In this study, we demonstrated that vaccination of IL-1R1 chimeric mice with LF + IL-1α induced significantly increased serum anti-LF antibody production in mice expressing IL-1R1 in either the stromal or bone marrow-derived cellular compartments. However, the responses induced by vaccination were not equal across the three chimeric groups; IL-1R1 KO→WT mice produced significantly less antigen-specific serum IgG, serum IgG subclasses, and LeTx-neutralizing antibodies than WT→KO and WT→WT mice. In addition, KO→WT mice had significantly lower antibody avidity than WT→KO and WT→WT mice. Therefore, maximal adjuvant activity of IL-1α after nasal immunization was maintained in mice expressing IL-1R1 on only hematopoietic cells, although T and B cells and mast cells were not required to be IL-1 responsive. Interestingly, DC expression of MyD88 was required for maximal IL-1α adjuvant activity. Conversely, IL-1α-induced innate cytokine and chemokine production required IL-1-responsive stromal cells, indicating that IL-1α-induced cytokine production was not required for IL-1α-induced anti-LF adaptive immune responses. In addition, although previous reports by our lab[16
] and others[70
] have suggested that CT may mediate its adjuvant activity by inducing IL-1, our data clearly show that CT maintains mucosal adjuvant activity in Il1r1−/−
mice, demonstrating that CT-induced IL-1 is not required for the mucosal adjuvant activity of CT.
Many studies have examined the early (0–48 hours) cytokine and chemokine profiles induced by specific adjuvants[25
], but few have compared these profiles with the adjuvant-induced adaptive immune response[48
]. Due to the ability of the chimeric model to assign adjuvant-induced responses to an adjuvant-responsive compartment, we examined the cytokine and chemokine profiles induced by nasal vaccination with IL-1α. Other studies have examined early serum cytokine production following i.m.,s.c., or i.p. administration of vaccine adjuvants to mice and have shown different profiles of cytokine/chemokine induction[25
]. In keeping with the results in the literature, we demonstrated an adjuvant-specific profile of cytokine production following intranasal vaccination, as IL-1α induced the production of IL-6, G-CSF, KC, and MCP-1. However, similar to the other studies, which observed peak cytokine production at 3–6 hr, IL-1α-induced cytokine/chemokine production peaked at 3 hr after vaccination. It is noteworthy that the timing of sample collection therefore affects which cytokines are detected. Although IL-1α induced some cytokines/chemokines that were similar to the other adjuvants described, its profile was distinct.
To our knowledge, no other group has directly investigated the manner in which IL-1α mediates its adjuvant activity, although others have evaluated the role of endogenous IL-1 in the induction of host immune responses. Such studies have demonstrated that IL-1β and IL-1R1 have activities on CD4+ T cell expansion [65
], and IL-1 is known to induce neutrophil migration [21
]. In our study, the neutrophil chemokine KC and the granulopoiesis cytokine G-CSF were produced primarily by the nonhematopoietic cells in the serum and nasal lavage. While G-CSF and KC remained increased in the nasal lavage of WT→KO mice, which did have anti-LF immune responses similar to WT→WT mice, their presence in KO→WT mice did not correlate with anti-LF adaptive immune responses equivalent to those measured in WT→WT mice. Although neutrophils have been shown to be important in the clearance of many pathogens [75
], they were recently demonstrated to inhibit antigen presentation and antigen-specific immune responses following vaccination with several antigens (i.e., HEL, OVA, or lysteriolysin O) and adjuvants (i.e., IFA, CFA, alum) when comparing neutrophil-depleted or G-CSFR−/− mice to WT [77
]. Therefore, it is unlikely that G-CSF, KC, or neutrophils play a large role in the nasal adjuvant activity of IL-1α.
Despite the significant induction of cytokines in serum and mucosal secretions after nasal immunization with IL-1α, our studies demonstrated that adjuvant-dependent early cytokine and chemokine production did not correlate with the induction of adaptive immune responses induced by vaccination. Our results are in agreement with others that adjuvant-induced cytokine production was not required for the induction of adaptive immunity. Sanders et al. recently demonstrated that, when vaccinating mice i.p. with flagellin, serum cytokine and chemokine induction and splenic DC maturation were largely impaired in TLR5−/− mice, though serum IgG responses to flagellin were not impaired[71
]. A recent study by Longhi et al. examined the ability of poly I:C to induce serum cytokine production when given i.p., and although they demonstrated increased production of six cytokines (IL-6, IL-12(p40), TNFα, IFNα, IFNβ, IFNγ), only IFNα and IFNβ correlated with antigen-specific CD4+ T cell development[74
]. Although we vaccinated mice i.n., our results and the published data from others suggest that many of the frequently used markers of innate immunity induction do not correlate with the induction of adaptive immune responses to the vaccine antigen. Therefore, studies of adjuvant mechanism should also focus on other areas of cooperation between the innate and adaptive immune response.
Several other studies have also used the bone marrow chimeric mouse model to evaluate the adaptive immune responses to many different antigens following codelivery with adjuvant. These studies have demonstrated that the induction of antigen-specific adaptive immune responsesis dependent upon both an adjuvant-responsive hematopoietic compartment and an adjuvant-responsive nonhematopoietic compartment[48
]. Two recent studies evaluated the requirements for TLR3 and MDA5 in poly I:C-induced antigen-specific IFNγ+ CD4+ T cell [74
] and CD8+ T cell[78
] responses when delivered i.p. to mice with HIV gag p24 or OVA, respectively. Unlike IL-1, which can only signal through IL-1R1, poly I:C signals through both MDA5 and TLR3, but it is unclear which receptor(s) mediate poly I:C-induced immune responses [78
]. Together, the two studies indicate that poly I:C requires signaling capabilities in both compartments: TLR3 on hematopoietic cells and MDA5 on stromal cells. Remarkably, all of these studies used different routes of challenge/immunization but still demonstrated the requirement for antigen/adjuvant responsiveness in both compartments. In contrast to those studies, our results demonstrate that only IL-1R1 expression on hematopoietic cells was sufficient to induce maximal adaptive immune responses. However, we primarily evaluated humoral immune responses and did not examine CTL or T cell proliferative responses, which may explain the discrepancy between the studies.
IL-1 has been shown to play a role in the development of Th immune responses, including increased production of Th2 cytokines in Il1r1−/−
mice when compared to WT mice [38
]. However, we did not observe any significant differences in antigen-specific Thcytokine production between chimeric mice vaccinated with LF + CT or LF + IL-1α. It is possible that this is a reflection of the route of delivery, as they delivered antigen subcutaneously and it has been shown that different routes of immunization induce different Th biases [79
]. Additionally, serum antigen-specific IgM, IgG1, and IgG2a titers have not been shown to significantly differ between WT and Il1r1−/−
]. Our results following immunization with LF + CT agree with the data of both studies, as we did not observe any significant differences in anti-LF IgG subclass production between chimeric groups. However, we did demonstrate that IL-1R1 chimerism significantly impacted the ability of IL-1α to enhance anti-LF IgG subclass production, indicating that the addition of exogenous IL-1 may have effects not seen when comparing the effects of endogenous levels of IL-1 in WT and Il1r1−/−
Other studies have demonstrated the importance of direct DC stimulation with the adjuvant CpG to induce the production of antigen-specific IgG2b and IgG2a/c after systemic immunization[36
]. Similar to the results in our present study, the production of proinflammatory cytokines by non-DCs was able to partially compensate for the lack of TLR ligand responsiveness, inducing DC maturation [68
] and, in one study, the production of significantly greater amounts of total IFNγ+ CD4+ T cells compared to Myd88−/−
]. These studies demonstrate that adjuvant-responsive DCs were required for maximal adjuvant activity after systemic immunization. Our results also demonstrate that the direct activation of DCs by IL-1α is required for maximal antibody production after nasal immunization using IL-1α as the adjuvant. Similar to the previously published studies, we observed significantly impaired production of antigen-specific IgG2b and IgG2c. However, further studies are required to determine the exact role of IL-1 stimulation of DCs in B cell antibody production following vaccination. Additionally, these studies do not indicate whether IL-1-responsive DCs are sufficient for IL-1 adjuvant activity. However, we must note that the CD11c-Cre mutation used to generate the DC-Myd88−/−
mice also results in a loss of MyD88 expression on alveolar macrophages. Nonetheless, due to the small volume used for vaccination (5 μl/nostril), it is unlikely that any of the vaccine formulation reached the alveolae[82
In summary, this study demonstrated that IL-1-responsive hematopoietic cells are sufficient for the maximal induction of antigen-specific adaptive immunity following vaccination with the adjuvant IL-1α and that the direct action of IL-1α on DCs is required for maximal adjuvant activity. Although the Il1r1+/+ stromal (radiation-resistant) cell compartment was able to produce cytokines/chemokines after nasal immunization with IL-1α and enhance the induction of antigen-specific adaptive responses, it was not sufficient for inducing maximal anti-LF adaptive immune responses in the absence of IL-1R1 on hematopoietic cells. Future studies are planned to optimize the nasal delivery of IL-1α-adjuvanted vaccines to maximize local adjuvant activity by direct activation of DCs.