-13.

BACKGROUND: Eosinophilic esophagitis (EoE) is an increasingly common inflammatory condition of the esophagus; however, the underlying immunologic mechanisms remain poorly understood. The epithelium-derived cytokine IL-33 is associated with type 2 immune responses and elevated in esophageal biopsy specimens from patients with EoE. OBJECTIVE: We hypothesized that overexpression of IL-33 by the esophageal epithelium would promote the immunopathology of EoE. METHODS: We evaluated the functional consequences of esophageal epithelial overexpression of a secreted and active form of IL-33 in a novel transgenic mouse, EoE33. EoE33 mice were analyzed for clinical and immunologic phenotypes. Esophageal contractility was assessed. Epithelial cytokine responses were analyzed in three-dimensional organoids. EoE33 phenotypes were further characterized in ST2-/-, eosinophil-deficient, and IL-13-/- mice. Finally, EoE33 mice were treated with dexamethasone. RESULTS: EoE33 mice displayed ST2-dependent, EoE-like pathology and failed to thrive. Esophageal tissue remodeling and inflammation included basal zone hyperplasia, eosinophilia, mast cells, and TH2 cells. Marked increases in levels of type 2 cytokines, including IL-13, and molecules associated with immune responses and tissue remodeling were observed. Esophageal organoids suggested reactive epithelial changes. Genetic deletion of IL-13 in EoE33 mice abrogated pathologic changes in vivo. EoE33 mice were responsive to steroids. CONCLUSIONS: IL-33 overexpression by the esophageal epithelium generated immunopathology and clinical phenotypes resembling human EoE. IL-33 may play a pivotal role in the etiology of EoE by activating the IL-13 pathway. EoE33 mice are a robust experimental platform for mechanistic investigation and translational discovery.

Promoting (and targeting) alternative expression.

Lineage-specific effects of upstream promoters affect ST2 expression and effector function in TH1cells.

Outbreak caused by pandrug-resistant blaOXA-69/blaOXA-23/blaGES harboring Acinetobacter baumannii ST2 in an intensive care unit.

Acinetobacter baumannii has emerged as a main nosocomial pathogen exhibiting high rates of resistance to clinically relevant antibiotics. Six pandrug-resistant A. baumannii (PDR-A. baumannii) were recovered from three patients in a Tunisian Intensive Care Unit (ICU) between 10th and 16th of May 2018 resulting in one fatal case and raising the possibility of an outbreak. On 18th of May environmental screening of ICU surfaces was carried out. On 22nd of May a fourth patient was infected with PDR-A. baumannii and died. A second investigation was carried out for environmental screening and PDR-A. baumannii was isolated from the respirator. Antimicrobial susceptibility testing was performed according to EUCAST (2019) guidelines. MIC of colistin was determined by broth microdilution method. PCR was used to detect 14 beta-lactamases/carbapenemases and mcr (mcr-1 to mcr-5) genes. The genetic relatedness of PDR-A. baumannii isolates was determined by PFGE and MLST. Seven PDR-A. baumannii isolates were recovered from four patients, one MDR strain from wash basin, a PDR strain from hand sanitizer bottle and another PDR strain from respirator. All PDR-A. baumannii (n = 9) harbored blaOXA-69 gene and none carried mcr. Moreover, seven carried blaGES and blaOXA-23 genes. PFGE identified four pulsotypes (A, B, C, and D) with the pulsotype A gathering seven PDR-A. baumannii isolates: six from three patients and one from hygiene sample. MLST revealed that all PDR-A. baumannii isolates of pulsotype A belonged to the pandemic clone ST2. Systematic screening of MDR and PDR-A. baumannii is highly recommended to limit dissemination of such strains in ICUs.

Nosocomial surveillance of multidrug-resistant Acinetobacter baumannii: a genomic epidemiological study.

Acinetobacter baumannii is a major opportunistic pathogen causing hospital-acquired infections, and it is imperative to comprehend its evolutionary and epidemiological dynamics in hospitals to prevent and control nosocomial transmission. Here, we present a comprehensive genomic epidemiological study involving the genomic sequencing and antibiotic resistance profiling of 634 A. baumannii strains isolated from seven intensive care units (ICUs) of a Chinese general hospital over 2 consecutive years. Our study reveals that ST2 is highly dominant (90.54%) in the ICUs, with 98.90% of the ST2 exhibiting multidrug resistant or extensively drug resistant. Phylogenetic analyses of newly sequenced genomes and public data suggest that nosocomial isolates originated outside the hospital but evolved inside. The major lineages appear to be stable, with 9 of the 28 identified nosocomial epidemic clones infecting over 60% of the affected patients. However, outbreaks of two highly evolved clones have been observed in different hospitals, suggesting significant inter-hospital transmission chains. By coupling patient medical records and genomic divergence of the ST2, we found that cross-ward patient transfer played a crucial role in pathogen's nosocomial transmission. Additionally, we identified 831 potential adaptive evolutionary loci and 44 associated genes by grouping and comparing the genomes of clones with different prevalence. Overall, our study provides a comprehensive and contemporary survey on the epidemiology and genomic evolution of A. baumannii in a large Chinese general hospital. These findings shed light on the nosocomial evolution and transmission of A. baumannii and offers valuable information for transmission prevention and antibiotic therapy.IMPORTANCEThis study delved into the genomic evolution and transmission of nosocomial Acinetobacter baumannii on a large scale, spanning both an extended time period and the largest sample size to date. Through molecular epidemiological investigations based on genomics, we can directly trace the origin of the pathogen, detecting and monitoring outbreaks of infectious diseases in a timely manner, and ensuring public health safety. In addition, this study also collects a large amount of genomic and antibiotic resistance detection data, which is helpful for phenotype prediction based on genomic sequencing. It enables patients to receive personalized antibiotic treatment quickly, helps doctors select antibiotics more accurately, and contributes to reducing the use of antibiotics and lowering the risk of antibiotic resistance development.

A type 1 immunity-restricted promoter of the IL-33 receptor gene directs antiviral T-cell responses.

The pleiotropic alarmin interleukin-33 (IL-33) drives type 1, type 2 and regulatory T-cell responses via its receptor ST2. Subset-specific differences in ST2 expression intensity and dynamics suggest that transcriptional regulation is key in orchestrating the context-dependent activity of IL-33-ST2 signaling in T-cell immunity. Here, we identify a previously unrecognized alternative promoter in mice and humans that is located far upstream of the curated ST2-coding gene and drives ST2 expression in type 1 immunity. Mice lacking this promoter exhibit a selective loss of ST2 expression in type 1- but not type 2-biased T cells, resulting in impaired expansion of cytotoxic T cells (CTLs) and T-helper 1 cells upon viral infection. T-cell-intrinsic IL-33 signaling via type 1 promoter-driven ST2 is critical to generate a clonally diverse population of antiviral short-lived effector CTLs. Thus, lineage-specific alternative promoter usage directs alarmin responsiveness in T-cell subsets and offers opportunities for immune cell-specific targeting of the IL-33-ST2 axis in infections and inflammatory diseases.

Recovery and genetic characterization of clinically-relevant ST2 carbapenem-resistant Acinetobacter baumannii isolates from untreated hospital sewage in Zhejiang Province, China.

The global transmission of carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant and grave threat to human health. To investigate the potential relationship between hospital sewage and the transmission of CRAB within healthcare facilities, isolates of Acinetobacter spp. obtained from untreated hospital sewage samples were subjected to antimicrobial susceptibility tests, genome sequencing, and bioinformatic and phylogenetic tree analysis, and that data were matched with those of the clinical isolates. Among the 70 Acinetobacter spp. sewage isolates tested, A. baumannii was the most prevalent and detectable in 5 hospitals, followed by A. nosocomialis and A. gerneri. Worryingly, 57.14 % (40/70) of the isolates were MDR, with 25.71 % (18/70) being resistant to carbapenem. When utilizing the Pasteur scheme, ST2 was the predominant type among these CRAB isolates, with Tn2006 (ΔISAba1-blaOXA-23-ATPase-yeeB-yeeA-ΔISAba1) and Tn2009 (ΔISAba1-blaOXA-23-ATPase-hp-parA-yeeC-hp-yeeB-ΔISAba1) being the key mobile genetic elements that encode carbapenem resistance. Seven A. gerneri isolates which harbored Tn2008 (ISAba1-blaOXA-23 -ATPase) and the blaPER-1 gene were also identified. Besides, an A. soil isolate was found to exhibit high-level of meropenem resistance (MIC ≥128 mg/L) and harbor a blaNDM-1 gene located in a core genetic structure of ISAba125-blaNDM-1-ble-trpF-dsbC-cutA. To investigate the genetic relatedness between isolates recovered from hospital sewage and those collected from ICUs, a phylogenetic tree was constructed for 242 clinical isolates and 9 sewage isolates. The results revealed the presence of two evolutionary clades, each containing isolates from both ICU and sewage water, suggesting that CRAB isolates in untreated sewage water were also the transmission clones or closely related evolutionary isolates recoverable in hospital settings. Findings in this work confirm that hospital sewage is a potential reservoir of CRAB.

Epidemiology and molecular characterization of CTX-M-type ESBLs producing Escherichia coli isolated from clinical settings.

OBJECTIVES: Recently, blaCTX-Ms have become the dominant ESBLs for E. coli strains worldwide. We aim to provide a systematic study on the relationships between sequence types (STs), clinical origins, and the blaCTX-Ms genotypes of E. coli strains. METHODS: Totally, 1005 complete sequences of clinical E. coli were collected from NCBI. Multilocus sequence typing (MLST) and antibiotic resistance genes screening were performed. RESULTS: Faeces (26.27%), urine (16.02%), and blood (8.26%) were shown to be the main sources of clinical E. coli isolates. The isolates belong to 153 STs and 26 clonal complexes (CCs). The most prevalent STs were ST2 (11.3%), ST43 (8.6%), and ST8 (5.7%). The positive rate for blaCTX-Ms was 34.7%. Different samples showed significantly different blaCTX-Ms positive rates (P<0.05). The main genotypes were blaCTX-M-55-like (47.6%), blaCTX-M-1-like (31.8%), and blaCTX-M-2-like (22.1%). The majority of ST2 strains had blaCTX-M-55-like genes. In ST8 strains, there was a homogeneous distribution of blaCTX-M-9, blaCTX-M-65, blaCTX-M-55, blaCTX-M-2, and blaCTX-M-1. Only ST43 strains exhibited the presence of blaCTX-M-79. The blaCTX-Ms showed a pattern of cross-continental transmission with intra-regional spread. Among the 349 blaCTX-Ms-producing E. coli strains, 148 strains also carried carbapenem resistance genes, including blaNDM (119, 34.1%), blaKPC (16, 4.6%), blaOXA-48 (9, 2.6%) and blaIMP (4, 1.1%). Also, 81 strains carried the mcr gene (23.2%). CONCLUSIONS: E. coli has become increasingly rich in blaCTX-Ms genotypes. Our findings about the connection between E. coli STs and blaCTX-Ms can be utilized to identify E. coli strains with high potential to spread drug resistance in the future.

DHT and Insulin Upregulate Secretion of the Soluble Decoy Receptor of IL-33 From Decidualized Endometrial Stromal Cells.

Interleukin 33 (IL-33) signaling regulates most of the key processes of pregnancy, including decidualization, trophoblast proliferation and invasion, vascular remodeling, and placental growth. Accordingly, dysregulation of IL-33, its membrane-bound receptor (ST2L, transducer of IL-33 signaling), and its soluble decoy receptor (sST2, inhibitor of IL-33 signaling) has been linked to a wide range of adverse pregnancy outcomes that are common in women with obesity and polycystic ovary syndrome, that is, conditions associated with hyperandrogenism, insulin resistance, and compensatory hyperinsulinemia. To reveal if androgens and insulin might modulate uteroplacental IL-33 signaling, we investigated the effect of dihydrotestosterone (DHT) and/or insulin on the expression of ST2L and sST2 (along with the activity of their promoter regions), IL-33 and sIL1RAP (heterodimerization partner of sST2), during in vitro decidualization of endometrial stromal cells from 9 healthy women. DHT and insulin markedly upregulated sST2 secretion, in addition to the upregulation of its messenger RNA (mRNA) expression, while the proximal ST2 promoter, from which the sST2 transcript originates, was upregulated by insulin, and in a synergistic manner by DHT and insulin combination treatment. On the other hand, sIL1RAP was slightly downregulated by insulin and IL-33 mRNA expression was not affected by any of the hormones, while ST2L mRNA expression and transcription from its promoter region (distal ST2 promoter) could not be detected or showed a negligibly low level. We hypothesize that high levels of androgens and insulin might lead to subfertility and pregnancy complications, at least partially, through the sST2-dependent downregulation of uteroplacental IL-33 signaling.

Regulatory T cell-derived IL-1Ra suppresses the innate response to respiratory viral infection.

Regulatory T (Treg) cell modulation of adaptive immunity and tissue homeostasis is well described; however, less is known about Treg cell-mediated regulation of the innate immune response. Here we show that deletion of ST2, the receptor for interleukin (IL)-33, on Treg cells increased granulocyte influx into the lung and increased cytokine production by innate lymphoid and γδ T cells without alteration of adaptive immunity to influenza. IL-33 induced high levels of the interleukin-1 receptor antagonist (IL-1Ra) in ST2+ Treg cells and deletion of IL-1Ra in Treg cells increased granulocyte influx into the lung. Treg cell-specific deletion of ST2 or IL-1Ra improved survival to influenza, which was dependent on IL-1. Adventitial fibroblasts in the lung expressed high levels of the IL-1 receptor and their chemokine production was suppressed by Treg cell-produced IL-1Ra. Thus, we define a new pathway where IL-33-induced IL-1Ra production by tissue Treg cells suppresses IL-1-mediated innate immune responses to respiratory viral infection.

Comparative genotypic characterization related to antibiotic resistance phenotypes of clinical carbapenem-resistant Acinetobacter baumannii MTC1106 (ST2) and MTC0619 (ST25).

BACKGROUND: The prevalence of Acinetobacter baumannii in nosocomial infections and its remarkable ability to develop antimicrobial resistance have been a critical issue in hospital settings. Here, we examined the genomic features related to resistance phenotype displayed by carbapenem-resistant A. baumannii (CRAB) MTC1106 (ST2) and MTC0619 (ST25). RESULTS: Resistome analysis of both strains revealed that MTC1106 possessed higher numbers of antimicrobial resistance genes compared to MTC0619. Some of those genetic determinants were present in accordance with the susceptibility profile of the isolates. The predicted ISAba1 region upstream of blaOXA-23 gene was related to carbapenem resistance since this IS element was well-characterized to mediate overexpression of carbapenemase genes and eventually provided capability to confer resistance. Unlike MTC0619 strain, which only carried class B and D ß-lactamase genes, MTC1106 strain also possessed blaTEM-1D, a class A ß-lactamase. Regarding to aminoglycosides resistance, MTC0619 contained 5 related genes in which all of them belonged to three groups of aminoglycosides modifying enzyme (AME), namely, N-acetyltransferase (AAC), O-nucleotidyltransferase (ANT), and O-phosphotransferase (APH). On the other hand, MTC1106 lacked only the AAC of which found in MTC0619, yet it also carried an armA gene encoding for 16S rRNA methyltransferase. Two macrolides resistance genes, mph(E) and msr(E), were identified next to the armA gene of MTC1106 isolate in which they encoded for macrolide 2'-phosphotransferase and ABC-type efflux pump, respectively. Besides acquired resistance genes, some chromosomal genes and SNPs associated with resistance to fluoroquinolones (i.e. gyrA and parC) and colistin (i.e. pmrCAB, eptA, and emrAB) were observed. However, gene expression analysis suggested that the genetic determinants significantly contributing to low-level colistin resistance remained unclear. In addition, similar number of efflux pumps genes were identified in both lineages with only the absence of adeC, a part of adeABC RND-type multidrug efflux pump in MTC0619 strain. CONCLUSIONS: We found that MTC1106 strain harbored more antimicrobial resistance genes and showed higher resistance to antibiotics than MTC0619 strain. Regarding genomic characterization, this study was likely the first genome comparative analysis of CARB that specifically included isolates belonging to ST2 and ST25 which were widely spread in Thailand. Taken altogether, this study suggests the importance to monitor the resistance status of circulating A. baumannii clones and identify genes that may contribute to shifting the resistance trend among isolates.

Characterization of a novel ArsR regulates divergent ars operon in Ensifer adhaerens strain ST2.

Microbes evolved resistance determinates for coping with arsenic toxicity are commonly regulated by a variety of transcriptional repressors (ArsRs). Ensifer adhaerens strain ST2 was previously shown tolerance to environmental organoarsenical methylarsenite (MAs(III)), which has been proposed to be a primordial antibiotic. In E. adhaerens strain ST2 chromosomal ars operon, two MAs(III) resistance genes, arsZ, encoding MAs(III) oxidase, and arsK, encoding MAs(III) efflux transporter, are controlled by a novel ArsR transcriptional repressor, EaArsR. It has two conserved cysteine pairs, Cys91-92 and Cys108-109. Electrophoretic mobility shift assays (EMSAs) demonstrate that EaArsR binds to two inverted-repeat sequences within the ars promoter between arsR and arsZ to repress ars operon transcription and that DNA binding is relieved upon binding of As(III) and MAs(III). Mutation of either Cys91 or Cys92 to serine (or both) abolished these mutants binding to the ars promoter. In contrast, both C108S and C109S mutants kept responsiveness to As(III) and MAs(III). These results suggest that cysteine pair Cys91-Cys92 and either Cys108 or Cys109 contribute to form arsenic binding site. Homology modeling of EaArsR indicates the binding site consisted of Cys91-Cys92 pair from one monomer and Cys108-Cys109 pair from the other monomer, which displays the diverse evolution of arsenic binding site in the ArsR metalloregulators.

The Role of the IL-33/ST2 Axis in CpG-Induced Macrophage Activation Syndrome.

Background: Macrophage activation syndrome (MAS) is a fatal inflammatory condition, which is often associated with the elevation of multiple proinflammatory cytokines and multiple organ dysfunction. Previous studies have shown that ST2 contributes to T cell overactivation and plays a detrimental role in mouse models of primary hemophagocytic lymphohistiocytosis. The purpose of this study was to investigate the role of the IL-33/ST2 axis in a mouse model of MAS induced by repeated injections of cytosine-phosphate-guanine (CpG). Methods: Serum cytokines were determined using the cytometric bead array by flow cytometry. IL-33 and ST2 were detected by immunohistochemistry and real-time quantitative PCR in the liver and spleen of mice. CD3 and F4/80 in the liver were detected by immunohistochemistry. Inflammatory macrophages and effector memory T lymphocytes were detected by flow cytometry. Result: The CpG-induced MAS model was successfully induced after repeated CpG injections, presenting with hypercytokinemia and hepatosplenomegaly. The numbers of IL-33 positive cells in the liver and spleen decreased significantly, while the expression of ST2 in the liver tended to increase in the mice with MAS. IL-33 and St2 knockout mice showed similar levels of hepatosplenomegaly, peripheral blood count, and cytokine storm when compared with wild-type (WT) mice after induction of MAS. There were also no significant differences in liver pathology (including inflammatory cell infiltration of CD3 and F4/80) and levels of splenic inflammatory macrophages and effector memory T cells between the WT and knockout mice. Conclusion: These results suggested that IL-33 decreased in the liver and spleen tissues of MAS mice. Further results suggest that IL-33 and St2 knockout mice have no treatment potential in CpG-induced MAS. Thus, the IL-33/ST2 axis has little effect on the prognosis of CpG-induced MAS.

TRAF4 is crucial for ST2+ memory Th2 cell expansion in IL-33-driven airway inflammation.

Tumor necrosis factor receptor-associated factor 4 (TRAF4) is an important regulator of type 2 responses in the airway; however, the underlying cellular and molecular mechanisms remain elusive. Herein, we generated T cell-specific TRAF4-deficient (CD4-cre Traf4fl/fl) mice and investigated the role of TRAF4 in memory Th2 cells expressing IL-33 receptor (ST2, suppression of tumorigenicity 2) (ST2+ mTh2 cells) in IL-33-mediated type 2 airway inflammation. We found that in vitro-polarized TRAF4-deficient (CD4-cre Traf4fl/fl) ST2+ mTh2 cells exhibited decreased IL-33-induced proliferation as compared with TRAF4-sufficient (Traf4fl/fl) cells. Moreover, CD4-cre Traf4fl/fl mice showed less ST2+ mTh2 cell proliferation and eosinophilic infiltration in the lungs than Traf4fl/fl mice in the preclinical models of IL-33-mediated type 2 airway inflammation. Mechanistically, we discovered that TRAF4 was required for the activation of AKT/mTOR and ERK1/2 signaling pathways as well as the expression of transcription factor Myc and nutrient transporters (Slc2a1, Slc7a1, and Slc7a5), signature genes involved in T cell growth and proliferation, in ST2+ mTh2 cells stimulated by IL-33. Taken together, the current study reveals a role of TRAF4 in ST2+ mTh2 cells in IL-33-mediated type 2 pulmonary inflammation, opening up avenues for the development of new therapeutic strategies.

IL-33/ST2 axis of human amnion fibroblasts participates in inflammatory reactions at parturition.

BACKGROUND: Inflammation of the fetal membranes is an indispensable event of labor onset at both term and preterm birth. Interleukin-33 (IL-33) is known to participate in inflammation via ST2 (suppression of tumorigenicity 2) receptor as an inflammatory cytokine. However, it remains unknown whether IL-33/ST2 axis exists in human fetal membranes to promote inflammatory reactions in parturition. METHODS: The presence of IL-33 and ST2 and their changes at parturition were examined with transcriptomic sequencing, quantitative real-time polymerase chain reaction, Western blotting or immunohistochemistry in human amnion obtained from term and preterm birth with or without labor. Cultured primary human amnion fibroblasts were utilized to investigate the regulation and the role of IL-33/ST2 axis in the inflammation reactions. A mouse model was used to further study the role of IL-33 in parturition. RESULTS: Although IL-33 and ST2 expression were detected in both epithelial and fibroblast cells of human amnion, they are more abundant in amnion fibroblasts. Their abundance increased significantly in the amnion at both term and preterm birth with labor. Lipopolysaccharide, serum amyloid A1 and IL-1ß, the inflammatory mediators pertinent to labor onset, could all induce IL-33 expression through NF-κB activation in human amnion fibroblasts. In turn, via ST2 receptor, IL-33 induced the production of IL-1ß, IL-6 and PGE2 in human amnion fibroblasts via the MAPKs-NF-κB pathway. Moreover, IL-33 administration induced preterm birth in mice. CONCLUSION: IL-33/ST2 axis is present in human amnion fibroblasts, which is activated in both term and preterm labor. Activation of this axis leads to increased production of inflammatory factors pertinent to parturition, and results in preterm birth. Targeting the IL-33/ST2 axis may have potential value in the treatment of preterm birth.