Retinoic Acid and Polyriboinosinic:Polyribocytidylic Acid Stimulate Robust Anti-Tetanus Antibody Production while Differentially Regulating
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免疫学杂志 2005年第12期
Abstract
Retinoic acid (RA), a bioactive retinoid, and polyriboinosinic:polyribocytidylic acid (PIC) are known to promote immunity in vitamin A-deficient animals. In this study, we hypothesized that RA, PIC, and the combination can provide significant immunoadjuvant activity even in the vitamin A-adequate state. Six-week-old C57BL/6 mice were immunized with tetanus toxoid (TT) and treated with RA and/or PIC at priming in three independent studies of short and long duration. RA and PIC differentially regulated both primary and secondary anti-TT IgG isotypes, whereas the combination of RA + PIC stimulated the highest level of anti-TT IgG production and, concomitantly, a ratio of IgG1 to IgG2a similar to that of the control group. The regulation of Ab response was strongly associated with type 1/type 2 cytokine gene expression. Whereas RA reduced type 1 cytokines (IFN- and IL-12), PIC enhanced both type 1 and type 2 cytokines (IL-4 and IL-12) and cytokine-related transcription factors. Despite the presence of PIC, the IL-4:IFN- ratio was significantly elevated by RA. In addition, RA and/or PIC modulated NK/NKT cell populations and the level of expression of the costimulatory molecules CD80/CD86, evident 3 days after priming. Notably, the NKT:NK and CD80:CD86 ratios were correlated with the IL-4:IFN- ratio, indicative of multiple converging modes of regulation. Overall, RA, PIC, and RA + PIC rapidly and differentially shaped the anti-tetanus Ig response. The robust, durable, and proportionate increase in all anti-TT IgG isotypes induced by RA + PIC suggests that this combination is promising as a means to enhance the Ab response to TT and similar vaccines.
Introduction
Soon after its discovery, vitamin A was characterized as the anti-infective vitamin due to the observation that vitamin A-deficient animals succumbed to infections that vitamin A-adequate animals survived (1). In humans, vitamin A deficiency is now recognized as a highly significant risk factor associated with increased mortality in children and pregnant women (2, 3). Intervention studies in at-risk populations have clearly demonstrated that providing vitamin A to children, ranging from newborn to 5 years of age, decreases child mortality rates by an average of 23%, with a 50% reduction observed in some studies (4, 5). Furthermore, vitamin A supplementation has reduced measles-related mortality and the severity of several infectious diseases, including measles, diarrhea, malaria, and HIV infection (6, 7, 8, 9). These encouraging results have prompted the distribution of vitamin A to children in at-risk populations, sometimes in concert with immunization programs, including vaccinations against measles and tetanus (10, 11, 12).
The anti-infective effect of vitamin A is thought to be attributable to immune stimulation and/or regulation. Vitamin A and its transcriptionally active metabolite, retinoic acid (RA),3 have been shown to modulate several indices of innate and adaptive immunity, such as dendritic cell (DC) maturation, cytokine production, T and B cell activation and Ab responses, as well as mucosal immunity (1, 13, 14). Whereas most immunological research has addressed the effects of vitamin A and RA in remediating vitamin A deficiency, it is also important to evaluate vitamin A and/or RA in the normal state, not only because these nutrients may potentially be useful in strategies to improve vaccine efficiency, but because many of the recipients of current vitamin A distribution-immunization programs are not themselves vitamin A deficient (10). Therefore, understanding the consequences of vitamin A and its metabolite RA on the development of Ag-specific Ab responses can aid in their appropriate use in both clinical and public health settings.
Polyriboinosinic:polyribocytidylic acid (PIC), a synthetic double-stranded polyribonucleotide and a mimetic of dsRNA viruses, is known for its ability to induce type I/type II IFNs and other cytokines (15), increase antiviral and antitumor reactions in several models (16, 17, 18), and activate both innate and adaptive immunity (16, 19, 20, 21). Multiple mechanisms appear likely in that PIC has promoted DC maturation, stimulated NK cell cytotoxicity (20, 21, 22), and also increased Ag-specific total IgG and IgG isotypes (21). Besides immune effects of PIC alone, it is of interest that combinations of IFNs and retinoids have shown promise in cancer therapy, which may in part be due to their regulatory effects on the immune system (23).
Tetanus toxoid (TT) is a classical T cell-dependent Ag and a clinically important vaccine especially in young children and women of child-bearing age (24). Previously, we demonstrated that the combination of RA and PIC synergistically increased anti-TT Ab production in vitamin A-deficient rats and mice (25), and elevated primary anti-TT IgG response in vitamin A-adequate rats (26). However, little is yet known of the independent and combined effects of these agents on the production of IgG subtypes, memory responses to TT, and cellular and molecular markers of type 1/type 2 immunity. The aim of the present study was to evaluate RA and PIC, alone and in combination, as modulators of molecular and cellular aspects of immunity in nonimmunocompromised vitamin A-sufficient mice.
Materials and Methods
Statistical analysis
Data are reported as mean ± SE. The main effects of RA and PIC, and the interaction of RA and PIC were evaluated by two-way ANOVA. Differences among groups were determined using Fisher’s protected least significant difference test (SuperAnova; Abacus Concepts). When group variances were unequal, data were subjected to log10 or square-root transformation before statistical analysis. Simple linear regression was determined using the same software. A value of p < 0.05 was considered statistically significant.
Results
Primary anti-TT Ab production is enhanced by RA and PIC
Primary anti-TT IgM and IgG production was determined 7 and 10 days after priming, respectively. IgM was elevated only by PIC alone and RA + PIC, each about 4-fold (Fig. 1a). Whereas RA and PIC alone significantly increased IgG, the combination of RA + PIC increased anti-TT IgG levels >80-fold compared with the response of normal control mice (Fig. 1b). Surprisingly, RA and PIC differentially regulated anti-TT IgG isotypes (Fig. 1, c–e). RA alone selectively increased IgG1 and IgG2b, and elevated the IgG1/IgG2a ratio, an indicator of type 1/type 2 balance. PIC alone strongly increased all anti-TT IgG isotypes (IgG1, IgG2a, and IgG2b), while the IgG1/IgG2a ratio was not different from control. Compared with PIC alone, RA + PIC more potently increased ant-TT IgG1, but attenuated the induction of IgG2a, thereby maintaining the IgG1/IgG2a ratio similar to control group (Fig. 1f). Two-way ANOVA confirmed that RA was a positive regulator for IgG1 and IgG2b, but a negative regulator for IgG2a, whereas PIC was a positive regulator for all of the IgG isotypes (Fig. 1, c–e). Hence, RA and PIC cooperatively promoted a robust primary anti-TT IgG response, but differentially regulated IgG isotypes in vitamin A-sufficient mice.
Type 1/Type 2 cytokines and Th1/Th2-related gene expression are regulated by RA and PIC
Having observed that RA and PIC differentially regulated anti-TT IgG isotypes, we next asked whether type 1/type 2 cytokines, the key regulators of Ig isotype switching, were also regulated. The effects of RA and PIC on type 1/type 2 cytokine mRNA levels were evaluated in two independent studies: an 11-day study with reimmunization of TT with and without RA, PIC, or RA + PIC 24 h before spleens were collected, and a 3-day study to examine effects in the early stage of priming. On day 11, RA alone selectively reduced the mRNA expression of type 1 cytokine genes (IFN- and IL-12; Fig. 2, a and b), and elevated the ratio of IL-4 to IFN- (IL-4/IFN-), a commonly used index of the balance of type1/type 2 responses (Fig. 2, a, b, and e). PIC alone significantly enhanced both type 1 cytokines (IFN- and IL-12) and type 2 cytokines (IL-4 and IL-10; Fig. 2, c and d) without altering the ratio of IL-4 to IFN- compared with the ratio in the control group. The combination of RA + PIC abrogated the induction of IFN- and IL-12 by PIC (RA + PIC < PIC), thereby elevating the ratio of IL-4 to IFN- (Fig. 2e). Moreover, RA and PIC also regulated several transcriptional factors and receptors involved in type 1/type 2 (Th1/Th2) responses. T-bet and GATA-3 are two major transcriptional factors essential for Th1 and Th2 differentiation, respectively (31). IFN regulatory factor-1 is such a factor involved in Th1 differentiation. IL-12R2, a subunit of IL-12R, is required for IL-12-induced Th1 differentiation (31, 32). Overall, RA slightly suppressed Th1-related gene expression, while PIC alone enhanced both Th1- and Th2-related gene expression, especially IFN regulatory factor-1 (IRF-1), which was significantly induced by PIC. The combination of RA + PIC tended to attenuate the induction of Th1-related genes by PIC (Table I). Despite weaker regulation of Th1/Th2-related transcription factors than cytokines, RA was still a negative regulator for each of these Th1-related genes, as shown by two-way ANOVA, whereas PIC was a positive regulator for both Th1- and Th2-related genes, patterns consistent with the regulation of type 1/type 2 cytokines.
Regression analysis indicated that anti-TT IgG1 titers were positively correlated with IL-4 (r = 0.47, p < 0.01) and IL-10 (r = 0.39, p < 0.05), while anti-TT IgG2a titers were highly correlated with the level of IL-12 (r = 0.60, p < 0.01). Interestingly, IgG2b titer was correlated with both IL-4 (r = 0.43, p < 0.05) and IL-12 (r = 0.58, p < 0.01). These data imply that RA and/or PIC in vivo differentially modulated type 1/type 2 responses, which in turn contributed to the regulation of anti-TT Ab response and IgG isotypes.
To further determine whether RA and PIC could regulate type 1/type 2 cytokines in the early stage of response to immunization (before primary Ab response and before or during Ig isotype switching), we also measured IL-4 and IFN- mRNA levels on day 3 after priming (Fig. 3). PIC alone selectively induced IFN- mRNA (Fig. 3a), whereas RA alone only slightly enhanced IL-4 mRNA (Fig. 3b). The combination of RA + PIC significantly increased IL-4, while it abrogated the induction of IFN-. In consequence, the ratio of IL-4:IFN- was significantly increased in mice that received RA + PIC compared with PIC alone (Fig. 3c). Two-way ANOVA confirmed that PIC was a positive regulator for IFN-, and RA was a positive regulator for IL-4, but a negative regulator for IFN-. Thus, RA and PIC were able to manipulate the expression of type 1/type 2 cytokine genes in the early stage of the response to immunization.
Lymphocyte populations and APC differentiation are regulated by RA and PIC
T cells, B cells, APCs, and NK/NKT cells are directly or indirectly involved in the regulation of thymus-dependent Ab production. Therefore, we wanted to determine whether RA and PIC modulate these cell types. The 3-day study showed that T and B cell populations were not affected by the treatments, except for CD8+ T cells, which were significantly reduced by PIC (Table II). RA and PIC differentially regulated NK and NKT cell populations. The proportion of NK cells (CD3–NK1.1+) was increased by PIC alone, whereas the proportion of NKT cells (CD3+NK1.1+) was increased by RA. Two-way ANOVA confirmed that RA was a positive regulator for the NKT cell population, and PIC was a positive regulator for the NK cell population (Table II).
Moreover, RA and PIC regulated populations and differentiation of APCs. We evaluated markers associated with three types of professional APC: DC (CD11c+), macrophages (CD11b+), and B cells (B220+). Treatment with RA and/or PIC for 3 days did not significantly affect B cell and DC populations (Table II and data not shown); however, RA and RA + PIC up-regulated CD11b, a major marker of macrophages (Table III). Regarding costimulatory molecules, the treatments did not affect MHC II and CD40 (data not shown). However, RA and RA + PIC induced the expression of the costimulatory molecule CD80 (B7-1), whereas PIC alone induced the expression of CD86 (B7-2). Two-way ANOVA indicated that RA was a positive regulator for CD80, but a negative regulator for CD86, while PIC was a positive regulator for CD86. Interestingly, RA and RA + PIC together up-regulated CD80 expression on CD11b+ cells, whereas PIC induced CD86 on CD11b– cells (Table III). These data indicated that RA and PIC affected different cell types and regulated the expression of CD80/CD86 differentially.
Simple regression showed that the ratio of IL-4 to IFN-, described above, was significantly correlated with both the ratio of NKT to NK cells (Fig. 4a) and the ratio of CD80 to CD86 (Fig. 4b). These results imply a possible functional association between the balance of NK/NKT cells, CD80/CD86 molecules, and type 1/type 2 cytokine responses.
Secondary anti-TT Ab production is enhanced by RA and/or PIC
Next, we wanted to determine whether the strong effects of RA and PIC on anti-TT Ab response observed in the primary response were durable, as would be crucial for a vaccine adjuvant. As for the primary response, RA and/or PIC given only with priming significantly enhanced secondary anti-TT IgG production (Fig. 5, a–d). Regarding anti-TT IgG isotypes, RA selectively enhanced IgG1 and IgG2b. Although PIC robustly increased all anti-TT IgG isotypes, it significantly reduced the IgG1 to IgG2a ratio as compared with the control group. The combination of RA + PIC stimulated the highest levels of IgG and IgG1, but suppressed induction of IgG2a by PIC, which in turn partially restored the ratio of IgG1 to IgG2a toward the control level (Fig. 5e).
Finally, the effects of RA and PIC on the secondary Ab response were further determined by measuring anti-TT ASCs. Consistent with the plasma Ab response, RA and PIC also differentially regulated splenic ASCs (Table IV). The number of ASCs was highly correlated with the corresponding plasma anti-TT IgG isotypes (IgG1, r = 0.727, p < 0.0001; IgG2a, r = 0.804, p < 0.0001; IgG2b, r = 0.73, p < 0.0001).
Discussion
The combination of RA and IFNs has been demonstrated to be an effective strategy for cancer chemoprevention and chemotherapy (33). However, the immunoregulatory effect of RA and IFNs combined has not yet been well characterized. DeCicco et al. (25, 30) reported that RA and PIC synergistically enhanced both primary and secondary anti-TT Ab responses in vitamin A-deficient rats, suggesting that RA and IFNs can interactively promote immune functions. However, whether RA and IFNs, especially in combination, can effectively modulate primary immune responses and promote long-term immunity in the healthy state had not been studied. Therefore, we evaluated the immunoregulatory effects of RA, PIC, and their combination in a model of normal immunocompetent mice. Several new findings resulted from these studies.
First, RA and/or PIC given at priming modulated important immune regulators within a few days of immunization. On day 3 after priming, RA and PIC differentially regulated mRNA levels of IFN- and IL-4, the signature cytokines of type 1 and type 2 immune responses, respectively. Previous studies showed that PIC selectively induces type 1 cytokines, such as IFN- and IL-12, while suppressing type 2 cytokines, such as IL-4 and IL-5 (21, 34). In contrast, RA suppressed Th1 cytokines, but enhanced Th2 cytokines in both vitamin A-deficient animals and in vitro culture (35, 36). In the present study, PIC significantly induced IFN- mRNA 3 days after priming. RA, in contrast, was a positive, albeit modest, regulator for IL-4, but a negative regulator for IFN-. In consequence, RA was a strong positive regulator of the ratio of IL-4 to IFN- gene expression. Therefore, RA, PIC, and their combination had already shaped the developing type 1/type 2 response within 3 days of immunization and treatment.
Retinoic acid and PIC also differentially regulated NK/NKT cell populations by 3 days after treatment and priming. NK cells are considered as an early source of IFN- (37), while NKT cells have been shown to secrete IL-4 and IL-10 and promote a Th2 (type 2) response (38, 39). In the present study, RA was a positive regulator for NKT cells, whereas PIC was a positive regulator for NK cells. The ratio of NKT cells to NK cells was positively correlated with the ratio of IL-4 to IFN- mRNAs, implying that RA and PIC treatments might regulate type 1/type 2 cytokines and anti-TT Ab response through modulating NK/NKT cell populations.
Furthermore, RA and PIC significantly affected APC characteristics within 3 days of immunization. PIC has been shown to induce DC maturation and expression of CD80 and CD86 (20). Several studies suggested that RA could regulate immune response by targeting APCs (40, 41). In the present study, RA and RA + PIC significantly induced expression of CD11b, consistent with our previous results in the human monocytic cell line THP-1 (29). Moreover, RA and PIC differentially regulated the expression of CD80 and CD86, the major costimulatory molecules for T cell activation. The regulation of CD80 and CD86 molecules was associated mostly with CD11b+ cells and CD11b– cells, respectively, suggesting the interesting and unexpected finding that these costimulatory molecules can be modulated individually. Although APC function was not assessed in the present study, the differences observed in cell markers imply that RA and PIC can modulate APC function, thereby affecting T cell activation as well as downstream Ab responses. Notably, the ratio of CD80/CD86 was positively correlated with the ratio of IL-4/IFN-. The differential regulation of CD80 and CD86 by RA and PIC is not readily explained at this time because the possibly distinct functions of CD80 and CD86 in type 1/type 2 responses have not yet been clarified. Kuchroo et al. (42) suggested that CD80 and CD86 are involved in the generation of Th1 and Th2 responses, respectively. Lang et al. (43) observed that CD86 was essential for both Th1 and Th2 responses, while CD80 provided a negative signal for Th1 response. Despite the present uncertainty about the individual roles of CD80 and CD86, the significant positive correlation observed between the ratio of IL-4 to IFN- and the ratio of CD80 to CD86 suggests that the early regulation of CD80 and CD86 molecules by RA/PIC might make a significant contribution to the ability of these treatments to rapidly modulate type 1/type2 cytokines.
Later, by days 10–12 after priming, the effects of RA and PIC on immune function became more dramatic as it was evident that RA and/or PIC robustly promoted the primary anti-TT IgG response. Compared with the control level of anti-TT IgG produced by normal mice, treated mice produced levels that were up to 80-fold higher. Moreover, RA and PIC differentially regulated anti-TT IgG isotypes. RA alone shifted anti-TT IgG production toward IgG1 and therefore elevated the ratio of IgG1/IgG2a. In contrast, PIC strongly boosted all of the IgG isotypes, without changing the ratio of IgG1 to IgG2a titers. Surprisingly, whereas RA + PIC synergistically enhanced IgG1, this combination attenuated IgG2a production as compared with PIC alone. Therefore, RA + PIC not only potently stimulated total anti-TT IgG production, but also kept the balance of IgG1/IgG2a Abs similar to that in control mice.
At the same time, the regulation of type 1/type 2 cytokines by RA and PIC was also very significant. PIC, which was expected to induce type 1 cytokines, significantly induced both type 1 and type 2 cytokines as well as Th1/Th2-related genes. The enhancement of type 2 cytokines by PIC was probably due to its ability to induce IFN- (19), which has been shown to reduce type 1 cytokines (e.g., IL-12), but increase type 2/regulatory cytokines (e.g., IL-10) as observed in treatment of multiple sclerosis (44, 45). Thus, PIC appears to be a relatively indiscriminant, but potent inducer of immune responses, with very broad inducing effects on both type 1 and type 2 immunity. The enhancement of type 1/type 2 cytokines by PIC was well correlated with the increased production of all anti-TT IgG isotypes. Oppositely, RA inhibited type 1 cytokines and Th1-related genes, confirming previous reports (35, 36). Interestingly, this inhibition occurred despite presence of PIC and was strongly correlated with the attenuation of IgG2a, suggesting that RA could abolish part of PIC-induced IgG2a production by suppressing type 1 cytokine expression. Although RA did not significantly induce type 2 cytokines, it consistently suppressed type 1 cytokines and therefore skewed the balance in the type 2 direction, which most likely enhanced the production of anti-TT IgG1. Nevertheless, RA combined with PIC manipulated type 1/type 2 cytokine expression, which in turn contributed to the enhancement of anti-TT Ab response and directed Ig isotype switching toward a nearly normal balance.
Because a strong memory response is a hallmark of successful vaccination, it was important to determine whether the immunoregulatory effects of RA and PIC were durable. Indeed, providing RA and PIC (only at the time of priming) greatly enhanced the secondary anti-TT IgG response. Consistent with increased plasma Ig isotypes, RA and PIC also up-regulated the number of splenic anti-TT ASCs. These data provided insight that RA and PIC enhanced secondary anti-TT IgG responses by regulating the clonal expansion of memory B cells and the differentiation of B cells into effector ASCs.
Formulation of vaccines with potent adjuvants is an important approach for improving vaccine efficiency. When incorporated into vaccines, adjuvants can accelerate, prolong, or enhance the quality of specific immune response to vaccine Ags. In the past decades, many adjuvants have been developed and tested; however, few of them are used for human vaccines because of potential toxicity and adverse effects (46). Therefore, developing effective adjuvants for human vaccines remains a challenge for the vaccine industry. The present study has demonstrated that RA and/or PIC treatments can robustly and durably enhance anti-TT Ab response in vitamin A-sufficient mice, suggesting that a simple nutritional intervention, RA, coupled with PIC can effectively improve vaccine performance. This outcome appears to involve multiple mechanisms, including early regulation of NK/NKT cell and APC populations and shaping of type 1/type 2 cytokine gene expression. Compared with RA or PIC alone, RA + PIC not only was more potent in increasing the TT-specific IgG response, but also maintained the balance of IgG1/IgG2a. Thus, RA + PIC may serve as a promising strategy for increasing vaccine efficiency in not only vitamin A-deficient, but also in healthy populations.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Financial support was provided by National Institutes of Health Grant DK-41479 and funds from the Dorothy Foehr Huck chair.
2 Address correspondence and reprint requests to Dr. A. Catharine Ross, Department of Nutritional Sciences, Pennsylvania State University, 126-S Henderson Building, University Park, PA 16802. E-mail address: acr6{at}psu.edu
3 Abbreviations used in this paper: RA, retinoic acid; ASC, Ab-secreting cell; DC, dendritic cell; PIC, polyriboinosinic:polyribocytidylic acid; TT, tetanus toxoid.
Received for publication February 14, 2005. Accepted for publication March 29, 2005.
References
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Hussey, G. D., M. Klein. 1990. A randomized, controlled trial of vitamin A in children with severe measles. N. Engl. J. Med. 323: 160-164.
Barreto, M. L., L. M. Santos, A. M. Assis, M. P. Araujo, G. G. Farenzena, P. A. Santos, R. L. Fiaccone. 1994. Effect of vitamin A supplementation on diarrhoea and acute lower-respiratory-tract infections in young children in Brazil. Lancet 344: 228-231.(Yifan Ma*, Qiuyan Chen an)
Retinoic acid (RA), a bioactive retinoid, and polyriboinosinic:polyribocytidylic acid (PIC) are known to promote immunity in vitamin A-deficient animals. In this study, we hypothesized that RA, PIC, and the combination can provide significant immunoadjuvant activity even in the vitamin A-adequate state. Six-week-old C57BL/6 mice were immunized with tetanus toxoid (TT) and treated with RA and/or PIC at priming in three independent studies of short and long duration. RA and PIC differentially regulated both primary and secondary anti-TT IgG isotypes, whereas the combination of RA + PIC stimulated the highest level of anti-TT IgG production and, concomitantly, a ratio of IgG1 to IgG2a similar to that of the control group. The regulation of Ab response was strongly associated with type 1/type 2 cytokine gene expression. Whereas RA reduced type 1 cytokines (IFN- and IL-12), PIC enhanced both type 1 and type 2 cytokines (IL-4 and IL-12) and cytokine-related transcription factors. Despite the presence of PIC, the IL-4:IFN- ratio was significantly elevated by RA. In addition, RA and/or PIC modulated NK/NKT cell populations and the level of expression of the costimulatory molecules CD80/CD86, evident 3 days after priming. Notably, the NKT:NK and CD80:CD86 ratios were correlated with the IL-4:IFN- ratio, indicative of multiple converging modes of regulation. Overall, RA, PIC, and RA + PIC rapidly and differentially shaped the anti-tetanus Ig response. The robust, durable, and proportionate increase in all anti-TT IgG isotypes induced by RA + PIC suggests that this combination is promising as a means to enhance the Ab response to TT and similar vaccines.
Introduction
Soon after its discovery, vitamin A was characterized as the anti-infective vitamin due to the observation that vitamin A-deficient animals succumbed to infections that vitamin A-adequate animals survived (1). In humans, vitamin A deficiency is now recognized as a highly significant risk factor associated with increased mortality in children and pregnant women (2, 3). Intervention studies in at-risk populations have clearly demonstrated that providing vitamin A to children, ranging from newborn to 5 years of age, decreases child mortality rates by an average of 23%, with a 50% reduction observed in some studies (4, 5). Furthermore, vitamin A supplementation has reduced measles-related mortality and the severity of several infectious diseases, including measles, diarrhea, malaria, and HIV infection (6, 7, 8, 9). These encouraging results have prompted the distribution of vitamin A to children in at-risk populations, sometimes in concert with immunization programs, including vaccinations against measles and tetanus (10, 11, 12).
The anti-infective effect of vitamin A is thought to be attributable to immune stimulation and/or regulation. Vitamin A and its transcriptionally active metabolite, retinoic acid (RA),3 have been shown to modulate several indices of innate and adaptive immunity, such as dendritic cell (DC) maturation, cytokine production, T and B cell activation and Ab responses, as well as mucosal immunity (1, 13, 14). Whereas most immunological research has addressed the effects of vitamin A and RA in remediating vitamin A deficiency, it is also important to evaluate vitamin A and/or RA in the normal state, not only because these nutrients may potentially be useful in strategies to improve vaccine efficiency, but because many of the recipients of current vitamin A distribution-immunization programs are not themselves vitamin A deficient (10). Therefore, understanding the consequences of vitamin A and its metabolite RA on the development of Ag-specific Ab responses can aid in their appropriate use in both clinical and public health settings.
Polyriboinosinic:polyribocytidylic acid (PIC), a synthetic double-stranded polyribonucleotide and a mimetic of dsRNA viruses, is known for its ability to induce type I/type II IFNs and other cytokines (15), increase antiviral and antitumor reactions in several models (16, 17, 18), and activate both innate and adaptive immunity (16, 19, 20, 21). Multiple mechanisms appear likely in that PIC has promoted DC maturation, stimulated NK cell cytotoxicity (20, 21, 22), and also increased Ag-specific total IgG and IgG isotypes (21). Besides immune effects of PIC alone, it is of interest that combinations of IFNs and retinoids have shown promise in cancer therapy, which may in part be due to their regulatory effects on the immune system (23).
Tetanus toxoid (TT) is a classical T cell-dependent Ag and a clinically important vaccine especially in young children and women of child-bearing age (24). Previously, we demonstrated that the combination of RA and PIC synergistically increased anti-TT Ab production in vitamin A-deficient rats and mice (25), and elevated primary anti-TT IgG response in vitamin A-adequate rats (26). However, little is yet known of the independent and combined effects of these agents on the production of IgG subtypes, memory responses to TT, and cellular and molecular markers of type 1/type 2 immunity. The aim of the present study was to evaluate RA and PIC, alone and in combination, as modulators of molecular and cellular aspects of immunity in nonimmunocompromised vitamin A-sufficient mice.
Materials and Methods
Statistical analysis
Data are reported as mean ± SE. The main effects of RA and PIC, and the interaction of RA and PIC were evaluated by two-way ANOVA. Differences among groups were determined using Fisher’s protected least significant difference test (SuperAnova; Abacus Concepts). When group variances were unequal, data were subjected to log10 or square-root transformation before statistical analysis. Simple linear regression was determined using the same software. A value of p < 0.05 was considered statistically significant.
Results
Primary anti-TT Ab production is enhanced by RA and PIC
Primary anti-TT IgM and IgG production was determined 7 and 10 days after priming, respectively. IgM was elevated only by PIC alone and RA + PIC, each about 4-fold (Fig. 1a). Whereas RA and PIC alone significantly increased IgG, the combination of RA + PIC increased anti-TT IgG levels >80-fold compared with the response of normal control mice (Fig. 1b). Surprisingly, RA and PIC differentially regulated anti-TT IgG isotypes (Fig. 1, c–e). RA alone selectively increased IgG1 and IgG2b, and elevated the IgG1/IgG2a ratio, an indicator of type 1/type 2 balance. PIC alone strongly increased all anti-TT IgG isotypes (IgG1, IgG2a, and IgG2b), while the IgG1/IgG2a ratio was not different from control. Compared with PIC alone, RA + PIC more potently increased ant-TT IgG1, but attenuated the induction of IgG2a, thereby maintaining the IgG1/IgG2a ratio similar to control group (Fig. 1f). Two-way ANOVA confirmed that RA was a positive regulator for IgG1 and IgG2b, but a negative regulator for IgG2a, whereas PIC was a positive regulator for all of the IgG isotypes (Fig. 1, c–e). Hence, RA and PIC cooperatively promoted a robust primary anti-TT IgG response, but differentially regulated IgG isotypes in vitamin A-sufficient mice.
Type 1/Type 2 cytokines and Th1/Th2-related gene expression are regulated by RA and PIC
Having observed that RA and PIC differentially regulated anti-TT IgG isotypes, we next asked whether type 1/type 2 cytokines, the key regulators of Ig isotype switching, were also regulated. The effects of RA and PIC on type 1/type 2 cytokine mRNA levels were evaluated in two independent studies: an 11-day study with reimmunization of TT with and without RA, PIC, or RA + PIC 24 h before spleens were collected, and a 3-day study to examine effects in the early stage of priming. On day 11, RA alone selectively reduced the mRNA expression of type 1 cytokine genes (IFN- and IL-12; Fig. 2, a and b), and elevated the ratio of IL-4 to IFN- (IL-4/IFN-), a commonly used index of the balance of type1/type 2 responses (Fig. 2, a, b, and e). PIC alone significantly enhanced both type 1 cytokines (IFN- and IL-12) and type 2 cytokines (IL-4 and IL-10; Fig. 2, c and d) without altering the ratio of IL-4 to IFN- compared with the ratio in the control group. The combination of RA + PIC abrogated the induction of IFN- and IL-12 by PIC (RA + PIC < PIC), thereby elevating the ratio of IL-4 to IFN- (Fig. 2e). Moreover, RA and PIC also regulated several transcriptional factors and receptors involved in type 1/type 2 (Th1/Th2) responses. T-bet and GATA-3 are two major transcriptional factors essential for Th1 and Th2 differentiation, respectively (31). IFN regulatory factor-1 is such a factor involved in Th1 differentiation. IL-12R2, a subunit of IL-12R, is required for IL-12-induced Th1 differentiation (31, 32). Overall, RA slightly suppressed Th1-related gene expression, while PIC alone enhanced both Th1- and Th2-related gene expression, especially IFN regulatory factor-1 (IRF-1), which was significantly induced by PIC. The combination of RA + PIC tended to attenuate the induction of Th1-related genes by PIC (Table I). Despite weaker regulation of Th1/Th2-related transcription factors than cytokines, RA was still a negative regulator for each of these Th1-related genes, as shown by two-way ANOVA, whereas PIC was a positive regulator for both Th1- and Th2-related genes, patterns consistent with the regulation of type 1/type 2 cytokines.
Regression analysis indicated that anti-TT IgG1 titers were positively correlated with IL-4 (r = 0.47, p < 0.01) and IL-10 (r = 0.39, p < 0.05), while anti-TT IgG2a titers were highly correlated with the level of IL-12 (r = 0.60, p < 0.01). Interestingly, IgG2b titer was correlated with both IL-4 (r = 0.43, p < 0.05) and IL-12 (r = 0.58, p < 0.01). These data imply that RA and/or PIC in vivo differentially modulated type 1/type 2 responses, which in turn contributed to the regulation of anti-TT Ab response and IgG isotypes.
To further determine whether RA and PIC could regulate type 1/type 2 cytokines in the early stage of response to immunization (before primary Ab response and before or during Ig isotype switching), we also measured IL-4 and IFN- mRNA levels on day 3 after priming (Fig. 3). PIC alone selectively induced IFN- mRNA (Fig. 3a), whereas RA alone only slightly enhanced IL-4 mRNA (Fig. 3b). The combination of RA + PIC significantly increased IL-4, while it abrogated the induction of IFN-. In consequence, the ratio of IL-4:IFN- was significantly increased in mice that received RA + PIC compared with PIC alone (Fig. 3c). Two-way ANOVA confirmed that PIC was a positive regulator for IFN-, and RA was a positive regulator for IL-4, but a negative regulator for IFN-. Thus, RA and PIC were able to manipulate the expression of type 1/type 2 cytokine genes in the early stage of the response to immunization.
Lymphocyte populations and APC differentiation are regulated by RA and PIC
T cells, B cells, APCs, and NK/NKT cells are directly or indirectly involved in the regulation of thymus-dependent Ab production. Therefore, we wanted to determine whether RA and PIC modulate these cell types. The 3-day study showed that T and B cell populations were not affected by the treatments, except for CD8+ T cells, which were significantly reduced by PIC (Table II). RA and PIC differentially regulated NK and NKT cell populations. The proportion of NK cells (CD3–NK1.1+) was increased by PIC alone, whereas the proportion of NKT cells (CD3+NK1.1+) was increased by RA. Two-way ANOVA confirmed that RA was a positive regulator for the NKT cell population, and PIC was a positive regulator for the NK cell population (Table II).
Moreover, RA and PIC regulated populations and differentiation of APCs. We evaluated markers associated with three types of professional APC: DC (CD11c+), macrophages (CD11b+), and B cells (B220+). Treatment with RA and/or PIC for 3 days did not significantly affect B cell and DC populations (Table II and data not shown); however, RA and RA + PIC up-regulated CD11b, a major marker of macrophages (Table III). Regarding costimulatory molecules, the treatments did not affect MHC II and CD40 (data not shown). However, RA and RA + PIC induced the expression of the costimulatory molecule CD80 (B7-1), whereas PIC alone induced the expression of CD86 (B7-2). Two-way ANOVA indicated that RA was a positive regulator for CD80, but a negative regulator for CD86, while PIC was a positive regulator for CD86. Interestingly, RA and RA + PIC together up-regulated CD80 expression on CD11b+ cells, whereas PIC induced CD86 on CD11b– cells (Table III). These data indicated that RA and PIC affected different cell types and regulated the expression of CD80/CD86 differentially.
Simple regression showed that the ratio of IL-4 to IFN-, described above, was significantly correlated with both the ratio of NKT to NK cells (Fig. 4a) and the ratio of CD80 to CD86 (Fig. 4b). These results imply a possible functional association between the balance of NK/NKT cells, CD80/CD86 molecules, and type 1/type 2 cytokine responses.
Secondary anti-TT Ab production is enhanced by RA and/or PIC
Next, we wanted to determine whether the strong effects of RA and PIC on anti-TT Ab response observed in the primary response were durable, as would be crucial for a vaccine adjuvant. As for the primary response, RA and/or PIC given only with priming significantly enhanced secondary anti-TT IgG production (Fig. 5, a–d). Regarding anti-TT IgG isotypes, RA selectively enhanced IgG1 and IgG2b. Although PIC robustly increased all anti-TT IgG isotypes, it significantly reduced the IgG1 to IgG2a ratio as compared with the control group. The combination of RA + PIC stimulated the highest levels of IgG and IgG1, but suppressed induction of IgG2a by PIC, which in turn partially restored the ratio of IgG1 to IgG2a toward the control level (Fig. 5e).
Finally, the effects of RA and PIC on the secondary Ab response were further determined by measuring anti-TT ASCs. Consistent with the plasma Ab response, RA and PIC also differentially regulated splenic ASCs (Table IV). The number of ASCs was highly correlated with the corresponding plasma anti-TT IgG isotypes (IgG1, r = 0.727, p < 0.0001; IgG2a, r = 0.804, p < 0.0001; IgG2b, r = 0.73, p < 0.0001).
Discussion
The combination of RA and IFNs has been demonstrated to be an effective strategy for cancer chemoprevention and chemotherapy (33). However, the immunoregulatory effect of RA and IFNs combined has not yet been well characterized. DeCicco et al. (25, 30) reported that RA and PIC synergistically enhanced both primary and secondary anti-TT Ab responses in vitamin A-deficient rats, suggesting that RA and IFNs can interactively promote immune functions. However, whether RA and IFNs, especially in combination, can effectively modulate primary immune responses and promote long-term immunity in the healthy state had not been studied. Therefore, we evaluated the immunoregulatory effects of RA, PIC, and their combination in a model of normal immunocompetent mice. Several new findings resulted from these studies.
First, RA and/or PIC given at priming modulated important immune regulators within a few days of immunization. On day 3 after priming, RA and PIC differentially regulated mRNA levels of IFN- and IL-4, the signature cytokines of type 1 and type 2 immune responses, respectively. Previous studies showed that PIC selectively induces type 1 cytokines, such as IFN- and IL-12, while suppressing type 2 cytokines, such as IL-4 and IL-5 (21, 34). In contrast, RA suppressed Th1 cytokines, but enhanced Th2 cytokines in both vitamin A-deficient animals and in vitro culture (35, 36). In the present study, PIC significantly induced IFN- mRNA 3 days after priming. RA, in contrast, was a positive, albeit modest, regulator for IL-4, but a negative regulator for IFN-. In consequence, RA was a strong positive regulator of the ratio of IL-4 to IFN- gene expression. Therefore, RA, PIC, and their combination had already shaped the developing type 1/type 2 response within 3 days of immunization and treatment.
Retinoic acid and PIC also differentially regulated NK/NKT cell populations by 3 days after treatment and priming. NK cells are considered as an early source of IFN- (37), while NKT cells have been shown to secrete IL-4 and IL-10 and promote a Th2 (type 2) response (38, 39). In the present study, RA was a positive regulator for NKT cells, whereas PIC was a positive regulator for NK cells. The ratio of NKT cells to NK cells was positively correlated with the ratio of IL-4 to IFN- mRNAs, implying that RA and PIC treatments might regulate type 1/type 2 cytokines and anti-TT Ab response through modulating NK/NKT cell populations.
Furthermore, RA and PIC significantly affected APC characteristics within 3 days of immunization. PIC has been shown to induce DC maturation and expression of CD80 and CD86 (20). Several studies suggested that RA could regulate immune response by targeting APCs (40, 41). In the present study, RA and RA + PIC significantly induced expression of CD11b, consistent with our previous results in the human monocytic cell line THP-1 (29). Moreover, RA and PIC differentially regulated the expression of CD80 and CD86, the major costimulatory molecules for T cell activation. The regulation of CD80 and CD86 molecules was associated mostly with CD11b+ cells and CD11b– cells, respectively, suggesting the interesting and unexpected finding that these costimulatory molecules can be modulated individually. Although APC function was not assessed in the present study, the differences observed in cell markers imply that RA and PIC can modulate APC function, thereby affecting T cell activation as well as downstream Ab responses. Notably, the ratio of CD80/CD86 was positively correlated with the ratio of IL-4/IFN-. The differential regulation of CD80 and CD86 by RA and PIC is not readily explained at this time because the possibly distinct functions of CD80 and CD86 in type 1/type 2 responses have not yet been clarified. Kuchroo et al. (42) suggested that CD80 and CD86 are involved in the generation of Th1 and Th2 responses, respectively. Lang et al. (43) observed that CD86 was essential for both Th1 and Th2 responses, while CD80 provided a negative signal for Th1 response. Despite the present uncertainty about the individual roles of CD80 and CD86, the significant positive correlation observed between the ratio of IL-4 to IFN- and the ratio of CD80 to CD86 suggests that the early regulation of CD80 and CD86 molecules by RA/PIC might make a significant contribution to the ability of these treatments to rapidly modulate type 1/type2 cytokines.
Later, by days 10–12 after priming, the effects of RA and PIC on immune function became more dramatic as it was evident that RA and/or PIC robustly promoted the primary anti-TT IgG response. Compared with the control level of anti-TT IgG produced by normal mice, treated mice produced levels that were up to 80-fold higher. Moreover, RA and PIC differentially regulated anti-TT IgG isotypes. RA alone shifted anti-TT IgG production toward IgG1 and therefore elevated the ratio of IgG1/IgG2a. In contrast, PIC strongly boosted all of the IgG isotypes, without changing the ratio of IgG1 to IgG2a titers. Surprisingly, whereas RA + PIC synergistically enhanced IgG1, this combination attenuated IgG2a production as compared with PIC alone. Therefore, RA + PIC not only potently stimulated total anti-TT IgG production, but also kept the balance of IgG1/IgG2a Abs similar to that in control mice.
At the same time, the regulation of type 1/type 2 cytokines by RA and PIC was also very significant. PIC, which was expected to induce type 1 cytokines, significantly induced both type 1 and type 2 cytokines as well as Th1/Th2-related genes. The enhancement of type 2 cytokines by PIC was probably due to its ability to induce IFN- (19), which has been shown to reduce type 1 cytokines (e.g., IL-12), but increase type 2/regulatory cytokines (e.g., IL-10) as observed in treatment of multiple sclerosis (44, 45). Thus, PIC appears to be a relatively indiscriminant, but potent inducer of immune responses, with very broad inducing effects on both type 1 and type 2 immunity. The enhancement of type 1/type 2 cytokines by PIC was well correlated with the increased production of all anti-TT IgG isotypes. Oppositely, RA inhibited type 1 cytokines and Th1-related genes, confirming previous reports (35, 36). Interestingly, this inhibition occurred despite presence of PIC and was strongly correlated with the attenuation of IgG2a, suggesting that RA could abolish part of PIC-induced IgG2a production by suppressing type 1 cytokine expression. Although RA did not significantly induce type 2 cytokines, it consistently suppressed type 1 cytokines and therefore skewed the balance in the type 2 direction, which most likely enhanced the production of anti-TT IgG1. Nevertheless, RA combined with PIC manipulated type 1/type 2 cytokine expression, which in turn contributed to the enhancement of anti-TT Ab response and directed Ig isotype switching toward a nearly normal balance.
Because a strong memory response is a hallmark of successful vaccination, it was important to determine whether the immunoregulatory effects of RA and PIC were durable. Indeed, providing RA and PIC (only at the time of priming) greatly enhanced the secondary anti-TT IgG response. Consistent with increased plasma Ig isotypes, RA and PIC also up-regulated the number of splenic anti-TT ASCs. These data provided insight that RA and PIC enhanced secondary anti-TT IgG responses by regulating the clonal expansion of memory B cells and the differentiation of B cells into effector ASCs.
Formulation of vaccines with potent adjuvants is an important approach for improving vaccine efficiency. When incorporated into vaccines, adjuvants can accelerate, prolong, or enhance the quality of specific immune response to vaccine Ags. In the past decades, many adjuvants have been developed and tested; however, few of them are used for human vaccines because of potential toxicity and adverse effects (46). Therefore, developing effective adjuvants for human vaccines remains a challenge for the vaccine industry. The present study has demonstrated that RA and/or PIC treatments can robustly and durably enhance anti-TT Ab response in vitamin A-sufficient mice, suggesting that a simple nutritional intervention, RA, coupled with PIC can effectively improve vaccine performance. This outcome appears to involve multiple mechanisms, including early regulation of NK/NKT cell and APC populations and shaping of type 1/type 2 cytokine gene expression. Compared with RA or PIC alone, RA + PIC not only was more potent in increasing the TT-specific IgG response, but also maintained the balance of IgG1/IgG2a. Thus, RA + PIC may serve as a promising strategy for increasing vaccine efficiency in not only vitamin A-deficient, but also in healthy populations.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Financial support was provided by National Institutes of Health Grant DK-41479 and funds from the Dorothy Foehr Huck chair.
2 Address correspondence and reprint requests to Dr. A. Catharine Ross, Department of Nutritional Sciences, Pennsylvania State University, 126-S Henderson Building, University Park, PA 16802. E-mail address: acr6{at}psu.edu
3 Abbreviations used in this paper: RA, retinoic acid; ASC, Ab-secreting cell; DC, dendritic cell; PIC, polyriboinosinic:polyribocytidylic acid; TT, tetanus toxoid.
Received for publication February 14, 2005. Accepted for publication March 29, 2005.
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