Mice vaccinated with allergen-pulsed myeloid dendritic cells are not protected from developing allergen-induced Th2 responses

BACKGROUND: Dendritic cells (DC) play a decisive role in the induction of allergen-induced Th1 and Th2 responses. Since the induction of allergen-specific Th1 responses has shown to inhibit allergen-induced Th2-type inflammation, in this study we investigated whether manipulated myeloid-derived DC pulsed with the specific allergen would predominantly induce allergen-specific Th1 responses thereby reducing the development of Th2 responses. METHODS: Murine bone marrow (BM)-DC were generated and pulsed with ovalbumin (OVA) and CpG oligodeoxynucleotides (CpG-ODN). Langerhans cells (LC) were also isolated and pulsed in vitro with OVA. Subsequently, mice were vaccinated intravenously with either CpG/OVA-pulsed BM-DC or OVA-pulsed LC, and the protocol to induce OVA-specific Th2 responses using OVA/alum sensitization was initiated. Airway inflammation and OVA-specific serum antibody levels were evaluated 6 days after the intranasal challenge with OVA. RESULTS: The application ofCpG/OVA-pulsed BM-DC was unable to reduce airway eosinophilia and inflammation in OVA/alum-immunized mice. OVA-specific IgG1 or IgE serum levels were also not reduced. The experiments using LC pulsed with OVA yielded similar results. However, mice vaccinated with CpG/OVA-pulsed BM-DC had greatly enhanced levels of serum OVA-specific IgG2a, suggesting the induction of allergen-specific Th1 responses in vivo. Moreover, allergen-induced mast cell degranulation was decreased using this approach. CONCLUSIONS: Taken together, our results demonstrated that the vaccination with OVA-pulsed BM-DC matured with CpG-ODN or OVA-pulsed LC did not result in a reduction in allergen-specific Th2 responses in a murine model of severe atopic asthma. Other DC-based vaccination strategies should be evaluated in order to prevent the development of allergic disorders.     

Interleukin-10-treated dendritic cells do not inhibit Th2 immune responses in ovalbumin/alum-sensitized mice

BACKGROUND: It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory function of human dendritic cells (DC) in vitro. Recently, we have shown that these IL-10 DC inhibit the production of T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines by T cells from atopic individuals in vitro. The current study was set out to analyze whether IL-10 DC also exert inhibitory effects in vivo in a murine model of allergy to ovalbumin adsorbed to the adjuvant aluminium hydroxide (OVA/alum). METHODS: OVA-pulsed or unpulsed bone marrow-derived DC, treated with IL-10 or left untreated during generation, were injected intravenously into BALB/c mice prior to and during OVA/alum sensitization, and sera and immune responses of mesenterial lymph node cells were analyzed. Additionally, bronchoalveolar lavage was performed after intranasal challenge with OVA. RESULTS: Treatment of BALB/c mice with OVA-pulsed DC led to a significantly enhanced proliferation as well as Th2 (IL-4, IL-5), Th1 (interferon-gamma) and IL-10 cytokine production after restimulation of lymph node cells with OVA in vitro compared with OVA immunization alone. In contrast, using OVA-pulsed IL-10 DC for transfer, proliferation and cytokine production by lymph node cells were not enhanced. OVA-specific immunoglobulin G1 (IgG1) and IgG2a production were significantly increased after transfer of OVA-pulsed DC and OVA-pulsed IL-10 DC, respectively, whereas anti-OVA IgE production and airway eosinophilia remained unchanged. CONCLUSIONS: Our data indicate that IL-10 treatment of DC decreases the Th1 and Th2 stimulatory capacity of DC but does not actually inhibit systemic (IgE) and local (airway inflammation) allergen-specific immune responses in a murine model of allergy.     

The primary responses of murine neonatal lymph node CD4+ cells are Th2-skewed and are sufficient for the development of Th2-biased memory

Exposure of neonatal mice to antigen often results in Th2-biased responses in later life. Examples of this Th2 tendency are (a) secondary antibody responses dominated by the Th2-associated IgG1 isotype and (b) Th2-mediated tolerance to alloantigens. We previously reported that neonates develop primary Th1 and Th2 function in the lymph nodes but exclusive Th2 primary splenic responses. Here, we have tested whether the Th2 bias of adults initially immunized as neonates is due to the early, primary Th2 polarization in the spleen. Surprisingly, removal of the spleen at birth had no affect on either IgG1-dominant secondary responses or the development of tolerance to alloantigens. Thus, neonatal lymph nodes are sufficient to generate Th2-biased function following neonatal antigen exposure. To understand how this could arise, we examined the primary Th1/Th2 responses of CD4+ lymph node cells. Unlike the balanced Th1/Th2 responses seen with total lymph node cells, the primary responses of isolated CD4+ cells were skewed to IL-4 producing function. These results suggest that the early development of Th2-dominant responses by lymph node CD4+ cells contributes substantially to the subsequent development of Th2-dominant memory in neonates.     

Effects of cytokine milieu secreted by BCG-treated dendritic cells on allergen-specific Th immune response

Bacillus Calmette-Guerin (BCG) is reported to suppress Th2 response and asthmatic reaction. Dendritic cells (DCs), the major antigen-presenting cells, infections with BCG are known to result in inducing various cytokines. Thus, DCs are likely to play a role in the effects of BCG on asthma. This study aims at investigating that cytokine milieu secreted by BCG-treated DCs directly enhances allergen-specific Th1 response and/or suppresses Th2 response in allergic asthma. DCs and CD3+ T cells were generated from Dermatophagoides farinae-sensitive asthmatics. DCs were cultured with and without BCG and subjected to flow cytometric analysis. IL-12 and IL-10 were determined from the culture supernatants. Some DCs were cocultured with T cells in the presence of D. farinae extracts after adding the culture supernatants from BCG-treated DCs, and IL-5 and IFN-gamma were determined. BCG-treated DCs enhanced significantly the expressions of CD80, CD86, and CD40, and the productions of IL-12 and IL-10. Addition of culture supernatants from BCG-treated DCs up-regulated production of IFN-gamma by T cells stimulated by DCs and D. farinae extracts (p<0.05), but did not down-regulate production of IL-5 (p>0.05). The cytokine milieu secreted by BCG-treated DCs directly enhanced allergen-specific Th1 response, although did not suppress Th2 response.     

菠萝蛋白酶诱导小鼠急性过敏性气道炎症的作用研究

探讨菠萝蛋白酶诱导小鼠急性过敏性气道炎症中2型固有淋巴细胞(group 2innate lymphoid cell,ILC2)及相关细胞因子的变化及意义。对未致敏BALB/c雌性小鼠在第1天和第3天给予菠萝蛋白酶滴鼻,ELISA检测12h内小鼠肺组织匀浆上清中IL-33变化,5d后处死小鼠,肺组织切片病理观察,瑞氏染色计数BALF中嗜酸性粒细胞含量,流式检测肺组织中ILC2数量变化,ELISA检测BALF中IL-5、IL-13水平。结果显示,菠萝蛋白酶诱导气道上皮细胞产生IL-33,3h时开始,6h时达高峰;菠萝蛋白酶滴鼻小鼠肺组织切片显示大量炎性细胞浸润和杯状细胞黏液分泌增加,BALF中嗜酸性粒细胞比例增加,IL-5和IL-13含量增加,流式检测肺组织中ILC2数量增多。研究表明,菠萝蛋白酶刺激小鼠气道上皮细胞释放IL-33,引起小鼠肺ILC2活化增殖并分泌大量Th2型细胞因子,引起嗜酸性粒细胞浸润等急性过敏性气道炎症表现,提示ILC2促进早期急性过敏性气道炎症的发生。     

CD8+ regulatory T cells are responsible for GAD-IgG gene-transferred tolerance induction in NOD mice

Our previous studies demonstrated that lipopolysaccharide (LPS)-stimulated splenocytes, retrovirally transduced with a glutamate decarboxylate 65 (GAD) and immunoglobulin G (IgG) fusion construct, can protect non-obese diabetic (NOD) mice from diabetes by inducing GAD-specific tolerance, and also that there are increased numbers of CD4(+) regulatory T cells (Tregs) in GAD-IgG-treated NOD mice. However, little is known about the role of CD8(+) Tregs in GAD-IgG gene-transferred tolerance induction in NOD mice. Here, we found that GAD-IgG-transduced splenocytes induced an increase in the number of CD8(+) Foxp3(+) Tregs in vitro. Using a T-cell depletion assay, we found that, compared with undepleted groups, NOD recipients transfused with CD8(-) or CD8(-) CD25(-) GAD-IgG-transduced splenocytes showed a decrease in the percentage of CD8(+) Foxp3(+) T cells, a high incidence of diabetes, serious insulitis, GAD-specific hyperresponsiveness at both the cellular and humoral levels, and changes in cytokine expression. These results indicate that CD8(+) Tregs, which were induced in vitro by GAD-IgG-transduced splenocytes, were also responsible for GAD-IgG gene-transferred tolerance induction in NOD mice.     

GM-CSF-producing CCR2+CCR6+ Th17 cells are pathogenic in dextran sodium sulfate-induced colitis model in mice

Although excessive immune responses by Th17 cells, a helper T cell subset, are implicated in the pathogenesis of inflammatory bowel disease (IBD), the mechanism by which its localization in an inflamed colon is regulated remains unclear. Chemokines and their receptors are involved in the pathogenesis of IBD, however, the relative significance of each receptor on Th17 cells remains unknown. We generated C–C motif chemokine receptor 2 (CCR2) knockout (KO) and CCR6 KO mice in the syngeneic background using the CRISPR/Cas9 system and found that the phenotypes of experimental colitis worsened in both mutant mice. Surprisingly, the phenotype of colitis in CCR2/CCR6-double knockout (CCR2/6 DKO) mice was opposite to that of the single-deficient mice, with significantly milder experimental colitis (p < .05). The same was true for the symptoms in CCR6 KO mice, but not in wild type mice treated with a CCR2 inhibitor, propagermanium. Colonic CCR2⁺CCR6⁺ Th17 cells produced a potentially pathogenic cytokine GM-CSF whose levels in the gut were significantly reduced in CCR2/6 DKO mice (p < .05). These results suggest that GM-CSF-producing CCR2⁺CCR6⁺ Th17 cells are pathogenic and are attracted to the inflamed colon by either CCR2 or CCR6 gradient, which subsequently exacerbates experimental colitis in mice.     

Resting B cells are not antigen-presenting cells in the induction of oral tolerance of specific Th2 immune responses in mice

BACKGROUND: It has been shown that antigen presentation by resting B cells can induce tolerance to intravenously administered protein antigens, but the role of resting B cells in the induction of oral tolerance is unclear. METHODS: Mice continuously treated since birth with rabbit anti-mouse IgM serum for 5 weeks were depleted of B cells. When 4 weeks old, B cell-depleted mice drank 10% chicken egg white (EW) for 5 days. Ten weeks later, they were immunized with 10 microgram of ovalbumin in alum and their T helper 2 (Th2) immune responses were tested. RESULTS: Th2 cell-mediated IgE and IgG1 antibody responses and spleen cell production of IL-4 and IL-5 were suppressed by prior EW feeding during anti-IgM treatment. When anti-IgM-treated spleen cells collected 1 week after EW ingestion were transferred to na?ve recipients, active suppression of Th2 immune responses was also demonstrated. CONCLUSIONS: Although resting small B cells aggregate in the mantle zone of follicles of intestinal Peyer's patches, the present data suggest that they are not antigen-presenting cells in the induction of oral tolerance of Th2 immune responses to oral antigens.     

Pulmonary CD103(+) dendritic cells prime Th2 responses to inhaled allergens

Allergic asthma stems largely from the actions of T helper 2 (Th2) cells, but the pathways that initiate Th2 responses to inhaled allergens are not fully understood. In the lung, there are two major subsets of dendritic cells (DCs), displaying CD11b or CD103. We found that after taking up inhaled ovalbumin in vivo, purified CD103(+) DCs from the lung or lung-draining lymph nodes primed Th2 differentiation ex vivo. Th2 induction by CD103(+) DCs was also seen when cockroach or house dust mite allergens were used. In contrast, CD11b(hi) DCs primed Th1 differentiation. Moreover, mice lacking CD103(+) DCs displayed diminished Th2 priming to various inhaled allergens and did not develop asthma-like responses following subsequent allergen challenge. Low-level antigen presentation by CD103(+) DCs was necessary, but not sufficient for Th2 priming. Together, these findings show that CD103(+) DCs have a significant role in priming Th2 responses to inhaled allergens.     

Increased allergen-specific Th2 responses in vitro in atopic subjects receiving subclinical allergen challenge

The study aimed to determine whether inhalation of subclinical allergen doses leads to a shift in the balance between T helper (Th) 1 and Th2 cells in asthmatic patients. Elevated IgE requires allergen-specific T cells producing cytokines such as interleukin (IL)-4 or 1L-13. Interferon-gamma (IFN-y) produced by Till cells counteracts the effects of IL-4. In nature, allergic persons are often exposed to low levels of allergen, leading to hyperreactivity, but not to acute allergic reactions. In this study, nine allergic persons inhaled low doses of allergen or placebo in a double-blind manner over seven consecutive weekdays. During the study, the bronchial responsiveness to histamine challenge increased, but no subject exhibited asthmatic symptoms. Blood was drawn on days 0,1, 4, and 9, and the number of IL-4– and IFN-γ-producing cells was measured by enzyme-linked immunospot (ELISPOT) assay after in vitro stimulation with a low-dose phytohemagglutinin (PHA) mixed with the relevant allergen or with PHA alone. In three of the four subjects receiving allergen, the IL-4/IFN-y ratio increased during the time of the study. No increase was seen in the placebo group. No increase was seen in serum IgE levels in any of the groups. We conclude that a shift in the balance between Thl and Th2 cells can be detected in subjects exposed to subclinical allergen doses.     

Selective maturation of dendritic cells by Nippostrongylus brasiliensis-secreted proteins drives Th2 immune responses

Helminth infections at mucosal and tissue sites strongly polarize towards Th2 immune responses, following pathways which have yet to be elucidated. We investigated whether dendritic cells (DC) exposed to gastrointestinal nematodes induce Th2 differentiation and, if so, whether this outcome reflects the absence of DC activation (the default hypothesis). We studied secreted proteins from the parasite Nippostrongylus brasiliensis, which induce Th2 development in vivo without live infection. Murine bone marrow-derived DC pulsed with N. brasiliensis excretory/secretory antigen (NES) can, on transfer to naive recipients, prime mice for Th2 responsiveness. Heat inactivation of NES abolishes both its ability to drive Th2 responses in vivo and its capacity to stimulate DC for Th2 induction. NES, but not heat-inactivated NES, up-regulates DC maturation markers associated with Th2 promotion (CD86 and OX40L), with little change to CD80 and MHC class II. Moreover, DC exposed to NES readily produce IL-6 and IL-12p40, but not IL-12p70. LPS induced high IL-12p70 levels, except in DC that had been pre-incubated with NES. These data contradict the default hypothesis, demonstrating that a helminth product (NES) actively matures DC, selectively up-regulating CD86 and OX40L together with IL-6 production, while blocking IL-12p70 responsiveness in a manner consistent with Th2 generation in vivo.     

Gr1+IL-4-producing innate cells are induced in response to Th2 stimuli and suppress Th1-dependent antibody responses

Alum is used as a vaccine adjuvant and induces T_h2 responsesand T_h2-driven antibody isotype production against co-injectedantigens. Alum also promotes the appearance in the spleen ofGr1+IL-4+ innate cells that, via IL-4 production, induce MHCII-mediated signaling in B cells. To investigate whether theseGr1+ cells accumulate in the spleen in response to other T_h2-inducingstimuli and to understand some of their functions, the effectsof injection of alum and eggs from the helminth, Schistosomamansoni, were compared. Like alum, schistosome eggs inducedthe appearance of Gr1+IL-4+ cells in spleen and promoted MHCII-mediated signaling in B cells. Unlike alum, however, schistosomeeggs did not promote CD4 T cell responses against co-injectedantigens, suggesting that the effects of alum or schistosomeeggs on splenic B cells cannot by themselves explain the T celladjuvant properties of alum. Accordingly, depletion of IL-4or Gr1+ cells in alum-injected mice had no effect on the abilityof alum to improve expansion of primary CD4 T cells. However,Gr1+ cells and IL-4 played some role in the effects of alum,since depletion of either resulted in antibody responses toantigen that included not only the normal T_h2-driven isotypes,like IgG1, but also a T_h1-driven isotype, IgG2c. These datasuggest that alum affects the immune response in at least twoways: one, independent of Gr1+ cells and IL-4, that promotesCD4 T cell proliferation and another, via Gr1+IL-4+ cells, thatparticipates in the polarization of the response.     

Ly-49 G2+ NK cells are responsible for mediating the rejection of H-2b bone marrow allog rafts in mice

NK cells can mediate the specific rejection of bone marrow butnot solid tissue allografts in lethally irradiated mice. NKcells are also responsible for the phenomenon of ‘hybridresistance’ in which F₁ hybrid H-2 heterozygous mice canreject parental H-2 homozygous bone marrow grafts. Ly-49C andLy-49 G2 are markers identified on subsets of NK cells. WhileLy-49C⁺ NK cells have been demonstrated to mediate the specificrejection of H-2^d bone marrow allografts, the role of the Ly-49G2⁺ NK subset is unclear because depletion of this subset invivo did not affect splenic NK activity against tumor targets.Through bone marrow transplantation typing studies, we demonstratethat Ly-49 G2⁺ NK cells complement Ly-49C⁺ NK cells in thatthey specifically mediate the rejection of H-2^b bone marrowallografts in lethally irradiated mice. In support of this,depletion of the Ly-49C⁺ NK subset in vivo also enhanced theability of the mice to reject H-2^b bone marrow cells suggestingthat the depletion was augmenting the ability of the Ly-49 G2⁺NK cells to reject the marrow allografts. Depletion of Ly-49G2⁺ NK cells in F₁ hybrid mice abrogated their ability to rejectparental H-2^b but not H-2^d bone marrow grafts. Therefore, Ly-49G2 denotes, a subset of NK cells that appears to play a criticalrole in the recognition of H-2^b bone marrow cells in allogeneicand F₁ hybrid mice.     

Profiling of differential gene expression in activated, allergen-specific human Th2 cells

Th2 cells play a pivotal role in the pathogenesis of allergic diseases, including asthma, but the molecular basis of the Th1/Th2 dichotomy and the precise molecular pathways leading to Th2-dominant immune responses are still unclear. To this end, we have combined suppression subtractive hybridization (SSH) and high throughput analysis of cDNA arrays spotted with IMAGE clones to determine the profile of differential gene expression in human allergen-specific Th2 cells. Allergen-stimulated Th2 cells were used as the tester, and either resting Th2 cells or stimulated Th1 cells were used as the driver. SSH was used to equalize different mRNA levels and remove common sequences between the tester and the driver. Comparison of cDNA arrays probed with subtracted tester and non-subtracted driver provided a profile of Th2-selective gene expression. Analysis of 77 sequence-confirmed and differentially expressed genes in Th2 cells showed predominant EST sequences, representing 80% of sequences analyzed. The pattern of gene expression in 19 selected sequences was further analyzed in additional Th1 and Th2 clones. A total of 15 sequences showed predominant expression in Th2 cells, while the remaining four EST sequences showed no detectable amplification signal. The database containing Th2-selective genes will further our understanding of Th2 cell function and the genetic basis of allergic diseases.     

Polarized Th2 like cells, in the absence of Th0 cells, are responsible for lymphocyte produced IL-4 in high IgE-producer schistosomiasis patients

Background  

Human resistance to re-infection with S. mansoni is correlated with high levels of anti-soluble adult worm antigens (SWAP) IgE. Although it has been shown that IL-4 and IL-5 are crucial in establishing IgE responses in vitro, the active in vivo production of these cytokines by T cells, and the degree of polarization of Th2 vs. Th0 in human schistosomiasis is not known. To address this question, we determined the frequency of IL-4 and IFN-γ or IL-5 and IL-2 producing lymphocytes from schistosomiasis patients with high or low levels of IgE anti-SWAP.     

Regulatory Th2 response induced following adoptive transfer of dendritic cells in prediabetic NOD mice

We previously demonstrated that immunotherapy with dendritic cells (DC) prevented diabetes development in prediabetic NOD mice and that this effect was optimal when using a stimulatory DC population generated from bone marrow cells cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. In this study, we have investigated the mechanism by which GM-CSF- and IL-4-cultured DC prevent diabetes in prediabetic NOD mice. Histological analysis of pancreatic tissue from DC-treated mice revealed a reduction in the severity of insulitis compared to controls. Analysisof the T cell response in DC-treated mice suggested a general shift towards a Th2-dominated response, as determined by cytokine production following either concanavalin A or anti-TCR stimulation. Furthermore, sorted CD45RB(lo) CD25+ CD4+ T cells from the spleen of DC-treated mice produced high amounts of Th2 cytokines following anti-TCR stimulation, suggesting that these cells are responsible for the apparent Th2 shift. We conclude that DC therapy may have corrected the immunoregulatory defect in the NOD mouse, thus restoring a balance between pathogenic Th1 cells and protective Th2 cells.     

Th2 cells shape the differentiation of developing T cell responses during interactions with dendritic cells in vivo

During priming, naive CD4(+) Th cells differentiate into cells that produce either IFN-gamma or IL-4. Even though the cascade of pathways that induces IL-4-producing Th2 cells has been determined in vitro, the signals promoting Th2 differentiation under physiological conditions remain enigmatic, especially the natural role of the single most important Th2-inducing signal,IL-4. Using Th2 and naive Th cells, each expressing a distinct transgenic TCR, here we show that Th2 cells migrate with the same dynamics as naive Th cells in draining lymph nodes and bind to the same DC, when driven by antigen in complete Freund's adjuvant (CFA). Th2-cell-derived IL-4 deviates CFA-induced Th1 development toward a Th2 phenotype, if both cell populations co-localize in the same T cell area, and are activated simultaneously. Thus, intranodal Th2 cells directly influence Th cell differentiation in vivo, but only under restricted conditions. These findings have implications for the design of cytokine-based therapies and explain the spreading of Th2 responses to multiple aeroallergens in allergic asthma, where naive Th and Th2 cells co-localize in lung-draining lymph nodes.