YCIMM3148S0008-8749(12)00230-410.1016/j.cellimm.2012.12.004Elsevier Inc.Fig. 1Oral administration of ovalbumin induces inhibition of specific anti-ova antibodies in serum. (A) Tolerization protocol and experimental groups. Groups of C57BL/6 mice were administered a 1:5 solution of egg white in drinking water for three consecutive days (days −10 to −7). Seven days later, the mice were immunized i.p. with 10μg of OVA in 1mg of Al(OH)3 (day 0) and boostered 14days thereafter with 10μg of OVA in PBS. (B) Blood serum was collected in the beginning of the experiment and after each immunization when the levels of anti-ova total Ig antibodies from immunized (■), tolerant (▴) and control (●) mice were assayed by ELISA. (C–F) Serum levels of anti-ova IgG1, IgE, IgM and IgA were assayed after booster immunization (day 21). Control mice received tap water and were sham immunized with saline. Data shown are means of five mice/group. (B) Statistical differences between tolerant and immunized mice by Student t test after first immunization and after booster. ∗∗=P<0.01; ∗∗∗=P<0.001. (C–F) Different letters represent statistical differences measured by ANOVA with Tukey post-test (P<0.05).Fig. 2Tolerant mice showed high levels of total serum IgM and IgA. Serum levels of total IgG1 (A), IgE (B), IgM (C) and IgA (D) were assayed by Elisa 7days after booster immunization as described in Fig. 1A. Data shown are means of five mice/group. Results are expressed as mean±SEM. Different letters represent statistical differences (P<0.01) measured by ANOVA with Tukey post-test.Fig. 3Similar lymphocyte activation and Treg generation in tolerant and immunized mice after booster immunization. Tolerant and Immunized mice present similar patterns of lymphocyte activation after booster immunization. Mice received either OVA (tolerant) or tap water (immunized and control) seven days before priming immunization. Fourteen days after mice received a booster immunization. Seven days after booster immunization mice were killed and their spleen were collected for phenotypical analysis of lymphocytes by flow cytometry. Control mice were sham immunized with saline. Percentage and absolute cell number of CD4+CD69+T cells (A and B); CD19+CD69+B cells (C and D); CD4+CD44hi T cells (E and F); CD4+CD25+CD45RBlow Treg cells (G, H); CD4+CD25+LAP+Treg cells (I and J) and CD4+Foxp3+Treg cells (K and L). For gating strategies and representatives dot plots see Supplementary Figs. 1 and 2. Different letters indicate statistical differences as measured by ANOVA with Tukey post test (P<0.05).Fig. 4Cytokines profile of tolerant and immunized mice after booster immunization. Tolerant mice received oral OVA by continuous feeding for three days and immunized mice received tap water. Seven days after both mice were i.p. immunized with 10μg of OVA in 1mg of Al(OH)3 and boostered 14days thereafter with 10μg of OVA in PBS. Control mice received tap water and were sham immunized with saline. Spleen cells were cultured and stimulated with 0.1μg/ml of anti-CD3 for 24–48h. Total mRNA was extracted from cell culture and submitted to qPCR to determine expression of IL-2, IL-4, IFN-γ, IL-10 and TGF-β (see Section 2). Results are expressed as fold changes of mRNA expression normalized by samples from control animals. Different letters indicate statistical differences as measured by ANOVA with Tukey post test (P<0.05).Fig. 5Tolerant mice displayed higher number of immunoglobulin secreting cells (ISC) in the spleen and the bone marrow as compared to immunized mice. Tolerant mice received oral OVA by continuous feeding for three days and immune mice received tap water. Seven days after both mice were i.p. immunized with 10μg of OVA in 1mg of Al(OH)3 and boostered 14days thereafter with 10μg of OVA in PBS. Control mice received tap water and were sham immunized with saline. Mice were killed after priming immunization or after booster immunization when spleen and bone marrow cells were cultured and total Ig-, IgM-, IgG- and IgA-secreting cells (ISC) were measured by ELISPOT. ISC assayed after priming immunization (A and F) and after booster immunization (B–E – spleen, G–J – bone marrow). Bars represent values (mean +/−SEM) of each group. Different letters indicate statistical differences as measured by ANOVA with Tukey post test (P<0.05).Fig. 6Tolerant mice showed earlier appearance of activated and regulatory T cells after priming immunization with OVA. Mice received oral OVA (Tolerant-dashed lines) or tap water (Immunized – solid lines) prior to immunization with OVA in Al(OH)3, after which animals were killed at several intervals (1, 2, 4 and 7days) – one group of tolerant animals were killed before immunization (Pre-0) – and spleen cells were harvested for flow cytometry analysis. We analyzed the kinetics of appearance (percentage and cell number) of early activated T (CD4+CD69+) and B (CD19+CD69+) cells; effector/memory CD4+CD44hi and CD4+CD25+LAP+and CD4+Foxp3+regulatory T cells. Asterisks represent statistical differences (ANOVA) between tolerant and immunized mice at each time point ∗=P<0.05; ∗∗=P<0.01; ∗∗∗=P<0.001. Statistical differences between distinct time points of the same treatment (tolerant or immunized) are indicated by # (#=P<0.05; ##=P<0.01; ###=P<0.001). Pre-immunization expansions are highlighted by dotted line rectangles.Fig. 7Cytokines kinetics of tolerant and immunized mice. Mice received oral OVA (Tolerant-dashed lines) or tap water (Immunized – solid lines) prior to immunization with OVA in Al(OH)3, after which animals were killed at several intervals (1, 2, 4 and 7days) (left panels). Spleen cells were cultured and stimulated with 0.1g/ml of anti-CD3 for 48h. Total mRNA was extracted from cell culture and submitted to qPCR to determine expression of IL-2, IL-4, IFN-γ, IL-10 and TGF-β (see Section 2). Data highlighted by doted rectangle are showed in separated graphs (right panels) in order to visualize the differences in the first two days of the kinetics (Tolerant-open symbols – □∇♢; Immunized – closed symbols ♦). Results are expressed as fold changes of mRNA expression normalized by samples from control animals. For left panels, asterisks represent statistical differences (ANOVA) between tolerant and immunized mice at each time point (∗=P<0.05; ∗∗=P<0.01; ∗∗∗=P<0.001). Statistical differences between distinct time points of the same treatment (tolerant or immunized) are indicated by # (#=P<0.05; ##=P<0.01; ###=P<0.001). For right panels # symbols indicate statistical differences between all groups as measured by ANOVA with Tukey post-test (#=P<0.05; ##=P<0.01; ###=P<0.001).Oral tolerance correlates with high levels of lymphocyte activityArchimedes BarbosaCastro-Juniora⁎archimedesjunior@yahoo.com.brBernardo CoelhoHortaaAna CristinaGomes-SantosaAndre PiresCunhaa1RaphaelSilva SteinbergbDanielle SantiagoNascimentocAna Maria CaetanoFariaaNelson MonteiroVazaaDepartamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, BrazilbDepartamento de Genética, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, BrazilcInstituto Nacional de Saúde da Mulher, da Criança e do Adolescente Fernandes Figueira, IFF, Fundação Oswaldo Cruz, Brazil⁎Corresponding author. Address: Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Bioquímica e Imunologia, Av. Antônio Carlos 6627, Pampulha 31270-901, Belo Horizonte, MG, Brazil. Fax: +55 31 3409 2640.1Present address: Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA.Highlights► Systemic immunological activity of orally-tolerant and immunized mice were compared. ► Tolerant and immunized mice had similar number of activated and regulatory T cells. ► Tolerant mice had higher numbers of Ig-secreting cells than immunized mice. ► Treg and activated lymphocytes appeared earlier in tolerant mice after immunization.AbstractOral tolerance is defined as an inhibition of specific immune responsiveness to a previously ingested antigen. Paradoxically, we found an increased lymphocyte activity in tolerant mice alongside the specific inhibition. Orally-tolerant mice presented higher number of immunoglobulin secreting cells (ISC) in spleen and bone marrow; showed a greater variety of Ig classes being produced: IgM and IgA in the spleen and IgG and IgM in the bone marrow. ISC from immunized mice produced mainly IgG. Despite having the same number of regulatory and activated T cells in the spleen after immunization, these cells appeared earlier in tolerant mice, right after the primary immunization. Also, tolerant mice showed a prompt expression of regulatory cytokines (TGF-β and IL-10) and a transient expression of effector cytokines (IL-2 and IFN-γ). Thus, in addition to an inhibited specific responsiveness, orally-tolerant mice displayed an early and widespread mobilization of activated and regulatory lymphocytes.KeywordsOral toleranceLymphocyte activationRegulatory T cellsImmunoglobulin-secreting cellsKineticsCytokines1IntroductionIn the first decades of last century, several independent experiments showed that the ingestion of immunogenic proteins had the unexpected consequence of reducing specific immune reactions to subsequent immunization [1–4]. In 1946, Chase described a way to inhibit experimental drug allergy (contact sensitization) by prior feeding of the sensitizing agents [5]. As “negative” phenomena, these puzzling findings remained unexamined up to the mid 1970s, when they were again observed as powerful interferences of ingested proteins with subsequent immunization [6–8], which also included a natural relative unresponsiveness to the autochthonous microbiota [9].Oral tolerance has been intensely investigated and is considered a special case of “peripheral tolerance”, i.e., as a subtractive phenomenon. First raised by Burnet and Fenner [10], the notion of “specific immune tolerance” became a central tenet of immunological theory [11] supported by experimental findings of Medawar and collaborators, who rendered neonatal mice “tolerant” to skin allografts and suggested that this depended on the deletion or permanent inhibition (anergy) of allo-reactive lymphocytes [12]. In line with this reasoning, “oral tolerance” was interpreted as necessary to ensure unresponsiveness to harmless antigens (food proteins and commensal bacteria), however, retaining the ability to eliminate pathogens [13]. Oral tolerance can be adoptively transferred to syngeneic recipients by T cells [14] and, therefore, is not a subtractive phenomenon, but rather has positive, systemic aspects. More specifically, the transfer of special T cell subsets with regulatory phenotypes, such as CD4+CD25+CD45RBlow or CD4+CD25+Foxp3+, demonstrated the participation of these cells in oral tolerance induction [15]. Also, CD4+CD25+LAP+regulatory T cells, that have a membrane bound form of TGF-β named latent associated peptide (LAP), have been implicated in oral tolerance [16–18]. These T cells would restrict the specific immunological reactivity to food proteins and the gut microbiota through immunosuppressive cytokines, such as IL-10 and TGF-β [19,20]. Although the generation of regulatory T cells has been described as a dominant mechanism for oral tolerance induction, the majority of studies focus only on inhibition of specific lymphocyte activity. Furthermore, the original experiments by Medawar’s group were replicated and contradicted. As measured by the acceptance of an allogeneic skin graft, allograft tolerance correlates with high levels of T and B lymphocyte activity, rather than their unresponsiveness [21]. Mice that accepted the allograft had many more activated lymphocytes in their spleen than mice that rejected a similar graft. This suggested dominant tolerant mechanisms as opposed to a recessive, deletional mechanisms [22] and proposed another way to understand tolerance, not only as an inhibition of specific immunological reactivity, but as a reflection of systemic phenomena that buffer antigen perturbations with more global lymphocyte activity. Little attention has been given to these results and even less to global immunological activities proposed to be involved in oral tolerance.Our aim in the present study was to analyze global profiles of lymphocyte activation of mice rendered orally-tolerant by the ingestion of ovalbumin (OVA) submitted to priming and booster immunizations. We showed that global lymphocyte activity in these tolerant animals was heighten, in spite of their reduced lymphocyte responsiveness to OVA. The frequency of immunoglobulin secreting cells (ISC) in the spleen and bone marrow of orally-tolerant mice was higher than that of immunized (non-tolerant) mice, with the prevalence of IgA and IgM (in spleen) or IgM and IgG (in bone marrow). Additionally, when compared to immunized mice after priming immunization, tolerant mice showed an earlier appearance of regulatory and activated T cells supported by a prompt expression of regulatory cytokine (IL-10 and TGF-β) alongside a transient expression of lymphocyte effector cytokines (IFN-γ and IL-2) when compared to immunized mice after priming immunization.2Materials and methods2.1Experimental animalsFemale 8week old C57BL/6 mice were obtained from our animal facility (CEBIO, Instituto de Ciências Biológicas, UFMG, Belo Horizonte, Brazil) and maintained in our animal breeding unit throughout the experiments. The animals were handled according to the rules established by the Local Ethical Committee for animal research (CETEA-UFMG, Brazil). Each experimental group contained 4–5 mice. The experiments were repeated at least twice.2.2Antigen and adjuvantCrystallized hen’s egg albumin (OVA, grade V, Sigma, St. Louis, MO) was used as antigen on immunization protocols. Aluminum hydroxide [Al(OH)3] was used as adjuvant.2.3Oral tolerance inductionOral tolerance to OVA was induced by enforcing mice to drink, ad libitum, a 1/5 solution of hen egg white in drinking water for 3 consecutive days. Oral treatment was discontinued 7days before parenteral immunization. The egg white solution was prepared in our laboratory from eggs bought at supermarkets. Daily average consumption was estimated at 20mg OVA/mouse and this resulted in significant levels of tolerance. Bottles were changed every day to avoid contamination. Control groups received filtered tap water.2.4Parenteral immunizationsMice were actively sensitized by an intraperitoneal (i.p.) injection of 0.2ml of saline (0.9% NaCl) containing 10μg of OVA (Sigma, St. Louis, MO) adsorbed in 1mg of aluminum hydroxide, Al(OH)3. Fourteen days later, the animals received the same dose of OVA in saline. Control animals received 0.2ml of sterile saline.2.5Analysis of Ig isotypes by ELISALevels of total and OVA-specific IgG1, IgE, IgM and IgA in the sera were determined by ELISA. Briefly, 96-well plates (Nunc, Roskild, Denmark) were coated overnight with 2μg of OVA in 100μl/well of sodium carbonate buffer, pH 9.6, at 4°C. To measure total levels of antibodies, plates were covered with unlabeled goat anti-mouse Ig (Southern Biotechnology). Plates were washed and blocked with 200μl of PBS contain 0.25% casein for 1h at room temperature (RT). The plates were then washed again, and incubated for 1h at RT with dilutions of mouse serum samples starting at 1/100 in PBS-casein. Plates were washed and then peroxidase-conjugated goat anti-mouse antibodies against mouse IgG1, IgE, IgM or IgA (Southern Biotechnology) were added, and plates were incubated for 1h at 37°C. Color reaction was developed at room temperature with 100μl/well of orthophenylenediamine (1mg/ml), 0.04% H2O2 substrate in sodium citrate buffer, pH 5.0 for 20min. Reaction was stopped by the addition of 20μl/well of 2N H2SO4. Absorbance was measured at 490nm by an ELISA reader (Bio-Rad Model 450 Microplate Reader). ELISA scores (Elisa∗) anti-OVA were computed by running sums of optical densities between 1:100 and 1:12,800 of serum dilutions in individual mice. Each score is shown as a mean±SEM of respective group.2.6Cell culture and isolation of total RNASpleen cells were cultured in 24-well plates at 5×106cells/ml in RPMI medium supplemented with 0.1mM nonessential amino acids, 1mM sodium pyruvate, 2mM L-glutamine, 100U/ml penicillin, 100U/ml streptomycin, 10% heat-inactivated fetal bovine serum (GIBCO) and 5×10−5M 2-ME (Sigma). Cells were stimulated with 0.1μg/ml of anti-CD3 and maintained for 24–48h in 5% of CO2 humidified chamber. Then total RNA was extracted from cultured cells using TRIzol (Gibco BRL, USA) derived from the single-step RNA isolation method developed by Chomczynski and Sacchi [23] according to manufacturer’s protocol.2.7qPCRThe PCR technique was used to amplify the synthesized cDNA from cell culture employing the SYBR Green PCR Master Mix (Applied Biosystems) on an ABI Prism 7500 fast sequence detection system (Applied Biosystems), as indicated by manufacturer. oligonucleotides pairs were synthesized as follows: for IL-2 – F: 5′-CTCCTGAGCAGGATGGAGAATT-3′ and R: 5′-CGCAGAGGTCCAAGTGTAGCT-3′; IL-4 F: 5′-CATCGGCATTTTGAACGAGGTCA-3′ and R: 5′-CTTATCGATGAATCCAGGCATCG-3′; IL-10 F: 5′-AAGCTGAGAACCAAGACCCAGACATCAAGGCG-3′ and R: 5′-AGCTATCCCAGAGCCCCAGATCCGATTTTGG-3′; TGF-B1 – F: 5′-GCAACATGTGGAACTCTACCAG-3′ and R: 5′-CAGCCACTCAGGCGTATCA-3′; INF-γ: F: 5′-CAGCAACAGCAAGGCGAAA-3′ and R: 5′-CGCTTCCTGAGGCTGGATT-3′. The β-actin gene was used as internal control in all reactions (F: 5′-TTGCTGACAGGATGCAGAAG-3′ and R: 5′-ACATCTGCTGGAAGGTGGAC-3′).2.8Flow cytometryThe immunophenotyping of murine spleen cells was analyzed by flow cytometry. Briefly, cells were washed twice in PBS containing 3% BSA and 0.1% sodium azide. Cells (3×105) were subsequently stained, according to standard methods. The mAbs used were as follows: rat anti-mouse CD4-PE, rat anti-mouse CD8-PE, rat anti-mouse CD45RB-PE, rat anti-mouse CD19-FITC, rat anti-CD25-biotin, rat anti-mouse CD69-PerCP, rat anti-mouse CD44-PE, all from BD Pharmingen. Rat anti-mouse LAP-biotin was purchased from eBioscience. Foxp3 staining and analysis were performed using the Foxp3 staining set (clone FJK-16s; eBioscience) according to the manufacturer’s instructions. For biotinylated antibodies an extra step with Cy5-conjugated streptavidin to stain cells. Stained cells were ressuspended in PBS containing 3% BSA and 0.1% sodium azide. Cell samples were analyzed on a FACScan Flow Cytometry (Becton Dickinson). Analyses were performed using FlowJo™software (Tree Star, Inc.) by gating the lymphocytes population on the basis of relative size (forward light scatter) and granularity (side angle scatter). Results were expressed as the percentage and absolute numbers of each lymphocyte population. The gating definition was based on fluorescence minus one (FMO) controls where cells are stained with all antibodies excluding the one of the interest for gating (see Supplementary Fig. 1) [47].2.9ELISPOT assayThe number of immunoglobulin-secreting cells (ISC) was determined in a modified version of Elisa spot assay described by Czerkinsky et al. [24]. Briefly, 96-well flat bottom microtitre plates (NUNC) were coated with anti-Ig heavy-chain specific antibody diluted in K1K2 buffer (pH 5.0). Spleen or bone marrow cells were harvested and plated at concentration of 50.000 cells in the first wells and submitted to serial dilutions (1:2) until 250 cells in final well. Plates were incubated at 37°C in 5% CO2 humidified cell chamber for 5h. The cells were then lysed with distilled water and the plates were washed with PBS to remove cell debris. Subsequently the plates were incubated for 2h with biotin-conjugated antibodies to mouse Ig heavy and light, μ, γ or α chains (Southern Biotechnology) at concentration of 1μg/ml in PBS-Casein 2%. Alkaline-phosphatase (AP) conjugated streptavidin were used for incubation before subsequent revelation. The plates were washed with PBS-Tween 0.05%, after which 100μl of substrate solution containing 5-bromo-4-chloro-3-indolylphosphate (BCIP) in 1mM AMP buffer (2-amino-2methyl-1-propanolol). The plates were incubated at 37°C until blue spots were visible, which were counted under a microscope (5-fold amplification).2.10Statistical methodsStandard error of the mean (SEM) and P values were determined using Prism™ software (GraphPad Software, Inc.). P values were calculated using appropriate 2-tailed t test or ANOVA analysis followed by Tukey post-test with a 95% confidence interval. The results are expressed as means±standard error of the mean (SEM).3Results3.1Inhibition of anti-ova antibodies in tolerant mice was parallel to augmented serum levels of total IgM and equal levels of total IgA as compared to immunized miceTo compare the activation profile of lymphocytes from tolerant and immunized animals, C57BL/6 mice were rendered tolerant to OVA by oral administration in the drinking bottle of an egg-white solution for 3days. Seven days after oral treatment mice were i.p. immunized with OVA in Al(OH)3 and 14days thereafter they received a booster immunization with OVA in saline. Animals not pre-treated with oral OVA were considered immunized mice. Control group consists of non-manipulated animals (Fig. 1A). Serum titers of specific antibodies (anti-OVA) were measured to evaluate tolerance induction. As shown in Fig 1B, immunized animals presented increasing levels of specific anti-OVA antibody responses while the levels of tolerant mice remained low, closer to those from control, naive mice. The formation of anti-ova antibodies of all isotypes, IgG1, IgE, IgM and IgA in tolerant mice was inhibited (Fig. 1C–F). These data confirm the tolerant status of the mice by previously feeding ovalbumin. Next, we analyzed the serum levels of total (non-specific) IgG1, IgE, IgM and IgA. Total (non-specific) IgG1 and IgE levels in tolerant mice were lower as seen with specific antibodies (Fig 2A and B). However higher levels of IgM (Fig 2C) and similar levels of IgA (Fig 2D) were found in tolerant mice as compared to immunized mice.3.2Tolerant and immunized mice present similar pattern of lymphocyte activation after booster immunization with ovalbuminAs the best time to verify the inhibition of specific activity in tolerant animals is after booster immunization we decided to analyze phenotype spleen lymphocyte at this time point. We examined the presence of activated lymphocyte and regulatory T cells (Fig. 3).Analysis of lymphocyte activation was unable to distinguish tolerant from immunized mice; both presented same frequency and number of CD4+CD69+T cells (Fig. 3A and B) as well as CD19+CD69+B cells (Fig. 3C and D). Tolerant mice showed higher frequency of CD4+CD44hi effector T cell but absolute numbers were similar when compared to immunized mice (Fig. 3E and F). Interestingly, immunized and tolerant mice also displayed similar frequency and number of regulatory T cells: CD4+CD25+CD45RBlow (Fig. 3G and H) and CD4+CD25+LAP+T cell at this time (Fig. 3I and J). Although no differences were found regarding the frequency of CD4+Foxp3+T cells between groups, tolerant and immunized mice showed elevated numbers of these cells as compared to control mice.We also measured the cytokines levels in cultured spleen cells by quantitative real time polymerase chain reaction (qPCR). Immunized mice had more IL-4 and IFN-γ (Fig. 4B and C) and tolerant mice presented higher levels of TGF-β (Fig. 4E). Tolerant mice presented IL-2 levels similar to those of immunized mice (Fig. 4A). Interestingly, both immunized and tolerant mice showed similar higher levels of IL-10 (Fig. 4D). These results indicate that in spite of specific inhibition of lymphocyte activity, tolerant mice also showed similar levels of global lymphocyte activity. Nevertheless this activity has a different outcome mediated by distinctive cytokine profile.3.3Tolerant mice displayed higher numbers of immunoglobulin secreting cells (ISC) in spleen and bone marrowWe assessed the production of immunoglobulin-secreting cells (ISC) in spleen (Fig. 5A–E) and bone marrow (Fig. 5F–J) of mice after priming and booster immunization by ELISPOT. After priming immunization, with antigen in adjuvant, tolerant and immunized mice presented similar number of ISC either in spleen (Fig. 5A) or bone marrow (Fig. 5F). After booster immunization, tolerant mice have higher number of ISC both in spleen (Fig. 5B) and in bone marrow (Fig. 5G) compared to immunized mice. Moreover immunoglobulin class composition of these ISC differed between tolerant and immunized mice. In the spleen, immunized mice had more IgG-secreting ISC (Fig. 5C), while tolerant mice had more IgA- and IgM-secreting cells (Fig 5D and E). As compared to immunized mice, the bone marrow of tolerant mice had higher number of IgG- and IgM-secreting cells (Fig. 5H and I). These results reinforce the preceding one showing elevated serum levels of total IgM and IgA in tolerant mice (Fig. 2C and D) and point to a high functional activity of B cells in tolerant mice, in spite of specific antibody inhibition.3.4Activated and regulatory T cells emerged with different kinetics in the spleen of orally tolerant miceAs we failed to distinguish tolerant from immunized mice in their percentage of Treg or activated lymphocyte in spleen 14days after booster immunization (Fig. 3), we analyzed the kinetics of appearance of these cells in tolerant and immunized mice after priming immunization, with OVA in Al(OH)3. This immunization is known to participate in the induction of oral tolerance and has an ideal time point following oral antigen, after which it is not possible to cause a tolerant state. In this kinetics we included a time point (time 0 in graphs – Fig. 7) concerning the tolerant mice without parenteral immunization, 7days after feeding. This would denote the effect of antigen feeding alone in lymphocyte populations in spleen and guide us to better description of what happens to lymphocytes after immunization. The others kinetic time points were at days 1, 2, 4 and 7 after immunization.Interestingly, orally tolerant mice already have higher frequency (Fig. 6A) and absolute number (Fig. 6B) of CD4+CD69+T cells in spleen compared to control, not manipulated mice, 7days after antigen feeding. Additionally, CD4+CD25+LAP+Treg cell number (but not frequency) were elevated in tolerant mice after feeding, even before immunization (Fig. 6H). One day after immunization these cells remained higher in tolerant mice. CD4+Foxp3+Treg cells appeared earlier in tolerant mice, at day 2 (Fig. 6I and J). Interestingly, we observed an increase in both regulatory cell types frequencies (CD4+Foxp3+and CD4+CD25+LAP+) in immunized mice at day 4 (Fig. 6G and I). While LAP+Treg cells increased similarly in tolerant and immunized mice regarding cell frequencies (Fig. 6G), the absolute number of these cells remained higher in tolerant mice (Fig. 6H). On the contrary, the frequencies and cell number of CD4+Foxp3+Treg cells were higher in immunized mice at the end of the kinetics (Fig 6I and J).The kinetics of recently activated B cells (CD19+CD69+) number did not show differences between tolerant and immunized mice. But the frequencies of these cells augmented just after immunization in both mice compared to control (Fig. 6C). CD4+CD44hi T cells of tolerant mice have increased in both frequency and number just after immunization, remaining higher than that of immunized mice until the fourth day (Fig. 6E and F).3.5Orally tolerant mice presented earlier production of regulatory cytokines and transient production of effector cytokines after primingDuring the kinetics we also analyzed cytokine production through their mRNA expression level assessed by quantitative polymerase chain reaction (qPCR) (Fig. 7). The major differences occurred at the end of the kinetics, when immunized animals showed higher levels of IL-2, IL-4, IFN-γ than tolerant mice (Fig. 7A–C). Immunized and tolerant mice had similar levels of IFN- γ at day 7 (Fig. 7C). Interestingly, immunized mice also presented higher levels of IL-10 and TGF-β than tolerant mice at day 7 (Fig. 7D and E). Nonetheless, some important differences were found in the early kinetics, between day 0 and 2, and we show these data on separate graphs (Fig. 7F–J). Tolerant mice presented higher levels of IL-2, IL-10 and TGF- β even before immunization, 7days after antigen feeding (Fig. 7F–J). After immunization there was a transient production of IFN-γ in tolerant mice on the first day (Fig. 7H) and another peak of IL-2 production at day 2 (Fig. 7F). IL-10 levels remained high in tolerant animals until second day after immunization (Fig. 7I). At this time, the first 2days of kinetics, immunized mice did not show not any increase in cytokine expression, indicating the protracted activation of their immune system compared to tolerant mice.4DiscussionThe vast majority of contacts of the organism with potentially immunogenic proteins occurs in the gut with food and microbiota antigens. Parenteral exposures to immunogens generally trigger an increase in specific antibody production and the development of immunological memory. These are considered to be basic elements of anti-infectious immunity. On the other hand, the consequences of oral exposures to immunogens are frequently described as inhibitory of further specific responsiveness. This has been repeatedly described as necessary to ensure unresponsiveness to harmless antigens from the diet and the gut microbiota [25].The specific suppression characteristic of “oral tolerance” has been described in cellular and molecular detail, but little is known about the global activities of the immune system underlying the physiological, daily contact with dietary antigens. Herein, we show that in spite of a reduction in specific antibody formation, tolerant mice have high frequencies of ISC in the spleen and the bone marrow. Secondly, in the spleen and bone marrow of tolerant mice T cells bearing both activated/memory and regulatory phenotypes emerge with a kinetics different from that of non-tolerant mice. Thus, the question we try to answer here is: how to conciliate this increased global activity with the inhibition of specific immune reactivity.In tolerant mice, splenic ISC cells expressed mainly IgA and IgM isotypes. In the bone marrow, both tolerant and immunized mice presented IgG- and IgM-secreting ISC, but these cells were more frequent in tolerant mice. Thus, although the number of activated B cells is similar in tolerant and immunized mice, they differ in the expression of Ig isotypes and the frequency of activated lymphocytes in systemic compartments. This phenomenon was not registered before.IgA is a predominant isotype at mucosal sites, and an association between oral tolerance and increased specific SIgA production was previously suggested, implying that systemic tolerance occurrs alongside with local immunization [26]. However, this is not an universal finding and secretory IgA antibodies responses have also been reported to be both normal or suppressed in tolerant mice [27–30]. In previous experiments from our laboratory, tolerant mice showed suppression of specific SIgA [29]. In the experiments reported here, we found no anti-ova SIgA after parenteral challenge either in tolerant or immunized mice. However, tolerant mice had lower specific serum IgA, as seen in others isotypes (Fig. 1F), but higher total serum IgA (Fig. 2D) consistently with a higher number of IgA-secreting cells in spleen (Fig. 5A). Production of non-specifc serum IgA in tolerant mice is compatible with the overall effects of oral tolerance. IgA has no inflammatory activity; it cannot fix complement, nor mediate the production of inflammatory cytokines [31]. This would assure the assimilation of the antigen, rather than triggering the inflammatory events which occur in immunization.Similarly, in tolerant mice specific IgM response was lower, but total IgM levels in serum were higher. Also after booster immunization tolerant mice had higher numbers of IgM-secreting cells both in spleen and bone marrow. IgM has been implicated in many physiological activities such as: clearance of apoptotic, tumor or senescent cells; cytokine actions; inflammation and in the regulation of B and T cells [32]; also, natural IgM antibodies modulate B and T cell activation [33]. Therefore, the increased production of IgM in spleen and bone marrow in tolerant mice may reflect changes in the physiologic activity of cells able to buffer inflammatory reactions triggered by parenteral immunization.Our results reinforce those of Bandeira et al. (1989) on transplantation allo-tolerance. In those experiments, mice were neonatally injected with allogeneic cells and received a skin allograft as adults; some accepted and some rejected the allograft; higher number of activated B and T cells were found in animals that accepted donor skin allograft [21]. These authors claimed that tolerance is an active phenomena and correlates with high levels of lymphocyte activation, rather than their inhibition.Many authors described systemic effects of oral tolerance that go beyond the limits of gut associated lymphoid tissue. For example, attempts to immunize tolerant animals with the tolerated antigen has profound inhibitory effects on immune responsiveness [34,35]. Carvalho and collaborators have shown that the injection of tolerated antigens may block a variety of immune phenomena such as: antibody responses [36] and DTH responses [37] to unrelated antigens; granuloma formation around Schistosoma mansoni eggs trapped in the lungs [38]; lethal GvH reactions [39]; the differentiation of eosinophils in the bone marrow [40] and inflammatory reactions triggered by injection of caraggenan [41]. These facts suggest that lymphocytes with regulatory properties, which are activated in mucosa, permeate the body and mediate anti-inflammatory influences when exposed to the specific antigen.Many subsets of regulatory T cells have been involved in oral tolerance: IL-10-producing Tr1 cells, TGF-β producing CD4+CD25+LAP+T cell and naturally arising Treg CD4+CD25+Foxp3+[42]. In the present work, tolerant mice showed higher expression of TGF-β but equal expression of IL-10, as compared to immunized mice. Interestingly, tolerant mice showed a high expression of IL-2 indicating a significant lymphocyte activation rates after booster immunization. This activation was qualitatively different from that of immunized mice, as tolerant animals did not express IL-4 and IFN-γ at this time.Antigen specific Tregs cells are observed in Peyer’s patches and mesenteric lymph nodes of the gut 48h after oral administration of antigen [43]. Additionally, antigen presenting cells (APC) that trigger this tolerogenic response exist constitutively in the GALT and can migrate to mesenteric lymph nodes in a CCR7-dependent manner and distal lymph nodes, such as inguinal lymph nodes, are isolated from this tolerogenic process [20]. The systemic effects of oral tolerance seems to depend on the mobility of antigen specific T cells that circulate the body after being activated in GALT by dietary antigens [44]. Nevertheless, priming with the antigen in adjuvant reinforces maintenance of oral tolerance [45,29].To examine systemic influences during oral tolerance induction we analyzed the kinetics of appearance of activated and regulatory lymphocytes in the spleen just after priming immunization and compared with the activated and regulatory profile in mice 7days after antigen feeding. Surprisingly, tolerant mice showed augmented number and frequencies of CD4+CD69+T cells after feeding (Fig. 6A and B) but after immunization the kinetic of these cells was similar in tolerant and immunized mice. This indicates that antigen feeding by itself, triggered their activation. However, primary immunization could not change the kinetics of CD4+CD69+lymphocyte frequency in tolerant and immunized mice. The immunization also induced similar degrees of expansion of CD19+CD69+ activated B cells frequencies in tolerant and immunized mice one day after immunization (Fig. 6C). However, no differences were seen in cell number kinetics (Fig. 6D).Two additional differences in the kinetics of tolerant and immunized mice were noted. The first is the rapid appearance of activated/memory CD4+CD44hi T cells of tolerant mice that remained elevated until day 4 after immunization (Fig. 7E and F). The second was the prompt expansion of regulatory T cells in tolerant mice soon after priming immunization. Importantly, regulatory CD4+T cells expressing LAP had already elevated numbers one week after feeding, remaining higher one day after immunization. CD4+Foxp3+Treg cells expanded after the second day in tolerant mice (Fig 7G–J). Additionally, immunized mice presented an delayed expansion of CD4+Foxp3+Treg cells, reaching a higher frequency and cell number at day 7. The LAP+ cells population also expanded in immunized mice but never exceeded that from tolerant animals (Fig. 7G and H).The rapid development of regulatory T cells in tolerant mice might be related to their preceding activation by the previous oral exposure to the antigen. This exposure activates gut lymphocytes, which then migrate systemically, altering further interactions with the antigen. In a mouse model of asthma using a transgenic mouse containing only one clone of CD4 T cells and one clone of B cells in a RAG−/− background, Mucida and coworkers also described an early expansion of Treg (CD4+CD25+CD45RBlow) in mesenteric lymph nodes of tolerant mice 4days after parenteral immunization [45]. In our study, we observed an expansion of CD4+CD25+Foxp3+T cells in the spleen of tolerant C57BL/6 mice two days after parenteral immunization. We also observed augmented Treg levels in immunized mice, but this occurred later. Tolerant mice presented increased levels of IL-2, IL-10 and TGF-β even before priming immunization (Fig. 7F–J) and another peak of IL-2 production two days after immunization. This might be related to the rapid induction of regulatory T cells in these animals. Additionally, tolerant mice showed a transient production of IFN-γ soon after priming but its expression is downregulated after booster immunization.In summary, a reduction in specific reactivity is the hallmark of tolerance, but few studies were concerned with what happens concomitantly with the non-specific “global” activity during tolerance induction. Herein, we show that tolerant mice have heightened lymphocyte activity, quantitatively similar to immunized mice, concomitantly with specific lymphocyte inhibition, and that there were also qualitative and temporal differences. Qualitatively, tolerant mice displayed widespread activation of B lymphocytes in systemic compartments, with more IgM and IgA-secreting cells in spleen and IgM and IgG-secreting cells in bone marrow, while IgG was more frequent in immunized mice. Temporally, tolerant mice showed an earlier expansion of regulatory T cells and a rapid reactivation of lymphocytes, as indicated by higher number of T cells with activated/memory phenotype, reaffirmed by the earlier and transient production of regulatory (IL-10, TGF-β) and effector (IL-2, IFN-γ) cytokines. Nonetheless, immunized animals also presented regulatory T cells (CD4+Foxp3+and CD4+LAP+) and regulatory cytokines (TGF-β and IL-10), but their protracted development might explain their inability to prevent the inflammatory outcome.The true nature and function of regulatory T cells is still open to discussion, and few studies have considered the role they could play in global immunological activity. Regulatory T cells might promote a more distributed lymphocyte activation in parallel with its known suppressive action on specific immunity. In support to this assumption, Winstead and colleagues recently demonstrated the role of regulatory T cells in driving clonal diversity of T cells during reconstitution from lymphopenia [46]. In this sense, oral tolerance (and other forms of tolerance) may be seen as a physiological non-inflammatory activity that induces specific unresponsiveness by privileging systemic activation of lymphocytes.AcknowledgmentsWe thank Ms. Ilda Marçal for her excellent technical assistance and Juliana Pfisterer for helping in qPCR experiments. This work was supported by research grants from FAPEMIG and CNPq (Brazil). Some of the authors are recipients of scholarships from CAPES (A.B.C.J.) and CNPq (A.C.G.S., B.C.H.) and fellowships from CNPq, Brazil (A.M.C.F.).Appendix ASupplementary dataSupplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cellimm.2012.12.004.Appendix ASupplementary dataSupplementary data 1Gating strategy for cytometry experiments. Spleen cells were harvested and submitted to flow cytometry analysis. The lymphocytes population was gated on the basis of relative size (forward light scatter) and granularity (side angle scatter). Depending on the antibodies pool cells were sequentially gated by individual staining (eg.CD4+ and CD69+) or by double staining (eg. CD4+CD25+) followed by single staining (eg. LAP+). The gating definition was based on fluorescence minus one (FMO) controls where cells are stained with all antibodies excluding the one of the interest for gating [47].Supplementary data 2Dot plots from results of Fig. 3. Mice received either OVA (tolerant) or tap water (immunized and control) seven days before priming immunization. Mice received a booster immunization 14 days after. Seven days after booster immunization mice were killed and their spleen were collected for phenotypical analysis of lymphocytes by flow cytometry. Control mice were sham immunized with saline. Cells were stained for CD4+CD69+ Tcells; CD19+CD69+ B cells; CD4+CD44hi T cells; CD4+CD25+CD45RBlow Treg cells; CD4+CD25+LAP+ Treg cells; CD4+Foxp3+ Treg.Supplementary data 3Dot plots from results of Fig. 7. Mice received oral OVA (Tolerant) or tap water (Immunized) prior to immunization with OVA in Al(OH)3, after which animals were killed at several intervals (1, 2, 4 and 7 days) – one group of tolerant animals were killed before immunization (Pre-0) – and spleen cells were harvested for flow cytometry analysis. The lymphocytes population was gated on the basis of relative size (forward light scatter) and granularity (side angle scatter). (A) CD4+CD69+ gated on CD4+ lymphocytes; (B) CD19+CD69+ gated on CD19+ lymphocytes; (C) CD4+CD44hi gated on lymphocytes; (D) CD4+CD25+LAP+ gated on CD4+CD25+ lymphocytes; (E) CD4+ Foxp3+ gated on lymphocytes.References[1]A.BesredkaDe. Sexiéme memoire de l’anaphylaxie latiqueAnn. Inst. Pasteur Lille231909166[2]H.G.WellsStudies on the chemistry of anaphylaxis III. Experiments with isolated proteins, especially those of the hen’s eggJ. Infect. Dis.91911147171[3]H.G.WellsT.B.OsborneThe biological reactions of the vegetable proteins I anaphylaxisJ. Infect. 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