The response of CD4+
T cells to protein antigens in vivo
can be dramatically enhanced by the administration of antigen in adjuvant. This increases T-cell expansion and prevents tolerance induction. Potent adjuvants enhance T-cell function by augmenting the clonal expansion of antigen-stimulated T cells by producing growth and/or survival signals (23
). They also regulate intracellular response pathways such as that controlled by nuclear factor-κB (NF-κB) and the Nalp3-containing inflammasome (25
). As a result, adjuvants have been frequently used in infectious and malignant vaccination strategies, attempts to ameliorate and/or augment immune responses in immunocompromised individuals and the elderly (23
), and attempts to use animals to model antigen-mediated chronic inflammatory human disorders. Previous studies from our laboratory and others have demonstrated that appropriately sized chitin fragments can induce innate inflammatory responses (6
). In these studies we add to our knowledge of the biology of chitin, adaptive immunity, adjuvants, and adjuvant responses by demonstrating that chitin is also a potent multifaceted adjuvant that induces adaptive Th2, Th1, and Th17 immune responses in the murine lung. These studies also demonstrate that the adjuvant properties of chitin are mediated by a pathway(s) that involves TLR-2, MyD88, and IL-17A and that the IL-17A response feeds back to inhibit IFN-γ production in this antigen-driven experimental system.
Innate immune activation is a critical step in the initiation of an adaptive immune response. As a result, activation of a class of innate pathogen receptors called pattern recognition receptors is a central feature of many adjuvant systems (26
). This can be readily appreciated in prior studies, which demonstrated that TLR activators, such as LPS, and NOD-like receptor activators, such as aluminum hydroxide, are effective immune adjuvants (15
). Our studies demonstrate that chitin is also an effective immune adjuvant and that the adjuvant properties of chitin are dependent on TLR-2 signaling. These findings are in accord with prior studies from our laboratory demonstrating that chitin is a PAMP that activates macrophages via TLR-2 (6
) and studies from others that highlight the ability of TLR-2 agonists to serve as effective adjuvants for Th2 responses (16
A number of lines of evidence suggest that adjuvants augment adaptive immune responses by stimulating the production of key proinflammatory cytokines. TNF and IL-1 enhance the expansion, persistence, and differentiation of responding CD4+
T cells. IL-1 and IL-6 also exhibit costimulatory effects on CD4+
), and IL-1 enhances the expression of CD40L and OX40, which are potent stimulators of CD4 cognate helper function (30
). Our studies demonstrate that chitin is a potent stimulator of the production of IL-17A. They also demonstrate that IL-17A plays an important role in the pathogenesis of the Th2 adjuvant effects of this polysaccharide. The finding that chitin is a potent adjuvant for IL-17A production is in accord with studies with a variety of other adjuvants, including poly (I:C) (31
), cholera toxin (32
), class I restricted peptides (33
), and Dectin-1 agonists (34
). They are also in accord with studies from our laboratory that demonstrated that chitin is a PAMP that activates IL-17A via TLR-2 (6
). The present studies, however, are the first to demonstrate that IL-17A plays a critical role in the adjuvant effects of chitin (or any other adjuvant) and thus plays a critical role in the pathogenesis of adaptive Th2 responses while inhibiting Th1 adaptive immunity. These findings add to studies from a number of other investigators highlighting the important role of IL-17A in Th2 responses and the ability of IL-17 to regulate Th1 cell differentiation (35
Interestingly, although IL-17A production was largely TLR-2 dependent, the contributions that each makes to the adjuvant effects of chitin were not identical. In the absence of TLR-2, both T-cell proliferation and cytokine elaboration were markedly diminished, suggesting the absence of T-cell priming to OVA. In contrast, in the absence of IL-17A, only cytokine production was affected. Th2 cytokines such as IL-5 and IL-13 were decreased, whereas the level of IFN-γ was augmented. In addition, T-cell proliferation was not altered. These results suggest that in IL-17 null mice, the priming of CD4+ T cells is still occurring; however, the polarization toward a Th2 profile is impaired.
Allergens comprise a vast collection of nonreplicating entities with diverse immunogenic potential capable of inducing specific immune-inflammatory responses (38
). How they accomplish these tasks is a subject of intense research. A great deal has been learned using OVA as a surrogate allergen. Because it is an archetypical innocuous antigen, conventional modeling has used aluminum hydroxide as an adjuvant. In contrast, it is now known that other antigens are immunogenic. This can be readily seen with house dust mite (HDM), which can induce sensitization and acute and chronic inflammation in the absence of an exogenous adjuvant (39
). HDM also allows sensitization to OVA antigen to occur through its “bystander” effect (38
). It is clear from these studies that some allergens have intrinsic adjuvant activity. The mechanisms that underlie these responses, however, are poorly defined. It is important to point out that chitin is a component of many antigen preparations, including HDM and cockroach. Because our data demonstrate that chitin is a potent multifaceted antigen, it is tempting to speculate that, in some cases, powerful antigens induce allergic sensitization via the adjuvant properties of this associated chitin.
Aluminum hydroxide is the most commonly used adjuvant in vaccines and models of human diseases (24
). This is nicely illustrated in the commonly used OVA mouse model of asthma-like Th2 inflammation. The present studies demonstrate that chitin is an important adjuvant, which, like aluminum hydroxide, induces adaptive Th2 immunity. Interestingly, in vivo
, the Th2 inflammatory responses that were induced by chitin and aluminum hydroxide were quantitatively and qualitatively similar. However, chitin and aluminum hydroxide also differed in a number of interesting ways. One of the most striking was the ability of chitin and the relative inability of aluminum hydroxide to stimulate IL-17A and Th1 responses. Another is the impressive dependence of chitin on TLR-2 versus the reported LPS TLR-4–dependence of aluminum hydroxide (25
) and the lesser role of TLR-2 in alum-driven responses. These observations suggest that chitin and aluminum hydroxide do not mediate their adjuvant effects via identical mechanisms. Recent studies from our laboratory demonstrated that chitin is a size-dependent PAMP that can also bind to Dectin-1 and activate Syk (6
). Interestingly, Dectin-1 agonists have been demonstrated to be adjuvants that augment Th1 and IL-17 responses (34
). This allows for the interesting hypothesis that the multifaceted adjuvant properties of chitin are the result of its ability to activate more than one innate immune receptor. It is also tempting to speculate that the adjuvant properties of chitin will vary with size and thus may vary after interaction with chitinases or metabolism via other pathways. Additional investigations will be required to address these possibilities.
In summary, our studies demonstrate that chitin is a multifaceted adjuvant that augments Th2, Th1, humoral, and IL-17 responses in vivo and in vitro. These novel effects were mediated by pathways involving TLR-2, MyD88, and IL-17A. It also shows that the induced IL-17A inhibits the Th1 stimulatory effects of this polysaccharide. These studies provide insights that are relevant to the pathogenesis of allergic and parasitic sensitization and help to clarify the relationships between Th2, Th1, and IL-17 antigen-induced tissue responses in diseases such as asthma, allergy, and parasitic infestation. Additional investigations of the mechanisms of these adjuvant effects and the consequences of interventions that alter them in asthma, allergy, and related disorders are warranted.