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Hemin, a substrate of heme oxygenase (HO)-1, induces HO-1 expression on a variety of cells to exert anti-oxidant and anti-inflammatory roles. However, the role of HO-1 in allergic diseases for dendritic cells (DCs) is not fully understood. Here, we report that HO-1 modulates asthmatic airway inflammation by hemin-treated DC-released extracellular vesicles (DCEVs). Following induction of bone marrow-derived DCs by hemin and then by house dust mite (HDM) in vitro, mouse CD4+ naïve T cells were cocultured with DCEVs to determine T helper (h) cell differentiation. C57BL/6 mice were sensitized by different stimuli-induced DCEVs and challenged with HDM to analyze the changes of inflammatory cells and cytokines in the lung and bronchoalveolar lavage fluid. The results showed that hemin-treated DCEVs (hemin-DCEVs) express phosphatidylserine (PS), CD81, heat shock protein 70, and HO-1, which facilitates regulatory T (Treg) cells differentiation in vitro and in vivo. In HDM-induced asthmatic mouse model, hemin-DCEVs inhalation reduced eosinophils infiltration and mucus secretion in the airway, decreased the levels of IL-4, IL-5, and IL-13 in the lung and the number of Th2 cells in mediastinal lymph nodes (MLNs), and increased the number of Treg cells in MLNs. Thus, our study demonstrated, for the first time, that EVs from HO-1-overexpressing DCs alleviate allergic airway inflammation of eosinophilic asthma by potentiating Treg cells differentiation and limiting proinflammatory cytokine secretion, which expands our understanding of HO-1 function, opening the door for HO-1 inducer-like hemin as a novel therapeutic strategy for asthma or other allergic diseases.

Gut microbiota is increasingly linked to the development of various pulmonary diseases through a gut–lung axis. However, the mechanisms by which gut commensal microbes impact trafficking and functional transition of immune cells remain largely unknown. Using integrated microbiota dysbiosis approaches, we uncover that the gut microbiota directs the migration of group 2 innate lymphoid cells (ILC2s) from the gut to the lung through a gut–lung axis. We identify Proteobacteria as a critical species in the gut microbiome to facilitate natural ILC2 migration, and increased Proteobacteria induces IL-33 production. Mechanistically, IL-33–CXCL16 signaling promotes the natural ILC2 accumulation in the lung, whereas IL-25–CCL25 signals augment inflammatory ILC2 accumulation in the intestines upon abdominal infection, parabiosis, and cecum ligation and puncture in mice. We reveal that these two types of ILC2s play critical but distinct roles in regulating inflammation, leading to balanced host defense against infection. Overall results delineate that Proteobacteria in gut microbiota modulates ILC2 directional migration to the lung for host defense via regulation of select cytokines (IL-33), suggesting novel therapeutic strategies to control infectious diseases.

Bitter receptors function primarily in sensing taste, but may also have other functions, such as detecting pathogenic organisms due to their agile response to foreign objects. The mouse taste receptor type-2 member 138 (TAS2R138) is a member of the G-protein-coupled bitter receptor family, which is not only found in the tongue and nasal cavity, but also widely distributed in other organs, such as the respiratory tract, gut, and lungs. Despite its diverse functions, the role of TAS2R138 in host defense against bacterial infection is largely unknown. Here, we show that TAS2R138 facilitates the degradation of lipid droplets (LDs) in neutrophils during Pseudomonas aeruginosa infection through competitive binding with PPARG (peroxisome proliferator-activated receptor gamma) antagonist: N-(3-oxododecanoyl)-l-homoserine lactone (AHL-12), which coincidently is a virulence-bound signal produced by this bacterium (P. aeruginosa). The released PPARG then migrates from nuclei to the cytoplasm to accelerate the degradation of LDs by binding PLIN2 (perilipin-2). Subsequently, the TAS2R138–AHL-12 complex targets LDs to augment their degradation, and thereby facilitating the clearance of AHL-12 in neutrophils to maintain homeostasis in the local environment. These findings reveal a crucial role for TAS2R138 in neutrophil-mediated host immunity against P. aeruginosa infection.

BackgroundEosinophils act as a secondary antigen-presenting cell (APC) to stimulate Th cell responses against antigens. IL-25 plays a significant role in eosinophil activation in allergic asthma. The role of IL-25 on the classic APC functions of dendritic cells has been elucidated. However, whether IL-25 facilitates eosinophils for antigen presentation is unknown.ObjectiveTo elucidate the role of IL-25 on eosinophils antigen presenting function during allergic asthma and its related mechanism.MethodsEosinophils from allergic asthma subjects were cultured with IL-25 and HDM to identify the co-stimulator molecules expression. Co-cultures of patient eosinophils and autologous naïve CD4+ T cells in the same culture system were to explore whether eosinophils had the capacity to promote Th cell differentiation in response to IL-25 engagement. In asthma mouse model, IL-25-/- mice were exposed to HDM to investigate the effect of IL-25 on eosinophils during the sensitization phase. The impact of IL-25 on the capacity for eosinophils taking up antigens was evaluated. Mouse bone marrow derived eosinophils (BmEOS) were co-cultured with naïve CD4+T cells sorted from spleens under HDM and IL-25 stimulation to identify T cell differentiation.ResultsIL-25 upregulated HLA-DR, PD-L1, and OX-40L expression on eosinophils from allergic asthma patients. IL-25 and HDM co-sensitized eosinophils promoted Th2 differentiation. In mouse model, IL-25-/- mice experienced restrained allergic pulmonary inflammation and reduced eosinophils recruitment and antigen uptake capacity during the early sensitization phase. In vitro, IL-25 promoted antigen uptake by eosinophils. In BmEOS and naïve CD4+T cells co-culture, IL-25 accreted the proportion of CD4+Th2 cells, which was absent in CD4+T cells culture alone.ConclusionOur data identify a novel role of IL-25 in enhancing eosinophils antigen uptake and co-stimulator molecules expression to induce Th2 priming in the context of allergic inflammation.

Gene-editing technologies, including the widespread usage of CRISPR endonucleases, have the potential for clinical treatments of various human diseases. Due to the rapid mutations of SARS-CoV-2, specific and effective prevention and treatment by CRISPR toolkits for coronavirus disease 2019 (COVID-19) are urgently needed to control the current pandemic spread. Here, we designed Type III CRISPR endonuclease antivirals for coronaviruses (TEAR-CoV) as a therapeutic to combat SARS-CoV-2 infection. We provided a proof of principle demonstration that TEAR-CoV-based RNA engineering approach leads to RNA-guided transcript degradation both in vitro and in eukaryotic cells, which could be used to broadly target RNA viruses. We report that TEAR-CoV not only cleaves SARS-CoV-2 genome and mRNA transcripts, but also degrades live influenza A virus (IAV), impeding viral replication in cells and in mice. Moreover, bioinformatics screening of gRNAs along RNA sequences reveals that a group of five gRNAs (hCoV-gRNAs) could potentially target 99.98% of human coronaviruses. TEAR-CoV also exerted specific targeting and cleavage of common human coronaviruses. The fast design and broad targeting of TEAR-CoV may represent a versatile antiviral approach for SARS-CoV-2 or potentially other emerging human coronaviruses.

Airway epithelial cells (AECs) participate in allergic airway inflammation by producing mediators in response to allergen stimulation. Whether ovalbumin (OVA) challenge promotes exosome release from AECs (OVA‐challenged AEC‐derived exosomes (OAEs)), thereby affecting airway inflammation, as well as the underlying mechanisms, is unknown. Our study showed that AECs released an increased number of exosomes after OVA challenge, and the expression of Plexin B2 (PLXNB2; a natural CD100 ligand) was increased by a massive 85.7‐fold in OAEs than in PBS‐treated AEC‐derived exosomes (PAEs). CD100+F4/80+ macrophages engulfed OAEs to trigger the transcription of pro‐inflammatory chemokines and cytokines. Plxnb2 transcripts increased in asthmatic lungs, and similarly, PLXNB2 protein was highly enriched in exosomes purified from asthmatic bronchoalveolar lavage (BAL) fluid. Furthermore, aspiration of PLXNB2 or OAEs increased the recruitment of lung neutrophils, monocytes, eosinophils and dendritic cells in OVA‐challenged mice. Mechanistically, OAE aspiration enhanced the cleavage of CD100 by MMP14, which manifested as an increase in the soluble CD100 (sCD100) level in BAL fluid and lung homogenates. Knockdown of Mmp14 in macrophages prevented the cleavage of CD100 and reduced Ccl2, Ccl5 and Cxcl2 transcription. These data indicate that PLXNB2‐containing OAEs aggravate airway asthmatic inflammation via cleavage of CD100 by MMP14, suggesting potential therapeutic targets of OAE‐mediated asthma exacerbations.

Background Exosomes have emerged as a vital player in cell-cell communication; however, whether airway epithelial cell (AEC)-generated exosomes participate in asthma development remains unknown. Objective Our aims were to characterize the AEC-secreted exosomes and the potentially functional protein(s) that may contribute to the proinflammatory effects of AEC exosomes in the dendritic cell (DC)-dominant airway allergic models and to confirm their clinical significance in patients with asthma. Methods Mice were treated with exosomes derived from house dust mite (HDM)-stimulated AECs (HDM-AEC-EXOs) or monocyte-derived DCs primed by HDM and/or contactin-1 (CNTN1). The numbers of DCs in the lung were determined by flow cytometry. Proteomic analysis of purified HDM-AEC-EXOs was performed. CNTN1 small interfering RNA was designed to probe its role in airway allergy, and γ-secretase inhibitor was used to determine involvement of the Notch pathway. Results HDM-AEC-EXOs facilitate the recruitment, proliferation, migration, and activation of monocyte-derived DCs in cell culture and in mice. CNTN1 in exosomes is a critical player in asthma pathology. RNA interference–mediated silencing and pharmaceutical inhibitors characterize Notch2 receptor as necessary for relaying the CNTN1 signal to activate TH2 cell/TH17 cell immune response. Studies of patients with asthma also support existence of the CNTN1-Notch2 axis that has been observed in cell and mouse models. Conclusion This study's findings reveal a novel role for CNTN1 in asthma pathogenesis mediated through exosome secretion, indicating a potential strategy for the treatment of allergic airway inflammation.

Asthma is thought to be caused by malfunction of type 2 T helper cell (Th2)–mediated immunity, causing excessive inflammation, mucus overproduction, and apoptosis of airway epithelial cells. Heme oxygenase-1 (HO-1) functions in heme catabolism and is both cytoprotective and anti-inflammatory. We hypothesized that this dual function may be related to asthma's etiology. Using primary airway epithelial cells (pAECs) and an asthma mouse model, we demonstrate that severe lung inflammation is associated with rapid pAEC apoptosis. Surprisingly, NOD-like receptor protein 3 (NLRP3) inhibition, retinoid X receptor (RXR) deficiency, and HO-1 induction were associated with abrogated apoptosis. MCC950, a selective small-molecule inhibitor of canonical and noncanonical NLRP3 activation, reduced RXR expression, leading to decreased pAEC apoptosis that was reversed by the RXR agonist adapalene. Of note, HO-1 induction in a mouse model of ovalbumin-induced eosinophilic asthma suppressed Th2 responses and reduced apoptosis of pulmonary pAECs. In vitro, HO-1 induction desensitized cultured pAECs to ovalbumin-induced apoptosis, confirming the in vivo observations. Critically, the HO-1 products carbon monoxide and bilirubin suppressed the NLRP3–RXR axis in pAECs. Furthermore, HO-1 impaired production of NLRP3–RXR–induced cytokines (interleukin [IL]-25, IL-33, thymic stromal lymphopoietin, and granulocyte-macrophage colony-stimulating factor) in pAECs and lungs. Finally, we demonstrate that HO-1 binds to the NACHT domain of NLRP3 and the RXRα and RXRβ subunits and that this binding is not reversed by Sn-protoporphyrin. Our findings indicate that HO-1 and its products are essential for pAEC survival to maintain airway epithelium homeostasis during NLRP3–RXR–mediated apoptosis and inflammation.

Specific JAK/STAT pathways play a critical role in the functional differentiation of distinct Th subsets. Previously, we showed that HO-1, a stress-inducible protein, inhibits Th17 cell differentiation and alleviates neutrophilic airway inflammation, but the responsible molecular basis remains unclear.We employed Th17-skewing differentiation and NEA mouse models to study the role of HO-1 in regulating IL-6-STAT3-RORγt/SOCS3 signaling pathway to control Th17 cell-mediated neutrophilic airway inflammation. The levels of cytokines and expressions of relative signaling molecules were measured by ELISA, western blot, and qPCR, respectively. Frequency of CD4+ IL-17A+ , CD4+ IL-6R+ , and CD4+ IL-23R+ cells was analyzed by FCM. The interaction between HO-1 and signaling pathway-related proteins was determined by co-immunoprecipitation and western blot.Here, we show that hemin-induced HO-1 overexpression is required to mediate this process. Specifically, HO-1 decreased STAT3 phosphorylation but not IL-6R/IL-23R expression or JAK1/JAK2 activation in CD4+ T cells. The effect was accompanied by co-inhibition of SOCS3, a negative feedback factor of STAT3 activation. HO-1 bound to three domains on STAT3 (DNA-binding, linker, and transactivation domains) to directly regulate STAT3 activation. Conversely, either forced expression of a constitutively active STAT3 mutant or application of small-interfering RNA (siRNA) for HO-1 reversed these effects.Our data suggest that HO-1 exerts its inhibitory effect on Th17 cell differentiation by directly associating and blocking STAT3 phosphorylation. We speculate that hemin may be a potential therapeutic candidate for the treatment of other types of immune and pulmonary inflammatory-related diseases.

SHIP-1 is an inositol phosphatase that hydrolyzes phosphatidylinositol 3-kinase (PI3K) products and negatively regulates protein kinase B (Akt) activity, thereby modulating a variety of cellular processes in mammals. However, the role of SHIP-1 in bacterial-induced sepsis is largely unknown. Here, we show that SHIP-1 regulates inflammatory responses during Gram-negative bacterium Pseudomonas aeruginosa infection. We found that infected-SHIP-1−/− mice exhibited decreased survival rates, increased inflammatory responses, and susceptibility owing to elevated expression of PI3K than wild-type (WT) mice. Inhibiting SHIP-1 via siRNA silencing resulted in lipid raft aggregates, aggravated oxidative damage, and bacterial burden in macrophages after PAO1 infection. Mechanistically, SHIP-1 deficiency augmented phosphorylation of PI3K and nuclear transcription of signal transducer and activator of transcription 5 (STAT5) to induce the expression of Trib1, which is critical for differentiation of M2 but not M1 macrophages. These findings reveal a previously unrecognized role of SHIP-1 in inflammatory responses and macrophage homeostasis during P. aeruginosa infection through a PI3K/Akt–STAT5–Trib1 axis.

The DNA repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which excises 8-oxo-7,8-dihydroguanine (8-oxoG) lesions induced in DNA by reactive oxygen species, has been linked to the pathogenesis of lung diseases associated with bacterial infections. A recently developed small molecule, SU0268, has demonstrated selective inhibition of OGG1 activity; however, its role in attenuating inflammatory responses has not been tested. Here, we report that SU0268 has a favorable effect on bacterial infection both in mouse alveolar macrophages (MH-S cells) and in C57BL/6 wild-type mice by suppressing inflammatory responses, particularly promoting type I interferon responses. SU0268 inhibited pro-inflammatory responses during P. aeruginosa (PA14) infection, which is mediated by the KRAS-ERK1-NF-κB signaling pathway. Furthermore, SU0268 induces the release of type I interferon by the mtDNA-cGAS-STING-IRF3-IFNβ axis, which decreases bacterial loads and halts disease progression. Collectively, our results demonstrate that the small-molecule inhibitor of OGG1 (SU0268) can attenuate excessive inflammation and improve mouse survival rates during PA14 infection. This strong anti-inflammatory feature may render the inhibitor as an alternative treatment for controlling severe inflammatory responses to bacterial infection.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (CRISPR-Cas) systems in prokaryotes function at defending against foreign DNAs, providing adaptive immunity to maintain homeostasis. CRISPR-Cas may also influence immune regulation ability in mammalian cells through alterations of pathogenic extent and nature. Recent research has implied that Type I CRISPR-Cas systems of Pseudomonas aeruginosa strain UCBPP-PA14 impede recognition by Toll-like receptor 4, and decrease pro-inflammatory responses both in vitro and in vivo. However, the molecular mechanism by which CRISPR-Cas systems affect host immunity is largely undemonstrated. Here, we explored whether CRISPR-Cas systems can influence autophagy to alter the activation of inflammasome. Using the wild-type PA14 and total CRISPR-Cas region deletion (∆TCR) mutant strain, we elucidated the role and underlying mechanism of Type I CRISPR-Cas systems in bacterial infection, and showed that CRISPR-Cas systems impacted the release of mitochondrial DNA and induction of autophagy. CRISPR-Cas deficiency led to an increase of mitochondrial DNA release, a decrease in autophagy, an increase of inflammasome activation and, ultimately, an elevation of pro-inflammatory response. Our findings illustrate a new important mechanism by which Type I CRISPR-Cas systems control their virulence potency to evade host defense.

Epithelial cells (ECs)‐derived cytokines are induced by different stimuli through pattern recognition receptors (PRRs) to mount a type‐2‐cell‐mediated immune response; however, the underlying mechanisms are poorly characterized. Here, we demonstrated asthmatic features in both primary bronchial epithelial cells (pBECs) and mouse model using several allergens including ovalbumin (OVA), house dust mite (HDM), or Alternaria alternata. We found that toll‐like receptor 2 (TLR2) was highly induced in ECs but not dendritic cells (DCs) by various allergens, leading to recruitment of circulating basophils into the lung via C‐C chemokine ligand‐2 (CCL2). TLR2 expression increased thymic stromal lymphopoietin (TSLP) production through the NF‐κB and JNK signaling pathways to extend the survival of recruited basophils and resident DCs in the lung, predisposing a type‐2‐cell‐mediated airway inflammation. Conversely, TLR2 deficiency impaired http://secretion of TSLP and CCL2, decreased infiltration of lung basophils, and increased resistance to Th2 response. Blocking TSLP also phenocopied these phenomena. Our findings reveal a pro‐inflammatory role of airway ECs through a TLR2‐dependent TSLP production, which may have implication for treating allergic asthma.

The anti‐inflammatory role of heme oxygenase‐1 (HO‐1) has been studied extensively in many disease models including asthma. Many cell types are anti‐inflammatory targets of HO‐1, such as dendritic cells and regulatory T cells. In contrast to previous reports that HO‐1 had limited effects on basophils, which participate in T helper type 2 immune responses and antigen‐induced allergic airway inflammation, we demonstrated in this study, for the first time, that the up‐regulation of HO‐1 significantly suppressed the maturation of mouse basophils, decreased the expression of CD40, CD80, MHC‐II and activation marker CD200R on basophils, blocked DQ‐ovalbumin uptake and promoted basophil apoptosis both in vitro and in vivo, leading to the inhibition of T helper type 2 polarization. These effects of HO‐1 were mimicked by exogenous carbon monoxide, which is one of the catalytic products of HO‐1. Furthermore, adoptive transfer of HO‐1‐modified basophils reduced ovalbumin‐induced allergic airway inflammation. The above effects of HO‐1 can be reversed by the HO‐1 inhibitor Sn‐protoporphyrin IX. Moreover, conditional depletion of basophils accompanying hemin treatment further attenuated airway inflammation compared with the hemin group, indicating that the protective role of HO‐1 may involve multiple immune cells. Collectively, our findings demonstrated that HO‐1 exerted its anti‐inflammatory function through suppression of basophil maturation and activation, but promotion of basophil apoptosis, providing a possible novel therapeutic target in allergic asthma.

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is a group of autoimmune diseases characterized by nonspecific inflammation in the gastrointestinal tract. Recent investigations suggest that activation of Th17 cells and/or deficiency of regulatory T cells (Treg) is involved in the pathogenesis of IBD. Heme oxygenase (HO)-1 is a protein with a wide range of anti-inflammatory and immune regulatory function, which exerts significantly protective roles in various T cell-mediated diseases. In this study, we aim to explore the immunological regulation of HO-1 in the dextran sulfate sodium-induced model of experimental murine colitis. BALB/c mice were administered 4% dextran sulfate sodium orally; some mice were intraperitoneally pretreated with HO-1 inducer hemin or HO-1 inhibitor stannum protoporphyrin IX. The results show that hemin enhances the colonic expression of HO-1 and significantly ameliorates the symptoms of colitis with improved histological changes, accompanied by a decreased proportion of Th17 cells and increased number of Tregs in mesenteric lymph node and spleen. Moreover, induction of HO-1 down-regulates retinoic acid-related orphan receptor γt expression and IL-17A levels, while promoting Treg-related forkhead box p3 (Foxp3) expression and IL-10 levels in colon. Further study in vitro revealed that up-regulated HO-1 switched the naive T cells to Tregs when cultured under a Th17-inducing environment, which involved in IL-6R blockade. Therefore, HO-1 may exhibit anti-inflammatory activity in the murine model of acute experimental colitis via regulating the balance between Th17 and Treg cells, thus providing a possible novel therapeutic target in IBD.

Antigen-induced allergic airway inflammation is mediated by T helper type 2 (Th2) cells and their cytokines, but the mechanism that initiates the Th2 immunity is not fully understood. Recent studies show that basophils play important roles in initiating Th2 immunity in some inflammatory models. Here we explored the role of basophils in ovalbumin (OVA) -induced airway allergic inflammation in BALB/c mice. We found that OVA sensitization and challenge resulted in a significant increase in the amount of basophils in blood and lung, along with the up-regulation of activation marker of CD200R. However, depletion of basophils with MAR-1 or Ba103 antibody attenuated airway inflammation, represented by the significantly decreased amount of the Th2 subset in spleen and draining lymph nodes, interlukin-4 level in lung and OVA-special immunoglobulin E (sIgE) levels in serum. On the other hand, adoptive transfer of basophils from OVA-challenged lung tissue to naive BALB/c mice provoked the Th2 immune response. In addition, pulmonary basophils from OVA-challenged mice were able to uptake DQ-OVA and express MHC class II molecules and CD40 in vivo, as well as to release interleukin-4 following stimulation by IgE-antigen complexes and promote Th2 polarization in vitro. These findings demonstrate that basophils may participate in Th2 immune responses in antigen-induced allergic airway inflammation and that they do so through facilitating antigen presentation and providing interleukin-4.

Terminal maturation of invariant NKT (iNKT) cells from stage 2 (CD44+NK1.1–) to stage 3 (CD44+NK1.1+) is accompanied by a functional acquisition of a predominant IFN-γ–producing (iNKT-1) phenotype; however, some cells develop into IL-17–producing iNKT (iNKT-17) cells. iNKT-17 cells are rare and restricted to a CD44+NK1.1– lineage. It is unclear how iNKT terminal maturation is regulated and what factors mediate the predominance of iNKT-1 compared with iNKT-17. The tumor suppressor tuberous sclerosis 1 (TSC1) is an important negative regulator of mTOR signaling, which regulates T cell differentiation, function, and trafficking. Here, we determined that mice lacking TSC1 exhibit a developmental block of iNKT differentiation at stage 2 and skew from a predominantly iNKT-1 population toward a predominantly iNKT-17 population, leading to enhanced airway hypersensitivity. Evaluation of purified iNKT cells revealed that TSC1 promotes T-bet, which regulates iNKT maturation, but downregulates ICOS expression in iNKT cells by inhibiting mTOR complex 1 (mTORC1). Furthermore, mice lacking T-bet exhibited both a terminal maturation defect of iNKT cells and a predominance of iNKT-17 cells, and increased ICOS expression was required for the predominance of iNKT-17 cells in the population of TSC1-deficient iNKT cells. Our data indicate that TSC1-dependent control of mTORC1 is crucial for terminal iNKT maturation and effector lineage decisions, resulting in the predominance of iNKT-1 cells.

Objective: Inflammatory bowel disease (IBD) is characterized by recurrent and severe gastrointestinal inflammation. Activation of inflammatory cells, such as T H 17 lymphocytes, and/or deficiency of regulatory T cells (T reg ) are responsible for the pathogenesis of IBD. As an acute phase reactant, heme oxygenase-1 (HO-1) has been shown to play an anti-inflammatory and immunomodulatory role in many disease processes. In this study, we used a dextran sulfate sodium (DSS)-induced murine colitis model to investigate the effect of upregulating HO-1 by hemin on the development of colonic inflammation. Materials and Methods: The mice were enterically challenged with 4% DSS. In addition, some mice were intraperitoneally administered with hemin or Sn-protoporphyrin (SnPP) on days 0, 1, and 6 after DSS treatment. The severity of colitis was evaluated by daily monitoring of weight change and diarrhea. At the end of the experiment, the colon, spleen, and mesenteric lymph nodes were harvested for histology and various immunological assays. Results: Compared to control groups, DSS challenge markedly induced HO-1 expression in the colon epithelium. Upregulation of HO-1 by hemin was further correlated with attenuation of DSS-induced colitis. In contrast, inhibition of endogenous HO-1 by SnPP aggravated the colitis. To further assess the anti-inflammatory mechanisms, we examined whether hemin enhanced the proliferation of T reg cells and suppressed the production of interleukin (IL)-17. Flow cytometry analysis revealed that hemin markedly expanded the CD4+CD25+Foxp3+ T reg population. Moreover, hemin attenuated IL-17 and T H 17-related cytokines. This inhibition coincided with the attenuation of DSS-induced colitis. Finally, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labeling assay showed that hemin treatment markedly reduced programmed cell death of colonic epithelium, indicating that hemin exerts a modulatory effect on the induction of T reg , IL-17, and apoptosis. Conclusions: These results demonstrate that upregulation of HO-1 by hemin ameliorated experimental colitis. Moreover, our study suggests a broader protective mechanism of hemin.

Cumulative evidence suggests the up-regulation of interleukin (IL)-10 and T-regulatory (Treg) cells is implicated in anti-inflammatory effect of heme oxygenase-1 (HO-1). Thus, we postulated that induction of HO-1 could augment IL-10 and transforming growth factor (TGF)-beta production and foxp3+CD4+CD25+ Treg cell function, thereby leading to attenuation of airway inflammation. In this study, CD4+CD25+ Treg cells isolated from mouse spleen were either transfected with a HO-1 expression vector (pcDNA3HO-1) or treated with a HO-1 inducer (hemin). Up-regulation of HO-1 enhanced foxp3 expression and IL-10 secretion in the Treg cells in vitro. Next, BALB/c, C57/B6.129, and IL-10-deficient B6.129P2-Il10tm1Cgn/J mice were challenged by ovalbumin to induce airway inflammation. Consistent with in vitro findings, hemin treatment resulted in induction of HO-1 and foxp3 and production of IL-10 and membrane-bound TGF-beta1 in vivo. This was further correlated with decrease of ovalbumin-specific immunoglobulin E level and eosinophil infiltration in bronchial alveolar lavage fluid from the asthmatic mice. Furthermore, hemin significantly enhanced the biological activity of CD4+CD25+ Treg cells. This protective effect was specifically blocked by Sn-protoporphyrin, a HO-1 enzymatic inhibitor. Finally, hemin failed to up-regulate the function of CD4+CD25+ Treg cells from IL-10-deficient mice. Our study indicates that HO-1 exerts its protective effect on asthma through a mechanism mediated by foxp3+CD4+CD25+ Treg cells, IL-10, and membrane-bound TGF-beta1.

Asthma is characterized by increased airway submucosal infiltration of T helper (Th) cells and myeloid cells that co-conspire to sustain a chronic inflammation. While recent studies have demonstrated that the myeloid basophils promote Th2 cells in response to various types of allergens, the underlying mechanisms are poorly understood. Here, we found for the first time that in a mouse model of allergic asthma basophils highly expressed OX40 ligand (OX40L) after activation. Interestingly, blockade of OX40-OX40L interaction suppressed basophils-primed Th2 cell differentiation in vitro and ameliorated ovalbumin (OVA)-induced allergic eosinophilic inflammation mediated by Th2 activation. In accordance, the adoptive transfer of basophils derived from mediastinal lymph nodes (MLN) of OVA-immunized mice triggered a robust Th2 response and eosinophilic inflammation in wild-type mice but largely muted in OX40(-/-) mice and mice receiving OX40L-blocked basophils. Taken together, our results reveal a critical role of OX40L presented by the activated basophils to initiate Th2 responses in an allergic asthma model, implicating OX40-OX40L signaling as a potential therapeutic target in the treatment of allergic airway inflammation.

Allergic asthma is conventionally considered as a Th2 immune response characterized by eosinophilic inflammation. Recent investigations revealed that Th17 cells play an important role in the pathogenesis of non-eosinophilic asthma (NEA), resulting in steroid-resistant neutrophilic airway inflammation. Heme oxygenase-1 (HO-1) has anti-inflammation, anti-oxidation, and anti-apoptosis functions. However, its role in NEA is still unclear. Here, we explore the role of HO-1 in a mouse model of NEA. HO-1 inducer hemin or HO-1 inhibitor tin protoporphyrin IX was injected intraperitoneally into ovalbumin-challenged DO11.10 mice. Small interfering RNA (siRNA) was delivered into mice to knock down HO-1 expression. The results show that induction of HO-1 by hemin attenuated airway inflammation and decreased neutrophil infiltration in bronchial alveolar lavage fluid and was accompanied by a lower proportion of Th17 cells in mediastinal lymph nodes and spleen. More importantly, induction of HO-1 down-regulated Th17-related transcription factor retinoic acid-related orphan receptor γt (RORγt) expression and decreased IL-17A levels, all of which correlated with a decrease in phosphorylated STAT3 (p-STAT3) level and inhibition of Th17 cell differentiation. Consistently, the above events could be reversed by tin protoporphyrin IX. Also, HO-1 siRNA transfection abolished the effect of hemin induced HO-1 in vivo. Meanwhile, the hemin treatment promoted the level of Foxp3 expression and enhanced the proportion of regulatory T cells (Tregs). Collectively, our findings indicate that HO-1 exhibits anti-inflammatory activity in the mouse model of NEA via inhibition of the p-STAT3-RORγt pathway, regulating kinetics of RORγt and Foxp3 expression, thus providing a possible novel therapeutic target in asthmatic patients.

Background Airway inflammation is mainly mediated by T helper 2 cells (Th2) that characteristically produce interleukin (IL)-4, IL-5, and IL-13. Epidemiological studies have revealed an inverse association between the dietary intake of vitamin A and the occurrence of asthma. Serum vitamin A concentrations are significantly lower in asthmatic subjects than in healthy control subjects. It has been reported that all-trans retinoic acid (ATRA), a potent derivative of vitamin A, regulates immune responses. However, its role in Th2-mediated airway inflammation remains unclear. We investigated the effects of ATRA in a mouse model of allergic airway inflammation. Results We found that ATRA treatment attenuated airway inflammation and decreased mRNA levels of Th2- and Th17-related transcription factors. The data showed that airway inflammation coincided with levels of Th2- and Th17-related cytokines. We also showed that ATRA inhibited Th17 and promoted inducible regulatory T-cell differentiation, whereas it did not induce an obvious effect on Th2 differentiation in vitro. Our data suggest that ATRA may interfere with the in vivo Th2 responses via T-cell extrinsic mechanisms. Conclusions Administration of ATRA dramatically attenuated airway inflammation by inhibiting Th2 and Th17 differentiation and/or functions. ATRA may have potential therapeutic effects for airway inflammation in asthmatic patients.

Invariant natural killer T (iNKT) cells play important roles in bridging innate and adaptive immunity via rapidly producing a variety of cytokines. A small subset of iNKT cells produces IL-17 and is generated in the thymus during iNKT-cell ontogeny. The mechanisms that control the development of these IL-17-producing iNKT-17 cells (iNKT-17) are still not well defined. Diacylglycerol kinase ζ (DGKζ) belongs to a family of enzymes that catalyze the phosphorylation and conversion of diacylglycerol to phosphatidic acid, two important second messengers involved in signaling from numerous receptors. We report here that DGKζ plays an important role in iNKT-17 development. A deficiency of DGKζ in mice causes a significant reduction of iNKT-17 cells, which is correlated with decreased RORγt and IL-23 receptor expression. Interestingly, iNKT-17 defects caused by DGKζ deficiency can be corrected in chimeric mice reconstituted with mixed wild-type and DGKζ-deficient bone marrow cells. Taken together, our data identify DGKζ as an important regulator of iNKT-17 development through iNKT-cell extrinsic mechanisms.

Background Antigen-specific immunotherapy (SIT) has been widely practiced in treating allergic diseases such as asthma. However, this therapy may induce a series of allergic adverse events during treatment. Peptide immunotherapy (PIT) was explored to overcome these disadvantages. We confirmed that multiple antigen peptides (MAPs) do not cause autoimmune responses, which led to the presumption that MAPs intervention could alleviate allergic airway inflammation without inducing adverse effects. Results In this study, synthesized OVA323-339MAP octamers were subcutaneously injected into ovalbumin (OVA)-sensitized and -challenged Balb/c mice to observe its effect on allergic airway inflammation, Th2 immune response, and immune regulating function. It was confirmed that OVA sensitization and challenge led to significant peritracheal inflammatory, cell infiltration, and intensive Th2 response. Treatment of OVA323-339MAP octomers in the airway inflammation mice model increased CD4+CD25+Foxp3+ T regulatory (Treg) cells and their regulatory function in peripheral blood, mediastinal draining lymph nodes, and the spleen. Furthermore, OVA323-339MAP increased IL-10 levels in bronchial alveolar lavage fluid (BALF); up-regulated the expression of IL-10, membrane-bound TGF-β1, as well as Foxp3 in lung tissues; and up-regulated programmed death-1 (PD-1) and cytotoxic T lymphocyte associated antigen 4 (CTLA-4) on the surface of Treg cells. These results were further correlated with the decreased OVA specific immunoglobulin E (sIgE) level and the infiltration of inflammatory cells such as eosinophils and lymphocytes in BALF. However, OVA323-339 peptide monomers did not show any of the mentioned effects in the same animal model. Conclusions Our study indicates that OVA323-339MAP had significant therapeutic effects on mice allergic airway inflammation by regulating the balance of Th1/Th2 response through Treg cells in vivo.

BackgroundExcessive accumulation of bilirubin contributes to neonatal hyperbilirubinemia in rats. Heme oxygenase (HO) is one of the rate-limiting enzymes in catabolizing heme to bilirubin. In the present study, we investigated whether suppression of rat HO-1 (rHO-1) expression by small interference RNAs (siRNAs) reduces bilirubin levels in hyperbilirubinemic rats.ResultsFour pairs of siRNA targeting rHO-1 mRNA were introduced into BRL cells and compared for their inhibitory effect on the expression ofrHO-1gene and production of rHO-1 protein. The siRNA exhibiting the most potent effect on HO-1 expression and activity was then administered intraperitoneally to 7 to 9-day-old rats with hyperbilirubinemia. The siRNA distributed mostly in the liver and spleen of neonatal rat. Serum bilirubin levels and hepatic HO-1 expression were further evaluated. Systemic treatment of siRNA targeting rHO-1 reduced hepatic HO-1 expression and decreased the serum bilirubin levels in a time- and dose-dependent manner, and siRNA decreased the indirect bilirubin levels more effectively than Sn-protoporphyrin (SnPP), an HO-1 inhibitor.ConclusionsiRNA targeting rHO-l attenuates hepatic HO-1 expression and serum bilirubin levels. Thus this study provides a novel therapeutic rationale for the prevention and treatment of neonatal hyperbilirubinemia.