Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity.

Patients with asthma, a major public health problem, are at high risk for serious disease from influenza virus infection, but the pathogenic mechanisms by which influenza A causes airway disease and asthma are not fully known. We show here in a mouse model that influenza infection acutely induced airway hyper-reactivity (AHR), a cardinal feature of asthma, independently of T helper type 2 (T(H)2) cells and adaptive immunity. Instead, influenza infection induced AHR through a previously unknown pathway that required the interleukin 13 (IL-13)-IL-33 axis and cells of the non-T cell, non-B cell innate lymphoid type called 'natural helper cells'. Infection with influenza A virus, which activates the NLRP3 inflammasome, resulted in much more production of IL-33 by alveolar macrophages, which in turn activated natural helper cells producing substantial IL-13.

Translating Research for the Radiotheranostics of Nanotargeted 188Re-Liposome.

Nanoliposomes are one of the leading potential nano drug delivery systems capable of targeting chemotherapeutics to tumor sites because of their passive nano-targeting capability through the enhanced permeability and retention (EPR) effect for cancer patients. Recent advances in nano-delivery systems have inspired the development of a wide range of nanotargeted materials and strategies for applications in preclinical and clinical usage in the cancer field. Nanotargeted 188Re-liposome is a unique internal passive radiotheranostic agent for nuclear imaging and radiotherapeutic applications in various types of cancer. This article reviews and summarizes our multi-institute, multidiscipline, and multi-functional studied results and achievements in the research and development of nanotargeted 188Re-liposome from preclinical cells and animal models to translational clinical investigations, including radionuclide nanoliposome formulation, targeted nuclear imaging, biodistribution, pharmacokinetics, radiation dosimetry, radiation tumor killing effects in animal models, nanotargeted radionuclide and radio/chemo-combination therapeutic effects, and acute toxicity in various tumor animal models. The systemic preclinical and clinical studied results suggest 188Re-liposome is feasible and promising for in vivo passive nanotargeted radionuclide theranostics in future cancer care applications.

CEACAM3 decreases asthma exacerbations and modulates respiratory syncytial virus latent infection in children.

BACKGROUND: Respiratory syncytial virus (RSV) is associated with childhood asthma. Nevertheless, not all children exposed to RSV develop asthma symptoms, possibly because genes modulate the effects of RSV on asthma exacerbations. OBJECTIVE: The purpose of this study was to identify genes that modulate the effect of RSV latent infection on asthma exacerbations. METHODS: We performed a meta-analysis to investigate differentially expressed genes (DEGs) of RSV infection from Gene Expression Omnibus datasets. Expression quantitative trait loci (eQTL) methods were applied to select single nucleotide polymorphisms (SNPs) that were associated with DEGs. Gene-based analysis was used to identify SNPs that were significantly associated with asthma exacerbations in the Taiwanese Consortium of Childhood Asthma Study (TCCAS), and validation was attempted in an independent cohort, the Childhood Asthma Management Program (CAMP). Gene-RSV interaction analyses were performed to investigate the association between the interaction of SNPs and RSV latent infection on asthma exacerbations. RESULTS: A total of 352 significant DEGs were found by meta-analysis of RSV-related genes. We used 38 123 SNPs related to DEGs to investigate the genetic main effects on asthma exacerbations. We found that eight RSV-related genes (GADD45A, GYPB, MS4A3, NFE2, RNASE3, EPB41L3, CEACAM6 and CEACAM3) were significantly associated with asthma exacerbations in TCCAS and also validated in CAMP. In TCCAS, rs7251960 (CEACAM3) significantly modulated the effect of RSV latent infection on asthma exacerbations (false-discovery rate <0.05). The rs7251960 variant was associated with CEACAM3 mRNA expression in lung tissue (p for trend=1.2×10-7). CEACAM3 mRNA was reduced in nasal mucosa from subjects with asthma exacerbations in two independent datasets. CONCLUSIONS: rs7251960 is an eQTL for CEACAM3, and CEACAM3 mRNA expression is reduced in subjects experiencing asthma exacerbations. CEACAM3 may be a modulator of RSV latent infection on asthma exacerbations.

Pulmonary IL-33 orchestrates innate immune cells to mediate respiratory syncytial virus-evoked airway hyperreactivity and eosinophilia.

BACKGROUND: Respiratory syncytial virus (RSV) infection is epidemiologically linked to asthma. During RSV infection, IL-33 is elevated and promotes immune cell activation, leading to the development of asthma. However, which immune cells are responsible for triggering airway hyperreactivity (AHR), inflammation and eosinophilia remained to be clarified. We aimed to elucidate the individual roles of IL-33-activated innate immune cells, including ILC2s and ST2+ myeloid cells, in RSV infection-triggered pathophysiology. METHODS: The role of IL-33/ILC2 axis in RSV-induced AHR inflammation and eosinophilia were evaluated in the IL-33-deficient and YetCre-13 Rosa-DTA mice. Myeloid-specific, IL-33-deficient or ST2-deficient mice were employed to examine the role of IL-33 and ST2 signaling in myeloid cells. RESULTS: We found that IL-33-activated ILC2s were crucial for the development of AHR and airway inflammation, during RSV infection. ILC2-derived IL-13 was sufficient for RSV-driven AHR, since reconstitution of wild-type ILC2 rescued RSV-driven AHR in IL-13-deficient mice. Meanwhile, myeloid cell-derived IL-33 was required for airway inflammation, ST2+ myeloid cells contributed to exacerbation of airway inflammation, suggesting the importance of IL-33 signaling in these cells. Local and peripheral eosinophilia is linked to both ILC2 and myeloid IL-33 signaling. CONCLUSIONS: This study highlights the importance of IL-33-activated ILC2s in mediating RSV-triggered AHR and eosinophilia. In addition, IL-33 signaling in myeloid cells is crucial for airway inflammation.

CPEB2-activated PDGFRα mRNA translation contributes to myofibroblast proliferation and pulmonary alveologenesis.

BACKGROUND: Alveologenesis is the final stage of lung development to form air-exchanging units between alveoli and blood vessels. Genetic susceptibility or hyperoxic stress to perturb this complicated process can cause abnormal enlargement of alveoli and lead to bronchopulmonary dysplasia (BPD)-associated emphysema. Platelet-derived growth factor receptor α (PDGFRα) signaling is crucial for alveolar myofibroblast (MYF) proliferation and its deficiency is associated with risk of BPD, but posttranscriptional mechanisms regulating PDGFRα synthesis during lung development remain largely unexplored. Cytoplasmic polyadenylation element-binding protein 2 (CPEB2) is a sequence-specific RNA-binding protein and translational regulator. Because CPEB2-knockout (KO) mice showed emphysematous phenotypes, we investigated how CPEB2-controlled translation affects pulmonary development and function. METHODS: Respiratory and pulmonary functions were measured by whole-body and invasive plethysmography. Histological staining and immunohistochemistry were used to analyze morphology, proliferation, apoptosis and cell densities from postnatal to adult lungs. Western blotting, RNA-immunoprecipitation, reporter assay, primary MYF culture and ectopic expression rescue were performed to demonstrate the role of CPEB2 in PDGFRα mRNA translation and MYF proliferation. RESULTS: Adult CPEB2-KO mice showed emphysema-like dysfunction. The alveolar structure in CPEB2-deficient lungs appeared normal at birth but became simplified through the alveolar stage of lung development. In CPEB2-null mice, we found reduced proliferation of MYF progenitors during alveolarization, abnormal deposition of elastin and failure of alveolar septum formation, thereby leading to enlarged pulmonary alveoli. We identified that CPEB2 promoted PDGFRα mRNA translation in MYF progenitors and this positive regulation could be disrupted by H2O2, a hyperoxia-mimetic treatment. Moreover, decreased proliferating ability in KO MYFs due to insufficient PDGFRα expression was rescued by ectopic expression of CPEB2, suggesting an important role of CPEB2 in upregulating PDGFRα signaling for pulmonary alveologenesis. CONCLUSIONS: CPEB2-controlled translation, in part through promoting PDGFRα expression, is indispensable for lung development and function. Since defective pulmonary PDGFR signaling is a key feature of human BPD, CPEB2 may be a risk factor for BPD.

Toll-like receptor 9-dependent interferon production prevents group 2 innate lymphoid cell-driven airway hyperreactivity.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are important mediators of allergic asthma. Bacterial components, such as unmethylated CpG DNA, a Toll-like receptor (TLR) 9 agonist, are known to possess beneficial immunomodulatory effects in patients with T cell-mediated chronic asthma. However, their roles in regulating ILC2s remain unclear. OBJECTIVE: We sought to determine the role of TLR9 activation in regulating ILC2 function and to evaluate the therapeutic utility of an immunomodulatory microparticle containing natural TLR9 ligand (MIS416). METHODS: We evaluated the immunomodulatory effects of CpG A in IL-33-induced airway hyperreactivity (AHR) and airway inflammation. The roles of interferons were examined in vivo and in vitro by using signal transducer and activator of transcription 1 (Stat1)-/- mice and neutralizing antibodies against IFN-γ and IFN-α/ß receptor subunit 1, and their cellular sources were identified. The therapeutic utility of MIS416 was investigated in the Alternaria alternata model of allergic asthma and in humanized NSG mice. RESULTS: We show that TLR9 activation by CpG A suppresses IL-33-mediated AHR and airway inflammation through inhibition of ILC2s. Activation of TLR9 leads to production of IFN-α, which drives IFN-γ production by natural killer cells. Importantly, IFN-γ is essential for TLR9-driven suppression, and IFN-α cannot compensate for impaired IFN-γ signaling. We further show that IFN-γ directly inhibits ILC2 function through a STAT1-dependent mechanism. Finally, we demonstrate the therapeutic potential of MIS416 in A alternata-induced airway inflammation and validated these findings in human subjects. CONCLUSION: TLR9 activation alleviates ILC2-driven AHR and airway inflammation through direct suppression of cell function. Microparticle-based delivery of TLR9 ligands might serve as a therapeutic strategy for asthma treatment.

A natural killer T-cell subset that protects against airway hyperreactivity.

BACKGROUND: Infection of suckling mice with influenza virus expands a CD4-CD8- double-negative (DN) natural killer T (NKT) cell subpopulation that protects the mice as adults against allergen-induced airway hyperreactivity (AHR). However, this NKT cell subset has not been characterized, and the underlying mechanisms of protection remain unknown. OBJECTIVE: We characterized this specific NKT cell subpopulation that developed during influenza infection in neonatal mice and that suppressed the subsequent development of AHR. METHODS: A cell-surface marker was identified by comparing the mRNA expression profile of wild-type CD4+ NKT cells with that of suppressive Vα14 DN NKT cells. The marker-enriched NKT cell subset was then analyzed for its cytokine profile and its suppressive in vitro and in vivo abilities. RESULTS: We showed that DN NKT cells with high CD38 expression produced IFN-γ, but not IL-17, IL-4, or IL-13, and inhibited development of AHR through contact-dependent suppression of helper CD4 T-cell proliferation. The NKT subset expanded in the lungs of neonatal mice after infection with influenza and also after treatment of neonatal mice with Nu-α-GalCer, which effectively increased DN CD38hi NKT cell numbers. CONCLUSION: These results suggest that early/neonatal exposure to infection or antigen challenge affects subsequent lung immunity by altering the cellular composition of cells in the lung and that some subsets of NKT cells suppress AHR. These results provide a possible mechanism by which prior infections can protect against the development of allergic asthma and might be further explored as a protective measure for young children.

Biodistribution, pharmacokinetics and radioimmunotherapy of 188Re-cetuximab in NCI-H292 human lung tumor-bearing nude mice.

Background Cetuximab is a fully humanized IgG1 subclass monoclonal that binds specifically to the human epidermal growth factor receptor (EGFR). Although EGFR is expressed in normal cells, the overexpression of EGFR is detected in many human cancers, such as colon, rectum and lung tumors. In this study, cetuximab with a combination of radiotherapy nuclear 188Re achieved better therapeutic effect on lung cancer. Methods188Re-cetuximab administered by the i.v. route in human NCI-H292 lung tumor-bearing mice was investigated. NanoSPECT/CT images were taken to evaluate the distribution and tumor targeting of 188Re-cetuximab in mice. The anti-tumor effect of 188Re-cetuximab was assessed by the tumor growth inhibition, survival ratio. Results For nanoSPECT/CT imaging, a significant uptake in tumor was observed at 24 and 48 h following the injection of 188Re-cetuximab. The anti-tumor effect of 188Re-cetuximab was assessed by tumor growth inhibition and the survival ratio. The tumor-bearing mice treated with 188Re-cetuximab showed a better mean tumor growth inhibition rate (MGI = 0.049) and longer median survival time and lifespan (62.50 d; 70.07%) than those treated with 188Re-perrhenate and cetuximab only by single injection. A synergistic effect of tumor growth inhibition was observed with the combination index exceeding one for 188Re-cetuximab (CI = 6.135 and 9.276). Conclusion The tumor targeting and localization of 188Re-cetuximab were confirmed in this study. Synergistic therapeutic efficacy was demonstrated for the radioimmunotherapy of 188Re-cetuximab. The results of this study reveal the potential advantage and benefit obtained from 188Re-cetuximab for diagnosis and therapy of oncology applications in the future.

Regulation of type 2 innate lymphoid cell-dependent airway hyperreactivity by butyrate.

BACKGROUND: Allergic asthma is characterized by airway hyperreactivity (AHR) and inflammation driven by aberrant TH2 responses. Type 2 innate lymphoid cells (ILC2s) are a critical source of the TH2 cytokines IL-5 and IL-13, which promote acute asthma exacerbation. Short-chain fatty acids (SCFAs) have been shown to attenuate T cell-mediated allergic airway inflammation. However, their role in regulation of ILC2-driven AHR and lung inflammation remains unknown. OBJECTIVE: We investigated the immunomodulatory role of SCFAs in regulation of ILC2-induced AHR and airway inflammation and delineated the mechanism involved. METHODS: We assessed the role of SCFAs in regulating survival, proliferation, and cytokine production in lung sorted ILC2s. The SCFA butyrate was administered through drinking water or intranasally in BALB/c mice to evaluate its role in the ILC2-driven inflammatory response in IL-33 and Alternaria alternata models of allergic inflammation. We further confirmed our findings in human ILC2s. RESULTS: We show that butyrate, but not acetate or propionate, inhibited IL-13 and IL-5 production by murine ILC2s. Systemic and local administration of butyrate significantly ameliorated ILC2-driven AHR and airway inflammation. We further demonstrate that butyrate inhibited ILC2 proliferation and GATA3 expression but did not induce cell apoptosis, likely through histone deacetylase (HDAC) inhibition, because trichostatin A, a pan-HDAC inhibitor, exerted similar effects on ILC2s. Importantly, cotreatment with trichostatin A and butyrate did not result in an additive effect. Finally, we show that butyrate reduces cytokine production in human ILC2s. CONCLUSION: Our findings identify butyrate as a critical regulator of ILC2 proliferation and function through its HDAC inhibitory activity and can serve as a potential therapeutic target for asthma.

Deficiency of CPEB2-Confined Choline Acetyltransferase Expression in the Dorsal Motor Nucleus of Vagus Causes Hyperactivated Parasympathetic Signaling-Associated Bronchoconstriction.

Cytoplasmic polyadenylation element binding protein 2 (CPEB2) is an RNA-binding protein and translational regulator. To understand the physiological function of CPEB2, we generated CPEB2 knock-out (KO) mice and found that most died within 3 d after birth. CPEB2 is highly expressed in the brainstem, which controls vital functions, such as breathing. Whole-body plethysmography revealed that KO neonates had aberrant respiration with frequent apnea. Nevertheless, the morphology and function of the respiratory rhythm generator and diaphragm neuromuscular junctions appeared normal. We found that upregulated translation of choline acetyltransferase in the CPEB2 KO dorsal motor nucleus of vagus resulted in hyperactivation of parasympathetic signaling-induced bronchoconstriction, as evidenced by increased pulmonary acetylcholine and phosphorylated myosin light chain 2 in bronchial smooth muscles. Specific deletion of CPEB2 in cholinergic neurons sufficiently caused increased apnea in neonatal pups and airway hyper-reactivity in adult mice. Moreover, inhalation of an anticholinergic bronchodilator reduced apnea episodes in global and cholinergic CPEB2-KO mice. Together, the elevated airway constriction induced by cholinergic transmission in KO neonates may account for the respiratory defect and mortality. SIGNIFICANCE STATEMENT: This study first generated and characterized cpeb2 gene-deficient mice. CPEB2-knock-out (KO) mice are born alive but most die within 3 d after birth showing no overt defects in anatomy. We found that the KO neonates showed severe apnea and altered respiratory pattern. Such respiratory defects could be recapitulated in mice with pan-neuron-specific or cholinergic neuron-specific ablation of the cpeb2 gene. Further investigation revealed that cholinergic transmission in the KO dorsal motor nucleus of vagus was overactivated because KO mice lack CPEB2-suppressed translation of the rate-limiting enzyme in the production of acetylcholine (i.e., choline acetyltransferase). Consequently, increased parasympathetic signaling leads to hyperactivated bronchoconstriction and abnormal respiration in the KO neonates.

Upregulation of Interleukin-33 in obstructive renal injury.

Interstitial fibrosis and loss of parenchymal tubular cells are the common outcomes of progressive renal diseases. Pro-inflammatory cytokines have been known contributing to the damage of tubular cells and fibrosis responses after renal injury. Interleukin (IL)-33 is a tissue-derived nucleus alarmin that drives inflammatory responses. The regulation and function of IL-33 in renal injury, however, is not well understood. To investigate the involvement of cytokines in the pathogenesis of renal injury and fibrosis, we performed the mouse renal injury model induced by unilateral urinary obstruction (UUO) and analyze the differentially upregulated genes between the obstructed and the contralateral unobstructed kidneys using RNA sequencing (RNAseq). Our RNAseq data identified IL33 and its receptor ST2 were upregulated in the UUO kidney. Quantitative analysis confirmed that transcripts of IL33 and ST2 were upregulated in the obstructed kidneys. Immunofluorescent staining revealed that IL-33 was upregulated in Vimentin- and alpha-SMA-positive interstitial cells. By using genetically knockout mice, deletion of IL33 reduced UUO-induced renal fibrosis. Moreover, in combination with BrdU labeling technique, we observed that the numbers of proliferating tubular epithelial cells were increased in the UUO kidneys from IL33-or ST2-deficient mice compared to wild type mice. Collectively, our study demonstrated the upregulation of IL-33/ST2 signaling in the obstructed kidney may promote tubular cell injury and interstitial fibrosis. IL-33 may serve as a biomarker to detect renal injury and that IL-33/ST2 signaling may represent a novel target for treating renal diseases.

α-Galactosylceramide but not phenyl-glycolipids induced NKT cell anergy and IL-33-mediated myeloid-derived suppressor cell accumulation via upregulation of egr2/3.

Strategies for cancer immunotherapy include activating immune system for therapeutic benefit or blockade of immune checkpoints. To harness innate immunity to fight cancer, α-galactosylceramide (α-GalCer) has been used to activate NKT cells. Unfortunately, administration of α-GalCer causes long-term NKT cell anergy, but the molecular mechanism is unclear. In this study, we showed that α-GalCer-triggered egr2/3, which induced programmed death 1 and cbl-b in NKT cells, leading to NKT cell anergy. We also uncovered the induction of the immunosuppressive myeloid-derived suppressor cells (MDSCs) in the spleen by α-GalCer that might attenuate its antitumor efficacy. The accumulation of MDSC was accompanied by 20-fold rise in their arg-1 mRNAs and enhanced expression of programmed death 1/programmed death ligand 1. Furthermore, α-GalCer-induced egr-2/3 in hepatic NKT cells upregulated their TRAIL in addition to Fas ligand (FasL) and induced alarm signaling molecule IL-33 in Kupffer cells, presumably because of liver damage triggered by TRAIL/FasL. We further demonstrated that IL-33-stimulated macrophages produce G-CSF, which in turn, boosted MDSCs. Thus, α-GalCer-induced FasL/TRAIL and IL-33 provided a novel mechanism underlying α-GalCer-induced hepatotoxicity and MDSC accumulation. In contrast, analogs of α-GalCer containing phenyl group in the lipid tail could neither induce NKT anergy nor enhance MDSCs accumulation. Furthermore, tumor-infiltrating MDSCs in mice injected repeatedly with α-GalCer were 2-fold higher than those treated with phenyl-glycolipids. These results not only revealed the induction of MDSC via IL-33 as a new mechanism for α-GalCer-elicited immunosuppression but also provided one of the mechanisms underlying the superior antitumor potency of phenyl-glycolipids. Our findings have important implications for the development of NKT-stimulatory glycolipids as vaccine adjuvants and anticancer therapeutics.

Innate lymphoid cells and asthma.

Asthma is a complex and heterogeneous disease with several phenotypes, including an allergic asthma phenotype characterized by TH2 cytokine production and associated with allergen sensitization and adaptive immunity. Asthma also includes nonallergic asthma phenotypes, such as asthma associated with exposure to air pollution, infection, or obesity, that require innate rather than adaptive immunity. These innate pathways that lead to asthma involve macrophages, neutrophils, natural killer T cells, and innate lymphoid cells, newly described cell types that produce a variety of cytokines, including IL-5 and IL-13. We review the recent data regarding innate lymphoid cells and their role in asthma.

Avidity of CD1d-ligand-receptor ternary complex contributes to T-helper 1 (Th1) polarization and anticancer efficacy.

Invariant natural killer T cell (NKT) cells (iNKT cells) produce both T-helper 1 (Th1) and T-helper 2 cytokines in response to α-Galactosylceramide (α-GalCer) stimulation and are thought to be the important effectors in the regulation of both innate and adaptive immunity involved in autoimmune disorders, microbial infections, and cancers. However, the anticancer effects of α-GalCer were limited in early clinical trial. In this study, several analogs of α-GalCer, containing phenyl groups in the lipid tails were found to stimulate murine and human iNKT cells to secrete Th1-skewed cytokines and exhibit greater anticancer efficacy in mice than α-GalCer. We explored the possibility of different Vß usages of murine Vα14 iNKT or human Vα24 iNKT cells, accounting for differential cytokine responses. However, T-cell receptor Vß analysis revealed no significant differences in Vß usages by α-GalCer and these phenyl glycolipid analogs. On the other hand, these phenyl glycolipids showed greater binding avidity and stability for iNKT T-cell receptor when complexed with CD1d. These findings suggest that CD1d-phenyl glycolipid complexes may interact with the same population of iNKT cells but with higher avidity and stability to drive Th1 polarization. Thus, this study provides a key to the rational design of Th1 biased CD1d reactive glycolipids in the future.

T-cell immunoglobulin and mucin domain 1 deficiency eliminates airway hyperreactivity triggered by the recognition of airway cell death.

BACKGROUND: Studies of asthma have been limited by a poor understanding of how nonallergic environmental exposures, such as air pollution and infection, are translated in the lung into inflammation and wheezing. OBJECTIVE: Our goal was to understand the mechanism of nonallergic asthma that leads to airway hyperreactivity (AHR), a cardinal feature of asthma independent of adaptive immunity. METHOD: We examined mouse models of experimental asthma in which AHR was induced by respiratory syncytial virus infection or ozone exposure using mice deficient in T-cell immunoglobulin and mucin domain 1 (TIM1/HAVCR1), an important asthma susceptibility gene. RESULTS: TIM1(-/-) mice did not have airways disease when infected with RSV or when repeatedly exposed to ozone, a major component of air pollution. On the other hand, the TIM1(-/-) mice had allergen-induced experimental asthma, as previously shown. The RSV- and ozone-induced pathways were blocked by treatment with caspase inhibitors, indicating an absolute requirement for programmed cell death and apoptosis. TIM-1-expressing, but not TIM-1-deficient, natural killer T cells responded to apoptotic airway epithelial cells by secreting cytokines, which mediated the development of AHR. CONCLUSION: We defined a novel pathway in which TIM-1, a receptor for phosphatidylserine expressed by apoptotic cells, drives the development of asthma by sensing and responding to injured and apoptotic airway epithelial cells.

SCUBE2 regulates adherens junction dynamics and vascular barrier function during inflammation.

AIMS: SCUBE2 (Signal peptide-CUB-epidermal growth factor-like domain-containing protein 2) is a secreted or membrane-bound protein originally identified from endothelial cells (ECs). Our previous work showed that SCUBE2 forms a complex with E-cadherin and stabilizes epithelial adherens junctions (AJs) to promote epithelial phenotypes. However, it remains unclear whether SCUBE2 also interacts with vascular endothelial (VE)-cadherin and modulates EC barrier function. In this study, we investigated whether and how SCUBE2 in ECs regulates vascular barrier maintenance. METHODS AND RESULTS: We showed that SCUBE2 colocalized and interacted with VE-cadherin and VE-protein tyrosine phosphatase (VE-PTP) within EC AJs. Furthermore, SCUBE2 knockdown disrupted EC AJs and increased EC permeability. Expression of EC SCUBE2 was suppressed at both mRNA and protein levels via the nuclear factor-κB (NF-κB) signaling pathway in response to pro-inflammatory cytokines or permeability-inducing agents. In line with these findings, EC-specific deletion of Scube2 (EC-KO) in mice impaired baseline barrier function and worsened vascular leakiness of peripheral capillaries after local injection of histamine or vascular endothelial growth factor. EC-KO mice were also sensitive to pulmonary vascular hyperpermeability and leukocyte infiltration in response to acute endotoxin- or influenza virus-induced systemic inflammation. Meanwhile, EC-specific SCUBE2-overexpressing mice were protected from these effects. Molecular studies suggested that SCUBE2 acts as a scaffold molecule enabling VE-PTP to dephosphorylate VE-cadherin, which prevents VE-cadherin internalization and stabilizes EC AJs. As such, loss of SCUBE2 resulted in hyperphosphorylation of VE-cadherin at tyrosine 685, which led to its endocytosis, thus destabilizing EC AJs and reducing barrier function. All of these effects were exacerbated by inflammatory insults. CONCLUSIONS: We found that SCUBE2 contributes to vascular integrity by recruiting VE-PTP to dephosphorylate VE-cadherin and stabilize AJs, thereby promoting EC barrier function. Moreover, our data suggest that genetic overexpression or pharmacological upregulation of SCUBE2 may help to prevent vascular leakage and edema in inflammatory diseases.

Staphylococcus aureus exacerbates dermal IL-33/ILC2 axis activation through evoking RIPK3/MLKL-mediated necroptosis of dry skin.

Atopic dermatitis (AD) is a persistent skin disease typified by symptoms of dry skin and recurrent eczema. Patients with AD are at heightened risk for Staphylococcus aureus infection. Group 2 innate lymphoid cells (ILC2s) are mainly activated by epithelial cell-derived cytokines IL-33 and involved in the pathogenesis of AD. However, little is known about the effect of skin delipidization on the epithelial cell-derived cytokines and dermal ILC2s in AD. In our study, we investigated the mechanism by which S. aureus infection modulates and exacerbates the pathogenesis of dry skin, leading to type 2 inflammation in the context of innate immunity. In vivo, we found that S. aureus infection aggravated delipidization-induced dermal IL-33 release and dermal ILC2 accumulation, which exacerbated skin inflammation. We also noticed that Il33fl/fl K14cre mice and Tlr2-/- mice exhibited attenuated skin inflammation. In vitro, treatment with necroptosis inhibitors reduced IL-33 release from S. aureus-infected keratinocytes. Mechanistically, we observed an increase in the necroptosis-associated kinases, MLKL and RIPK3, in S. aureus-infected mice, indicating that IL-33 release was associated with necroptotic cell death responses. Our results reveal that S. aureus infection-elicited keratinocyte necroptosis contributes to IL-33-mediated type 2 inflammation, which exacerbates the pathogenesis of dry skin.

Therapeutic efficacy of 188Re-liposome in a C26 murine colon carcinoma solid tumor model.

Nanoliposomes are good drug delivery systems that allow the encapsulation of drugs into vesicles for their delivery. The objective of this study is to investigate the therapeutic efficacy of a new radio-therapeutics of (188)Re-labeled pegylated liposome in a C26 murine colon carcinoma solid tumor model. The safety of (188)Re-liposome was evaluated before radiotherapy treatment. The anti-tumor effect of (188)Re-liposome was assessed by tumor growth inhibition, survival ratio and ultrasound imaging. Apoptotic marker in tumor was also evaluated by the TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling) method after injection of (188)Re-liposome. The group treated with (188)Re-liposome displayed slight loss in body weight and decrease in white blood cell (WBC) count 7 to 14 days post-injection. With respect to therapeutic efficacy, the tumor-bearing mice treated with (188)Re-liposome showed better mean tumor growth inhibition rate (MGI) and longer median survival time (MGI = 0.140; 80 day) than those treated with anti-cancer drug 5-FU (MGI = 0.195; 69 day) and untreated control mice (MGI = 0.413; 48 day). The ultrasound imaging showed a decrease in both tumor volume and number of blood vessels. There were significantly more apoptotic nuclei (TUNEL-positive) in (188)Re-liposome-treated mice at 8 h after treatment than in control mice. These results evidenced the potential benefits achieved by oncological application of the radio-therapeutics (188)Re-liposome for adjuvant cancer treatment.

Innate lymphoid cells responding to IL-33 mediate airway hyperreactivity independently of adaptive immunity.

BACKGROUND: Asthma has been considered an immunologic disease mediated by T(H)2 cells and adaptive immunity. However, clinical and experimental observations suggest that additional pathways might regulate asthma, particularly in its nonallergic forms, such as asthma associated with air pollution, stress, obesity, and infection. OBJECTIVES: Our goal was to understand T(H)2 cell-independent conditions that might lead to airway hyperreactivity (AHR), a cardinal feature of asthma. METHODS: We examined a murine model of experimental asthma in which AHR was induced with glycolipid antigens, which activate natural killer T (NKT) cells. RESULTS: In this model AHR developed rapidly when mice were treated with NKT cell-activating glycolipid antigens, even in the absence of conventional CD4(+) T cells. The activated NKT cells directly induced alveolar macrophages to produce IL-33, which in turn activated NKT cells, as well as natural helper cells, a newly described non-T, non-B, innate lymphoid cell type, to increase production of IL-13. Surprisingly, this glycolipid-induced AHR pathway required not only IL-13 but also IL-33 and its receptor, ST2, because it was blocked by an anti-ST2 mAb and was greatly reduced in ST2(-/-) mice. When adoptively transferred into IL-13(-/-) mice, both wild-type natural helper cells and NKT cells were sufficient for the development of glycolipid-induced AHR. CONCLUSION: Because plant pollens, house dust, and some bacteria contain glycolipids that can directly activate NKT cells, these studies suggest that AHR and asthma can fully develop or be greatly enhanced through innate immune mechanisms involving IL-33, natural helper cells, and NKT cells.

The modulation of pulmonary group 2 innate lymphoid cell function in asthma: from inflammatory mediators to environmental and metabolic factors.

A dysregulated type 2 immune response is one of the fundamental causes of allergic asthma. Although Th2 cells are undoubtedly central to the pathogenesis of allergic asthma, the discovery of group 2 innate lymphoid cells (ILC2s) has added another layer of complexity to the etiology of this chronic disease. Through their inherent innate type 2 responses, ILC2s not only contribute to the initiation of airway inflammation but also orchestrate the recruitment and activation of other members of innate and adaptive immunity, further amplifying the inflammatory response. Moreover, ILC2s exhibit substantial cytokine plasticity, as evidenced by their ability to produce type 1- or type 17-associated cytokines under appropriate conditions, underscoring their potential contribution to nonallergic, neutrophilic asthma. Thus, understanding the mechanisms of ILC2 functions is pertinent. In this review, we present an overview of the current knowledge on ILC2s in asthma and the regulatory factors that modulate lung ILC2 functions in various experimental mouse models of asthma and in humans.