Mouse Nasal-associated Lymphoid Tissue Research Articles

RmIL-33-stimulated nuocytes promote allergic inflammation in mouse model of allergic rhinitis

Objective:The study aimed to investigate the role of nuocytes in allergic rhinitis （AR） murine models. Method:After intranasal administration of recombinant （rm） interleukin （IL）-33 in BALB/c mice, nuocytes were sorted and purified from the mouse nasal-associated lymphoid tissue （NALT）. Then, we examined the response of nuocytes to rmIL-33 in vitro. After a murine model of AR was established using ovalbumin, we adoptively transferred the cultured NALT-derived nuocytes to mice models, and determined allergic responses in them. Result:rmIL-33 expanded nuocytes in NALT of mice compared with AR mice （t=3.66, P<0.01）, and increased production of IL-13 from these cells in vitro in comparison with unstimulated nuocytes （t=19.90, P<0.000 1）. After adoptive transfer of nuocytes, sneezing （t=9.89, P<0.000 1） ,numbers of eosinophils（t=8.17, P<0.000 1）, concentrations of IL-13 （t=40.47, P<0.000 1） and IL-33 （t=19.89, P<0.000 1） in nasal lavage fluid were all enhanced when compared with AR mice. Conclusion:Nuocytes promote allergic inflammation in a murine model of AR.

Intervention of Orai1 Influences the Response of Nuocytes From Allergic Rhinitis Mice to IL-33.

Nuocytes are essential in innate type-2 immunity and contribute to the exacerbation of allergic rhinitis (AR). This study aimed to evaluate the intervention of Orai1 on the response of nuocytes from AR mice to interleukin (IL)-33. We established a murine model of AR. Nuocytes were obtained from the mouse nasal-associated lymphoid tissue. Then, we assessed expressions of Orai1, Ca2+ mean fluorescence intensity (MFI) in nuocytes, and their cellular response to mouse recombinant (rm) IL-33. After that, we administered rmlentivirus vectors (lenti) that encoded small hairpin RNA (shRNA) against ORAI1 (lenti-ORAI1) into nuocytes cultures and again evaluated Orai1 and Ca2+ MFI in nuocytes and their response to rmIL-33. Finally, we adoptively transferred nuocytes alone or nuocytes transfected by lenti or lenti-ORAI1 to AR models to investigate their roles during allergic inflammation. We showed that Orai1 and Ca2+ MFI were upregulated in AR mice nuocytes. These cells were induced to produce more IL-5 and IL-13 by rmIL-33. However, the intervention of Orai1 by lenti-ORAI1 in nuocytes decreased Orai1 and Ca2+ MFI and reduced productions of aforementioned cytokines even after the administration of rmIL-33. Numbers of sneezing, nasal rubbing, and counts of eosinophils were all enhanced after the adoptive transfer of nuocytes. Concentrations of IL-5, IL-13, and IL-33 in the nasal lavage fluid (NLF) of allergic mice were also increased. However, the adoptive transfer of nuocytes transfected by lenti-ORAI1 decreased aforementioned parameters. These findings show that the intervention of Orai1 in nuocytes influences the response of nuocytes to rmIL-33.

CD4+ T cells induce productions of IL-5 and IL-13 through MHCII on ILC2s in a murine model of allergic rhinitis

ObjectiveCD4+ T cells play an important role not only in the induction of allergy but also in allergic inflammation. Group 2 innate lymphoid cells (ILC2s) also mediate type 2 immune responses in allergic rhinitis (AR). However, the relationships between CD4+ T cells and ILC2s in allergic condition are currently not well defined. The study aimed to evaluate the potential influences of CD4+ T cells on ILC2s in the murine model of AR. MethodsA murine model of AR was established using ovalbumin (OVA), and OVA-induced ILC2s were sorted and purified from the mouse nasal-associated lymphoid tissue (NALT), and cultured in vitro. Then, the expression of major histocompatibility complex class II (MHCII) on ILC2s was examined. CD4+ T cells were separated from AR mice peripheral blood mononuclear cells (PBMCs). After that, productions of IL-5 and IL-13 on ILC2s cultures were assessed when CD4+ T cells or plus anti-MHCII antibody or anti-CD4 antibody were administered into the cultures. Finally, we adoptively transferred ILC2s alone or ILC2s plus anti-MHCII antibody to the murine model of AR to investigate their roles in the nasal allergic inflammation. ResultsWe showed that ILC2s could be induced by OVA in the mouse NALT. The number and percentage of ILC2s in AR mice were increased. MHCII was expressed on ILC2s, and its protein and mRNA were all enhanced in allergic condition. IL-5 and IL-13 proteins and mRNAs were elevated after CD4+ T cells administration, and were reduced after these cells plus anti-MHCII antibody or anti-CD4 antibody application. Numbers of sneezing and nasal rubbing as well as counts of eosinophils in nasal lavage fluid (NLF) were all enhanced after the adoptive transfer of ILC2s when compared to AR mice. IL-5 and IL-13 in the NLF of allergic mice were also increased in comparison with AR group. However, above parameters were all decreased after the transfer of ILC2s plus anti-MHCII antibody versus AR mice or ILC2s-treated ones. ConclusionThese findings show that CD4+ T cells induce productions of IL-5 and IL-13 through MHCII on ILC2s in AR mice models.

Differentiation of naive T cells into Th2 cells induced by nuocyte cells in mice with allergic rhinitis

Objective:The aim of this study is to examine whether nuocytes induced differentiation of primary T cells into Th2 cells in AR mice in vitro. Method:A murine model of AR on the background of BALB/c was established using ovalbumin, and nuocytes were sorted and purified from the mouse nasal associated lymphoid tissue (NALT) and cultured in vitro. Then, we assessed the expression of IL-4 in these cells. Mouse peripheral blood mononuclear cells were isolated and Th2 cells and T cells were isolated for in vitro culture, and the percentage of T cells in Th2 cells was detected. Then, NALT-derived nuocyte cells cultured in vitro were added to the above-mentioned mouse T cell culture medium for co-culture. Result:Numbers of sneezing, nasal rubbing and eosinophils in nasal lavage fluid were all enhanced in AR mice compared to normal ones. We used flow cytometry analysis to identify nuocytes from mice NALT as CD3CD4CD8CD19CD11bCD11cFcεR1 (lineage)-ICOS+, and also found that the cells expressed IL-4, and its protein and mRNA were all increased in AR mice versus normal mice. After nuocytes were co-cultured with T-cell cultures, we determined the percentage of Th2 cells in total T cells, and found that the percentage was increased significantly. Conclusion:nuocyte cells may induce the differentiation of primary T cells into Th2 cells in AR mice through IL-4 pathway.

Nasopharyngeal colonization with Streptococcus pneumoniae triggers dendritic cell dependent antibody responses against invasive disease in mice.

Nasopharyngeal colonization with Streptococcus pneumoniae (Spn) is an important precondition for the development of pneumococcal pneumonia. At the same time, nasopharyngeal colonization with Spn has been shown to mount adaptive immune responses against Spn in mice and humans. Cellular responses of the nasopharyngeal compartment, including the nasal-associated lymphoid tissue, to pneumococcal colonization and their importance for developing adaptive immune responses are poorly defined. We show that nasopharyngeal colonization with S.pneumoniae led to substantial expansion of dendritic cells (DCs) both in nasopharyngeal tissue and nasal-associated lymphoid tissue of mice. Depletion of DCs achieved by either diphtheria toxin (DT) treatment of chimeric zDC+/DTR mice, or by use of FMS-like tyrosine kinase 3 ligand (Flt3L) KO mice exhibiting congenitally reduced DC pool sizes, significantly diminished antibody responses after colonization with Spn, along with impaired protective immunity against invasive pneumococcal disease. Collectively, the data show that classical DCs contribute to pneumococcal colonization induced adaptive immune responses against invasive pneumococcal disease in two different mouse models. These data may be useful for future nasopharyngeal vaccination strategies against pneumococcal diseases in humans.

Allergic inflammation is exacerbated by allergen-induced type 2 innate lymphoid cells in a murine model of allergic rhinitis.

Group 2 innate lymphoid cells (ILC2s) represent a new innate effector leukocyte population that mediates type-2 immune response. However, the contribution of ILC2s to allergic rhinitis (AR) is currently not well defined. To evaluate the potential existence and function of allergen-induced ILC2s in the experimental AR. We established a murine model of AR using ovalbumin (OVA) and aluminium hydroxide. The OVA-induced ILC2s were sorted and purified from the mouse nasal-associated lymphoid tissue (NALT). Then, we assessed ILC2s responses to mouse recombinant interleukin (rmIL)-25, anti-IL17RB antibody and CC chemokine ligand (CCL) 25 in the culture. After that, we adoptively transferred the NALT-derived ILC2s alone or plus rmIL-25 or anti-IL17RB antibody to the murine model of AR to investigate their role in the nasal allergic inflammation. We showed that ILC2s could be induced by OVA in the NALT of AR model. They were induced to secrete IL-5 and IL-13 by rmIL-25, and blocking of IL17RB contributed to the decreased production of these cytokines in the culture. We found that CCL25 induced the NALT-derived ILC2s migration through CC chemokine receptor 9 on ILC2s in vitro. Numbers of sneezing and nasal rubbing as well as counts of invasive eosinophils were all enhanced after the adoptive transfer of cultured ILC2s in vitro. The expressions of IL-5, IL-13, IL-25 and CCL25 in the NLF of allergic mice were also increased. These findings show that ILC2s play a proinflammatory role in the murine AR model, and also highlight ILC2s as a new target in the future AR therapy.

Induction of TGF-β1 and TGF-β1–dependent predominant Th17 differentiation by group A streptococcal infection

Recurrent group A Streptococcus (GAS) tonsillitis and associated autoimmune diseases indicate that the immune response to this organism can be ineffective and pathological. TGF-beta1 is recognized as an essential signal for generation of regulatory T cells (Tregs) and T helper (Th) 17 cells. Here, the impact of TGF-beta1 induction on the T-cell response in mouse nasal-associated lymphoid tissue (NALT) following intranasal (i.n.) infections is investigated. ELISA and TGF-beta1-luciferase reporter assays indicated that persistent infection of mouse NALT with GAS sets the stage for TGF-beta1 and IL-6 production, signals required for promotion of a Th17 immune response. As predicted, IL-17, the Th17 signature cytokine, was induced in a TGF-beta1 signaling-dependent manner in single-cell suspensions of both human tonsils and NALT. Intracellular cytokine staining and flow cytometry demonstrated that CD4(+) IL-17(+) T cells are the dominant T cells induced in NALT by i.n. infections. Moreover, naive mice acquired the potential to clear GAS by adoptive transfer of CD4(+) T cells from immunized IL-17A(+)/(+) mice but not cells from IL-17A(-)/(-) mice. These experiments link specific induction of TGF-beta1 by a bacterial infection to an in vivo Th17 immune response and show that this cellular response is sufficient for protection against GAS. The association of a Th17 response with GAS infection reveals a potential mechanism for destructive autoimmune responses in humans.

Characterization of Mouse Nasal-associated Lymphoid Tissue (NALT) in BALB/c and NOD (non-obese diabetic) Mice

Characterization of Mouse Nasal-associated Lymphoid Tissue (NALT) in BALB/c and NOD (non-obese diabetic) Mice

The early interferon response of nasal-associated lymphoid tissue toStreptococcus pyogenesinfection

Streptococcus pyogenes is a major causative agent of tonsillitis or pharyngitis in children. Streptococcus pyogenes can persist in tonsils, and one-third of children treated with antibiotics continue to shed streptococci and have recurrent infections. Mouse nasal-associated lymphoid tissue (NALT) is functionally analogous to human oropharyngeal lymphoid tissues, and serves as a model for characterization of the mucosal innate immune response to S. pyogenes. Wild-type S. pyogenes induces transcription of both type I and interferon-gamma (IFN-gamma)-responsive genes, proinflammatory genes and acute-phase response proteins 24 h after intranasal infection. Invasion of NALT and the induction of the interferon response were not dependent on expression of antiphagocytic M protein. Intranasal infection induces a substantial influx of neutrophils into NALT at 24 h, which declines by 48 h after infection. Infection of IFN-gamma(-/-) [IFN-gamma knock-out mouse (GKO)] C57BL/6 mice with wild-type S. pyogenes resulted in local dissemination of bacteria to draining lymph nodes (LN), but did not lead to systemic infection by 48 h after infection. Infected GKO mice had an increased influx of neutrophils into NALT compared with immunocompetent mice. Thus, IFN-gamma-induced responses are required to prevent local dissemination of streptococci to the draining LN.

Comparative studies of local antibody and cellular immune responses to influenza infection and vaccination with live attenuated reassortant influenza vaccine (LAIV) utilizing a mouse nasal-associated lymphoid tissue (NALT) separation method

The first and most significant barrier against influenza infection is the mucosal-associated lymphoid tissue of the upper airways and rodent nasopharyngeal-associated lymphoid tissue (NALT) is considered equivalent to the lymphoid tissue of human Valdryer's ring. This study is the first attempt to analyze and compare local and systemic cellular and antibody immune responses in NALT and spleen in a mouse model of experimental influenza infection and intranasal vaccination with LAIV (live attenuated reassortant influenza vaccine). It was shown that the vaccine strain completely inherited the ability to induce high-grade local antibody responses (secretory IgA + IgG + IgM), local cellular lymphoproliferative activity, CD4 +, CD8 + and CD19 + lymphocyte and cytokine production responses from the virulent parental strain but it had less capacity to stimulate production of serum IgG, accumulation of CD8 + cells and IFN-γ production in the spleen. Primary non-complicated influenza infection and primary vaccination were accompanied by a short-term (24 h) increase in the levels of lymphocyte apoptosis in both NALT and spleen. However, experimental data indicated that vaccination with LAIV and uncomplicated forms of influenza infection did not influence immune system apoptosis following a secondary immune response.

IgA antibody-forming cell responses in the nasal-associated lymphoid tissue of mice vaccinated by intranasal, intravenous and/or subcutaneous administration

Effects of a single intranasal (i.n.), subcutaneous (s.c.) or intravenous (i.v.) vaccination and their combined vaccination of priming and boosting on a primary and a secondary IgA antibody forming cell (AFC) response were examined in the nasal associated lymphoid tissue (NALT), spleen and popliteal lymph nodes (pLNs) of BALB c mice. Mice were primed with the vaccine prepared from A/Yamagata/120/86 (H1N1) together with a cholera toxin-adjuvant and boosted with the same vaccine 3 weeks later. Three days after boosting, IgA-AFC responses in each lymphoid tissue were measured as an index of the immunological memory that mediates a secondary IgA-AFC response. Single i.n. vaccination induced a greater primary IgA-AFC response in the NALT not only than that in the spleen or pLNs, but also than that induced by single i.v. or s.c. vaccination. The combination of i.n. priming and i.n. boosting afforded a greater anamnestic IgA-AFC response in the NALT not only than that in the spleen or pLNs, but also than that induced by any other combinations of priming and boosting (i.n.-i.v., i.n.-s.c., s.c.-i.n., s.c.-i.v., and s.c.-s.c.). These results showed that i.n. priming induced a greater primary IgA-AFC response in the NALT and simultaneously induced the immunological memory that mediated a greater secondary-type AFC response following i.n. boosting in the NALT.

Isolation and characterization of mouse nasal-associated lymphoid tissue

A method for isolation of mouse nasal-associated lymphoid tissue (NALT), which is a principal mucosal lymphoid tissue of the respiratory tract in rodents, was developed. The paired lymphoid organs could be separated from the upper jaw by peeling away the palate where NALT was localized bilaterally on the posterior side. About 3×10 5 lymphocytes could be obtained from one NALT fragment. The NALT lymphocyte fraction from normal BALB/c mice contained T- and B-cells in about equal numbers, and contained about 4 times as many CD4 + T-cells as CD8 + T-cells when analyzed with a FACScan fluorescence analyzer. The composition of the NALT lymphocytes was similar to that of the lymphocytes from the portion of the nasal cavity remaining after isolation of the NALT. The NALT lymphocyte fraction from mice infected 7 days previously with influenza virus was also characterized. The numbers of NALT T- and B-cells from the infected mice were approximately 2 and 3 times higher than those of non-infected mice, respectively. In parallel with the cell increase, NALT lymphocytes produced IFN-γ when cultured for 24 h and contained cells secreting influenza virus-specific IgA and IgG antibodies. The results suggest that this method can be successfully used for investigating cellular dynamics of mucosal immunology in the upper respiratory tract.