Short-chain fatty acids reverses gut microbiota dysbiosis-promoted progression of glioblastoma by up-regulating M1 polarization in the tumor microenvironment.

Glioblastoma (GBM), known as the most malignant and common primary brain tumor of the central nervous system, has finite therapeutic options and a poor prognosis. Studies have shown that host intestinal microorganisms play a role in the immune regulation of parenteral tumors in a number of different ways, either directly or indirectly. However, the potential impact of gut microbiota on tumor microenvironment, particularly glioma immunological milieu, has not been clarified exactly. In this study, by using an orthotopic GBM model, we found gut microbiota dysbiosis caused by antibiotic cocktail treatment boosted the tumor process in vivo. An obvious change that followed gut microbiota dysbiosis was the enhanced percentage of M2-like macrophages in the TME, in parallel with a decrease in the levels of gut microbial metabolite, short-chain fatty acids (SCFAs) in the blood and tumor tissues. Oral supplementation with SCFAs can increase the proportion of M1-like macrophages in the TME, which improves the outcomes of glioma. In terms of mechanism, SCFAs-activated glycolysis in the tumor-associated macrophages may be responsible for the elevated M1 polarization in the TME. This study will enable us to better comprehend the "gut-brain" axis and be meaningful for the development of TAM-targeting immunotherapeutic strategies for GBM patients.

Traditional Chinese medicine Zhusha Anshen Wan: protective effects on liver, kidney, and intestine of the individual drugs using 1H NMR metabolomics.

Introduction: Zhusha Anshen Wan (ZSASW) is a traditional Chinese medicine compound mainly composed of mineral drugs. In clinical practice, ZSASW did not show the toxicity of administering equal doses of cinnabar alone, suggesting that the four combination herbs in ZSASW can alleviate the damage of cinnabar. The effect of each herb on reducing the toxicity of cinnabar has not been fully explained. Methods: In our study, we utilized a metabonomics approach based on high-resolution 1H nuclear magnetic resonance spectroscopy to investigate the reduction of toxicity by each herb in ZSASW. Liver, kidney and intestinal histopathology examinations and biochemical analysis of the serum were also performed. Results: Partial least squares-discriminant analysis (PLS-DA) was conducted to distinct different metabolic profiles in the urine and serum from the rats. Liver and kidney histopathology examinations, as well as analysis of serum clinical chemistry analysis, were also carried out. The metabolic profiles of the urine and serum of the rats in the CGU (treated with cinnabar and Glycyrrhiza uralensis Fisch) and CCC (treated with cinnabar and Coptis chinensis French) groups were remarkably similar to those of the control group, while those of the CRG (treated with cinnabar and Rehmannia glutinosa Libosch) and CAS (treated with cinnabar and Angelica sinensis) groups were close to those of the cinnabar group. The metabolic profiles of the urine and serum of the rats in the CGU and CCC groups were remarkably similar to those of the control group, while those of the CRG and CAS groups were close to those of the cinnabar group. Changes in endogenous metabolites associated with toxicity were identified. Rehmannia glutinosa, Rhizoma Coptidis and Glycyrrhiza uralensis Fisch could maintain the dynamic balance of the intestinal flora. These results were also verified by liver, kidney and intestinal histopathology examinations and biochemical analysis of the serum. The results suggested that Discussion: The metabolic mechanism of single drug detoxification in compound prescriptions has been elucidated. Coptis chinensis and Glycyrrhiza uralensis serve as the primary detoxification agents within ZSASW for mitigating liver, kidney, and intestinal damage caused by cinnabar. Detoxification can be observed through changes in the levels of various endogenous metabolites and related metabolic pathways.

Neuromedin U regulates the anti-tumor activity of CD8+ T cells and glycolysis of tumor cells in the tumor microenvironment of pancreatic ductal adenocarcinoma in an NMUR1-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a poor prognosis, which is lethal in approximately 90% of cases despite advanced standard therapies. A typical feature of PDAC is the immunosuppressive tumor microenvironment with multiple immunosuppressive factors including neurotransmitters. Recently, neuromedin U (NMU), a highly conserved neuropeptide with many physiological functions, has attracted attention for its roles in tumorigenesis and metastasis in several types of cancers. However, whether NMU affects PDAC progression remains unclear. In this study, using an orthotopic mouse model of PDAC in combination with bioinformatics analysis, we found that NMU was upregulated in tumor tissues from the patients with PDAC and positively correlated with a poor prognosis of the disease. Interestingly, knockout of the Nmu gene in mice enhanced the anti-tumor functions of tumor-infiltrating CD8+ T cells in an NMU receptor 1-dependent manner. Additionally, NMU promoted the glycolytic metabolism of mouse PDAC tumors. The activities of pyruvate kinase (PK) and lactate dehydrogenase (LDH), pivotal enzymes involved in the regulation of lactate production, were markedly reduced in tumor tissues from NMU-knockout mice. In vitro the presence of LDHA inhibitor can reduce the production of lactic acid stimulated by NMU, which can increase the anti-tumor activity of CD8+ T cells. Moreover, treatment of the pancreatic cancer cells with a phosphoinositide 3-kinase (PI3K) inhibitor diminished NMU-induced lactate production and the activities of PK and LDH, suggesting that NMU might regulate glycolysis via the PI3K/AKT pathway.

Gut Lactobacillus contribute to the progression of breast cancer by affecting the anti-tumor activities of immune cells in the TME of tumor-bearing mice.

Studies have proven that gut microbiota dysbiosis may influence the carcinogenesis and outcomes of multiple cancers. However, it is still unclear whether gut microbiota dysbiosis affect the progression of breast cancer, especially triple-negative breast cancer. In the present study, by using gut microbiota dysbiosis murine model established by treatment of mice with streptomycin, we found Lactobacillus and the metabolite-lactic acid are the pivotal factors for 4T1 tumor progression. In fact, streptomycin-treated mice exhibited slower tumor growth, in parallel with less abundance of Lactobacillus in the gut. Supplementation with Lactobacillus resulted in a rapid tumor growth, following a decrease in the expression of mRNAs for anti-tumor-related factors but an increase in the M2 polarization. The elevated percentages of IFN-γ-producing CD4+T cells and CD8+T cells in the tumor microenvironment of streptomycin-treated tumor-bearing mice may be vanished by supplementation of Lactobacillus. It seems likely that lactobacillus-mediated pro-tumor effect is related to the production of lactic acid. A decrease in the levels of lactic acid in the cecal feces and tumor tissues were observed in streptomycin-treated tumor bearing mice. However, supplementation of Lactobacillus can restore streptomycin-reduced concentration of lactic acid in the tumor tissues, suggesting that gut Lactobacillus are the source of lactic acid. Bioinformatics analysis result suggests high concentration of lactic acid in tumor sites may be related to the diminished anti-tumor immunity in the TME. This study reveals a correlation between gut Lactobacillus and tumor progression in a murine 4T1 tumor model, providing experimental evidence for clinical treatment of breast cancer.

Neuromedin U Induces Pulmonary ILC2 Activation via the NMUR1 Pathway during Acute Respiratory Syncytial Virus Infection.

Activated group 2 innate lymphoid cells (ILC2s) play a crucial role in respiratory syncytial virus (RSV)-induced airway inflammation and allergy-like symptoms because of their ability to secrete large quantities of type 2 cytokines. Cytokines such as IL-33, IL-25, and thymic stromal lymphopoietin are activators of ILC2s. Besides, a regulatory effect of neurotransmitters on ILC2 activation has been reported recently. However, whether and how RSV infection induces neurotransmitter production in the lungs and regulates pulmonary ILC2 activation remains unclear. In this study, using a murine model established by intranasal infection with RSV, we found that acute RSV infection induced the production of a neurotransmitter, neuromedin U (NMU), in the lungs of RSV-infected mice and upregulated the expression of NMUR1 (neuromedin U receptor 1) on ILC2s. Moreover, in vivo administration of NMU exacerbated RSV-induced airway inflammation by promoting the proliferation and activation of pulmonary ILC2s via the NMUR1 pathway, which involved PI3K, mitogen-activated protein kinase kinase, and NFAT signaling proteins. Furthermore, pulmonary neurons responded to the stimulation of RSV infection and secreted NMU in a Toll-like receptor 4- and Toll-like receptor 7-dependent manner. Collectively, our data suggest that NMU is a powerful neuropeptide to activate ILC2s, highlighting the critical regulatory effects of neurotransmitters on antiviral, inflammatory, and tissue homeostasis at the mucosal barrier during a viral respiratory infection.

Group 2 innate lymphoid cells mediate the activation of CD4+ T cells and aggravate Th1/Th2 imbalance via MHC II molecules during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection induces the activation of CD4+ T cells. However, the underlying mechanism of CD4+T-cell activation induced by RSV infection is not fully understood. In the present study, we found that depletion of CD4+ T cells can obviously reduce airway inflammation caused by RSV infection. Meanwhile, adoptive transfer of group 2 innate lymphocytes (ILC2s) significantly enhanced the number of CD4+ T cells and promoted their differentiation to Th2 in lung. In fact, RSV infection increased the expression of major histocompatibility complex-II (MHC II) molecules on the surface of pulmonary ILC2s. In vitro coculture experiments showed that ILC2s may act as promoters to promote the expansion and differentiation of RSV-infected CD4+ T cells. However, blocking the interaction between CD4+ T cells and ILC2s with anti-MHC-II mAbs significantly reduced CD4+T-cell expansion. These results suggest that pulmonary ILC2s may function as antigen-presenting cells to induce the activation of CD4+ T cells through the MHC II pathway during RSV infection.

Oral administration of Clostridium butyricum rescues streptomycin-exacerbated respiratory syncytial virus-induced lung inflammation in mice.

Changes in the intestinal microbiota indirectly impact the health of mucosa distal to the intestine, particularly the respiratory tract. However, the effects of intestinal microbiota dysbiosis on the regulation of respiratory syncytial virus (RSV) infection are not clear. In this study, we examined the effects of altering the intestinal microbiota on the pulmonary immune response against RSV infection. BALB/c mice were treated with streptomycin before infection with RSV to study the altered immune response. The ingestion of streptomycin led to a marked alteration in the intestinal microbiota with a reduced abundance of Lactobacillus and Clostridium genera, followed by greatly aggravated pulmonary inflammation in response to RSV infection. This aggravated inflammation was associated with a dysregulated immune response against RSV infection, characterized by the increased expression of IFN-γ and IL-17 and increased pulmonary M1-like macrophage polarization, and decreased expression of IL-5. Supplementation of Clostridium butyricum (CB) prevented aggravated inflammation and the dysregulated immune response characterized by greater M2 polarization of pulmonary macrophages and decreased release of IFN-γ and IL-17 as well as increased IL-5 levels. Furthermore, in vitro and in vivo experiments identified that butyrate, the main metabolite produced by CB, promoted M2 polarization of macrophages in RSV-infected mice exposed to streptomycin. Together, these results demonstrate the mechanism by which intestinal microbiota modulate the pulmonary immune response to RSV infection.

Resveratrol attenuates TNBC lung metastasis by down-regulating PD-1 expression on pulmonary T cells and converting macrophages to M1 phenotype in a murine tumor model.

Triple-negative breast cancer (TNBC) is an invasive breast cancer with the characteristics of easy to develop distant metastasis. Immune escape is one of the main reasons for TNBC growth and metastasis. Enhancement of T cell-mediated anti-tumor activity may benefit to inhibit tumor metastasis and improve the efficacy of cancer therapy. As a natural bioactive substance, resveratrol shows potential capability to prevent or suppress the development of a variety of cancers through direct or indirect effects, including immunoregulatory effect. However, whether resveratrol might affect lung metastasis of TNBC, and whether the effect of resveratrol might be associated with resveratrol-regulated immune responses in tumor microenvironment is still unknown. In this study, by using an experimental metastatic mouse 4 T1 tumor model, we identified that resveratrol may suppress TNBC lung metastasis by elevating local anti-tumor immunity. Indeed, an increase in the cytotoxic activity of CD8+T cells as well as the levels of type 1 cytokine IFN-γ and IL-2 in the lungs of resveratrol-treated tumor bearing mice were observed. The enhanced CD8+T cell activity and Th1 immune responses by resveratrol administration might be related to the down-regulated PD-1 expression on pulmonary CD8+T cells and CD4+T cells. Resveratrol may also convert macrophages to M1 phenotype in the lungs of tumor bearing mice. However, it seems likely resveratrol has no effect on pulmonary myeloid-derived suppressor cell activation. Our results provide an evidence that resveratrol might be a promising candidate agent for adjuvant therapy in the process of TNBC metastasis.

Group 2 innate lymphoid cells promote TNBC lung metastasis via the IL-13-MDSC axis in a murine tumor model.

Group 2 innate lymphoid cells (ILC2s) are reportedly associated with the progression of many tumors. However, the role of ILC2s in triple-negative breast cancer (TNBC) lung metastasis remains unclear. In this study, we found that ILC2s may be a key element in the process of TNBC lung metastasis since the adoptive transfer of pulmonary ILC2s increased the numbers of metastatic lung nodules and reduced the survival of tumor-bearing mice. ILC2-promoted 4 T1 lung metastasis appears to be related to ILC2-derived IL-13. An expansion of IL-13-producing ILC2s and an elevated expression of IL-13 mRNA in pulmonary ILC2s were determined in tumor-bearing mice, in parallel with an increase in the levels of local IL-13 by ILC2 transfer. The neutralization of IL-13 reduced the increased pulmonary metastatic nodules and improved the decreased survival rate caused by ILC2-adoptive transfer. Interestingly, adoptive transfer of ILC2s elevated IL-13Ra1 expression in myeloid-derived suppressor cells (MDSCs). Treatment of ILC2-transferred tumor-bearing mice with anti-IL-13 antibodies significantly diminished the number of pulmonary MDSCs and inhibited MDSC activation. Moreover, when pulmonary MDSCs were cocultured with ILC2s in the presence of an anti-IL-13 mAb, the number and activation of MDSCs were reduced. Depletion of MDSCs may promote the proliferation of CD4+ T cells and CD8+ T cells, but reduce the expansion of regulatory T cells (Tregs) in the lungs of ILC2-transferred tumor-bearing mice. Our results suggest that pulmonary ILC2s may promote TNBC lung metastasis via the ILC2-derived IL-13-activated MDSC pathway.

Role of gut microbiome in the outcome of cancer immunotherapy.

Nearly 3 × 1013 types of bacteria colonize the human intestine. These colonized bacteria help in maintaining intestinal homeostasis by establishing a complex relationship with the intestinal epithelium and lymphoid tissue. Alteration in the composition of the intestinal microbiota is associated with susceptibility to various pathological conditions, such as autoimmune disorders, diabetes, inflammation and cancer. Of late, several researchers have focused on examining the effects of gut microbiota on the outcome of various cancer treatment protocols. Side effects and complications of traditional chemotherapy and allogeneic hematopoietic cell transplantation are associated with intestinal dysbiosis. Gut microbiota affects the efficacy of immune checkpoint inhibitor-based immunotherapy. The gut is inhabited by diverse resident bacteria, of which, few enhance, while others inhibit the host response to immunotherapy. This review focuses on the correlation between intestinal microbiota and the outcome of tumor immunotherapy. Additionally, the molecular mechanisms underlying the effects of gut microbiota on the efficacy of cancer immunotherapy have been reviewed. Further studies are needed for the identification of distinct gut microbiota and their efficacy in tumor immunotherapy as certain types of intestinal bacteria could function as novel adjuvant drugs to enhance the effectiveness of antitumor therapy in humans.

Resveratrol-primed exosomes strongly promote the recovery of motor function in SCI rats by activating autophagy and inhibiting apoptosis via the PI3K signaling pathway.

Spinal cord injury (SCI) is a traumatic condition of the central nervous system (CNS) that can cause paralysis of the limbs. The molecular mechanisms of neural repair following SCI remain unclear and no effective treatment for SCI currently exists, since drugs have difficulty crossing the blood-brain barrier (BBB). The present study aimed to investigate whether exosomes could be used as specific carriers of resveratrol for induction of neuronal autophagy both in vitro and in vivo for the treatment of SCI. The results indicate that exosomes are able to enhance the solubility of resveratrol and enhance penetration of the drug through the BBB, thereby increasing its concentration in the CNS. Exosomes derived from resveratrol-treated primary microglia (Exo + Res) assisted the rehabilitation of paralyzed limbs in rats. Restoration of neural function following SCI was mediated through increased induction of autophagy and inhibition of apoptosis of neurons both in vitro and in vivo via activation of the PI3K signaling pathway. The mechanism of action of Exo + Res may be associated with the PI3K inhibitor 3-methyladenine (3-MA) in primary spinal neurons. The results suggest that Exo + Res are highly effective at crossing the BBB with good stability, suggesting they have potential for enhancing targeted drug delivery and the recovery of neuronal function in SCI therapy, likely associated with the induction of autophagy and inhibition of apoptosis via the PI3K signaling pathway.

Essential role of CD4+ T cells for the activation of group 2 innate lymphoid cells during respiratory syncytial virus infection in mice.

Aim: To investigate whether and how CD4+ T cells contribute to ILC2 activation during respiratory syncytial virus (RSV) infection. Methods: The methods of flow cytometry, quantitative PCR and ELISA were used in the present study. Results: Depletion of CD4+ T cells diminished the numbers of lung ILC2s as well as their ability to produce type 2 cytokines. CD4+ T cell-mediated ILC2 activation is related to IL-2. The main cellular source of IL-2 was CD4+ T cells. Depletion of CD4+ T cells decreased IL-2 levels in the lungs of RSV-infected mice. IL-2 can directly stimulate ILC2 proliferation and promote ILC2s to produce cytokines. Treatment of mice with neutralizing anti-IL-2 monoclonal antibodies diminished ILC2 activation. Conclusion: These results suggest that CD4+ T cells contribute to RSV-induced ILC2 activation partly via producing IL-2.

Critical role of OX40/OX40L in ILC2-mediated activation of CD4+T cells during respiratory syncytial virus infection in mice.

CD4+T cells are crucial cellular source of type 2 cytokines and responsible for RSV-induced asthma-like symptoms and asthma exacerbations. However, the mechanism for regulating the activation of CD4+T cells during RSV infection is not clear completely. We show in this study that infection with RSV may induce an expansion and activation of CD4+T cells in the lungs of BALB/c mice. RSV-induced CD4+T cell expansion and activation seems to depend upon the pulmonary group 2 innate lymphoid cells (ILC2s), since adoptive transfer of lung ILC2s can enhance not only the numbers of CD4+T cells but also the cytokine production by CD4+T cells. Interestingly, blockade of the contact between ILC2s and CD4+T cells, may significantly diminish the CD4+T cell expansion and cytokine production, suggesting that membrane molecules may be involved in ILC2-regulated CD4+T cell activation. In fact, infection with RSV resulted in an increase in the numbers of OX40+CD4+T cells as well as OX40L+ILC2s in the lungs of mice. Moreover, the mRNA expressions of OX40 and OX40L as well as the levels of OX40 and OX40L proteins in the lung CD4+T cells and ILC2s were elevated respectively. When co-culture of CD4+T cells with ILC2s in the presence of anti-OX40L antibody, the cytokine productions by CD4+T cells were reduced markedly, suggesting that lung ILC2s may regulate RSV-induced CD4+T cell expansion and activation perhaps via OX40/OX40L interaction.

Respiratory syncytial virus prevents the subsequent development of ovalbumin-induced allergic responses by inhibiting ILC2 via the IL-33/ST2 pathway.

AIM: How respiratory syncytial virus (RSV) influences the development of ovalbumin (OVA)-induced asthma remains elusive. As potent T helper (Th)2 cytokine producers, group 2 innate lymphoid cells (ILC2s) are known to serve important functions in the pathogenesis of allergic inflammation. However, how RSV infection affects innate immunity, especially with regard to the function of ILC2s in OVA-induced allergic airway inflammation, is largely unknown. MATERIALS & METHODS: RSV was used to infect adult BALB/c mice intranasally prior to sensitization and subsequent challenge with OVA. ILC2 frequencies and Th2 cytokine production by ILC2s were assessed by flow cytometry. Cytokine levels were detected both by real-time PCR and ELISA. RESULTS: Previous infection with RSV attenuated airway inflammation and decreased Th2 cytokine production in mice sensitized and challenged with OVA. Furthermore, previous infection with RSV inhibited the influx of ILC2s into the lung, and constrained their Th2 cytokine production. Adoptive transfer of ILC2s increased asthma-associated airway inflammation in mice previously infected with RSV. These results indicate that previous infection with RSV prevents OVA-induced asthma development via inhibition of ILC2s. Previous infection with RSV attenuated IL-33 production in lung tissue and reduced relative ST2L expression in lung ILC2s, meaning that previous infection with RSV may alter ILC2 function via the IL-33/ST2 signaling pathway. CONCLUSION: These results demonstrate that previous infection with RSV attenuates OVA-induced airway inflammation by inhibiting the recruitment and Th2 cytokine production of ILC2s via the IL-33/ST2 pathway.

Natural helper cells mediate respiratory syncytial virus-induced airway inflammation by producing type 2 cytokines in an IL-33-dependent manner.

AIM: Type 2 cytokine production during respiratory virus infection is considered to be linked with asthma exacerbation. As potent Th2 cytokine producers, natural helper (NH) cells play a key role in influenza virus-induced airway hyper-responsiveness. However, it is unclear whether NH cells contribute to respiratory syncytial virus (RSV)-induced airway inflammation, and how the cytokine profile in NH cells is changed during RSV infection. METHODS: BALB/c mice were infected intranasally with RSV. The number of NH cells in lungs was detected by flow cytometry. The expression of cytokine mRNAs was performed by real-time RT-PCR. Cytokines levels were determined by ELISA. RESULTS: Following intranasal infection with RSV, BALB/c mice showed an increase in the expression of mRNAs for Th2-like cytokines in NH cells. Furthermore, adoptive transfer of pulmonary NH cells resulted in a massive infiltration of mononuclear cells, in particular eosinophils and neutrophils in lungs, in parallel with an augmented production of Th2-associated cytokines, such as IL-4, IL-5 and IL-10 in bronchoalveolar lavage fluids, providing convincing evidence that NH cells contribute to RSV-induced lung pathogenesis by producing type 2 cytokines. It should be noted that blocking IL-33 with antibody can diminish the absolute number of pulmonary NH cells and the relative expression of mRNAs for type 2 cytokines in pulmonary NH cells, suggesting that IL-33 is necessary for activating Th2-type NH cells. CONCLUSION: These results reveal that pulmonary NH cells might participate in RSV-induced airway inflammation by producing large quality of type 2 cytokines in an IL-33-dependent manner.

Role of γδ T cells in exacerbated airway inflammation during reinfection of neonatally primed mice in adulthood.

Age at primary infection with respiratory syncytial virus (RSV) is a crucial factor in determining the outcome of reinfection. However, how neonatal RSV infection affects the immune system and renders the host more susceptible to reinfection in later life is poorly understood. In the present study, by using BALB/c mice that were first infected with RSV as neonates, the role of γδ T cells in the development of airway inflammation during reinfection in adulthood was investigated. We found that neonatal RSV infection resulted in an aggravated infiltration of mononuclear cells in bronchoalveolar lavage (BAL) fluids, in parallel with a significant increase in the levels of type 2 cytokines in lungs on day 4 after reinfection. Since the numbers of total γδ T cells as well as activated γδ T cells, particularly IL-4-, IL-5-, and IL-13-producing γδ T cells, were enhanced markedly in the lungs of neonatally primed mice, we speculate that γδ T cells might participate in the augmented airway inflammation seen during reinfection. Indeed, depletion of γδ T cells attenuated the severity of lung histopathology during reinfection. Meanwhile, treatment of neonatal mice with anti-TCRδ mAb diminished not only the numbers of neutrophils, eosinophils, and lymphocytes, but also the levels of IL-4, IL-5, and IL-13 in the lungs after reinfection in adulthood, suggesting that γδ T cells, particularly Th2-type γδ T cells might play a critical role in exacerbating the pulmonary tissue pathology during reinfection of adult mice that were first infected as neonates.

Macrophages produce IL-33 by activating MAPK signaling pathway during RSV infection.

It has been reported that RSV infection can enhance IL-33 production in lung macrophages. However, little is known about specific signaling pathways for activation of macrophages during RSV infection. In the present study, by using real-time RT-PCR as well as western blot assay, it became clear that RSV infection can enhance not only the expression of mRNAs for MAPK molecules (including p38, JNK1/2, and ERK1/2), but also the levels of MAPK proteins in lung macrophages as well as RAW264.7 cells. Furthermore, infection with RSV resulted in an increased level of phosphorylated MAPK proteins in RAW264.7 cells, suggesting that MAPK signaling pathway may participate in the process of RSV-induced IL-33 secretion by macrophages. In fact, the elevated production of IL-33 in RAW264.7 was attenuated significantly by pretreatment of the cells with special MAPK inhibitor before RSV infection, further confirming the function of MAPKs pathway in RSV-induced IL-33 production in macrophages. In contrast, the expression of NF-κB mRNA as well as the production of NF-κB protein in lung macrophages and RAW264.7 cells was not enhanced markedly after RSV infection. Moreover, RSV infection failed to induce the phosphorylation of NF-κB in RAW264.7 cells, suggesting that NF-κB signaling pathway may be not involved in RSV-induced IL-33 production in macrophages. Conclusion, these results indicate that RSV-induced production of IL-33 in macrophages is dependent on the activation of MAPK signaling pathway.

The significance of the levels of IL-4, IL-31 and TLSP in patients with asthma and/or rhinitis.

AIM: To investigate the clinical significance of the levels of IL-4, IL-33 and thymic stromal lymphopoietin (TLSP) in patients with asthma and/or rhinitis, then do the simple verification in animals. METHODS: Levels of IL-4 IL-31, IL-33 and TLSP were detected by ELISA and real-time PCR in 64 asthma patients (sIgE[+]: 32 cases, sIgE[-]: 32 cases), 64 rhinitis patients (sIgE[+]: 32 cases, sIgE[-]: 32 cases), 64 asthma complicated with allergic rhinitis patients (sIgE[+]: 32 cases, sIgE[-]: 32 cases) and 32 healthy controls. Then we detected the IL-4, IL-31, IL-33 and TLSP in the sensitized mice. RESULTS: Results showed that levels of IL-4, IL-31, IL-33 and TSLP in asthma and rhinitis patients, and those complicated with allergic rhinitis, had significant differences compared with the control group (p < 0.05). It was found that the indicators of mugwort and dust mite allergic patients were significantly higher than that of other allergic patients (p < 0.05). We got the same tendency in in vivo experiments. CONCLUSION: IL-4, IL-31, IL-33 and TSLP may be involved in the pathogenesis of asthma and rhinitis; dust mite and mugwort allergy could increase them significantly.

Natural helper cells are associated with the exacerbated airway inflammation seen during RSV reinfection of neonatally primed mice.

Infection with respiratory syncytial virus (RSV) in neonatal mice causes more aggressive airway disease when the mice are reinfected with the same virus as adults. However, the underlying mechanisms responsible for this phenomenon are not entirely defined. Natural helper (NH) cells are considered a key factor for virus-induced or exacerbated airway inflammation and airway hyper-responsiveness by producing type 2 cytokines. To confirm whether NH cells are involved in the aggravated lung pathology seen during reinfection, BALB/c mice were initially infected as neonates and reinfected in adulthood. We observed that neonatal RSV infection resulted in an enhanced infiltration of eosinophils and neutrophils in the lungs, in parallel with a significant increase in the levels of IL-5 and IL-13 in bronchoalveolar lavage fluids on day 2 after reinfection. It seems likely that pulmonary NH cells may play a role in the occurrence, since mice first infected at 1wk of age developed an additional increase in the number of NH cells as well as IL-5- and IL-13-producing NH cells in the lungs than those first infected as young adults. In fact, an elevated expression of mRNAs for IL-5 and IL-13 in pulmonary NH cells was detected in mice first infected as neonates. Furthermore, adoptive transfer of NH cells into neonatal mice was able to boost eosinophilic infiltration as well as the production of type 2 cytokines in the lungs after reinfection at adulthood. In contrast, the expression of mRNA for the type 1 cytokine IFN-γ was down-regulated markedly by adoptive transfer of NH cells. Thus, these results suggest that Th2-type NH cells may play a role in the exacerbated airway inflammation seen during RSV reinfection of neonatally primed mice.

Respiratory macrophages and dendritic cells mediate respiratory syncytial virus-induced IL-33 production in TLR3- or TLR7-dependent manner.

Respiratory syncytial virus (RSV) infection can increase the production of IL-33 in lungs of mice. However, little is known about cellular source of IL-33, particularly the types of IL-33-producing cells in innate immune cells during RSV infection. In this study, by using BALB/c mice that were infected intranasally with RSV, it became clear that RSV infection can enhance not only the number of IL-33(+)-alveolar macrophages (AMs) and dendritic cells (DCs), but also the expression of IL-33 mRNA in these cells, suggesting that innate immune cells participate in the production of IL-33. Indeed, in vitro experiments by using murine cell lines found that RSV infection results in more expression of IL-33 mRNA in AMs and DCs, further confirming that these cell types may be an important source of IL-33 during RSV infection. It should be noted that the expression of mRNA for TLR3 and TLR7 was up-regulated in pulmonary AMs during RSV infection. Blockade of TLRs by TLR3 or TLR7 antagonist significantly reduces the levels of IL-33 mRNA in AMs and DCs, suggesting that RSV-induced IL-33 production might be TLRs-dependent manner. Although the expression of TLRs mRNA in pulmonary interstitial macrophages (IMs) was enhanced after RSV infection, stimulation with agonists or inactivated RSV cannot alter the expression of IL-33 mRNA in IMs, suggesting that pulmonary IMs may not be a source of IL-33 during RSV infection. Thus, these results demonstrate that during RSV infection, respiratory macrophages and dendritic cells mediate the production of IL-33 in a TLR-dependent manner.