Poly-γ-glutamic acid (γPGA) derived from Bacillus subtilis is a promising biomaterial with potent immunomodulatory activities. Previously, it has been shown that such activities of γPGA are mediated by dendritic cells (DCs) in a TLR4-dependent manner. As DCs show considerable heterogeneity in cytoki...
Poly-γ-glutamic acid (γPGA) derived from Bacillus subtilis is a promising biomaterial with potent immunomodulatory activities. Previously, it has been shown that such activities of γPGA are mediated by dendritic cells (DCs) in a TLR4-dependent manner. As DCs show considerable heterogeneity in cytokine profiles, in chapter I, the experiments were set up to know whether NK1.1 expressing DCs (so called natural killer dendritic cells (NKDCs)) other than NK1.1- DCs contributed to anti-tumor immune responses upon γPGA stimulation. We found that γPGA stimulated NK1.1- DCs to produce IL12 but activated NKDCs to predominantly produce IFNγ. Moreover, NKDCs activated by γPGA displayed increased cytotoxicity against tumor cells via up-regulating the expression of cytolytic/pro-apoptotic molecules including perforin, TRAIL, and Fas ligand (FasL). Since mutual cross-talks between DCs and other innate lymphocytes (e.g., NK and NKT cells) can result in synergistic cellular Th1 immune responses, we investigated whether such interactions are required for optimal γPGA-induced Th1 immune responses. We showed that γPGA induced DC maturation to produce IL12 and consequently such DC-derived IL12 activated iNKT cells to release IFNγ, implying that DC/IL12/iNKT axis is the key pathway for generating an optimal Th1 response (e.g., enhanced anti-tumor immunity). On the other hand, we have previously demonstrated that before the onset of atopic dermatitis (AD) repeated γPGA treatment enhanced Th1 responses resulting in effective suppression of AD-like skin inflammation in NC/Nga mice, a murine model of AD. In addition, it has demonstrated that pro-inflammatory cytokines IFNγ and TNFα promoted apoptosis of basophils indispensable in papain-elicited allergic immune responses. Based on these previous studies, in chapter II, we investigated the cellular mechanism of γPGA-mediated suppression of allergic immune responses by employing the papain-elicited basophil-dependent model. We found that in vivo γPGA treatment dramatically reduced basophils which are totally dependent on TLR4, DC, and IL12. Importantly, the cellular target of γPGA/TLR4/DC/IL12 axis proved to be iNKT cells since iNKT cell-deficient CD1d KO mice had no effect on basophil depletion post-γPGA treatment. Furthermore, iNKT cell-mediated basophil reduction was mainly attributed to Th1 cytokines such as IFNγ and TNF, which is consistent with aforementioned studies. Thus, these results demonstrated that γPGA-induced iNKT cell polarization toward the Th1 phenotype induces apoptotic basophil depletion, leading to the suppression of Th2 immune responses. In summary, my PhD thesis studies have demonstrated that NKDCs and iNKT cells activated by DC-derived IL12 upon stimulation with γPGA were critically important as the source of IFNγ for Th1 responses at early time point post-γPGA injection and increased levels of IFNγ from these cells might lead to either enhance anti-tumor immune responses or prevent allergic responses via controlling Th1/Th2 imbalance.
Poly-γ-glutamic acid (γPGA) derived from Bacillus subtilis is a promising biomaterial with potent immunomodulatory activities. Previously, it has been shown that such activities of γPGA are mediated by dendritic cells (DCs) in a TLR4-dependent manner. As DCs show considerable heterogeneity in cytokine profiles, in chapter I, the experiments were set up to know whether NK1.1 expressing DCs (so called natural killer dendritic cells (NKDCs)) other than NK1.1- DCs contributed to anti-tumor immune responses upon γPGA stimulation. We found that γPGA stimulated NK1.1- DCs to produce IL12 but activated NKDCs to predominantly produce IFNγ. Moreover, NKDCs activated by γPGA displayed increased cytotoxicity against tumor cells via up-regulating the expression of cytolytic/pro-apoptotic molecules including perforin, TRAIL, and Fas ligand (FasL). Since mutual cross-talks between DCs and other innate lymphocytes (e.g., NK and NKT cells) can result in synergistic cellular Th1 immune responses, we investigated whether such interactions are required for optimal γPGA-induced Th1 immune responses. We showed that γPGA induced DC maturation to produce IL12 and consequently such DC-derived IL12 activated iNKT cells to release IFNγ, implying that DC/IL12/iNKT axis is the key pathway for generating an optimal Th1 response (e.g., enhanced anti-tumor immunity). On the other hand, we have previously demonstrated that before the onset of atopic dermatitis (AD) repeated γPGA treatment enhanced Th1 responses resulting in effective suppression of AD-like skin inflammation in NC/Nga mice, a murine model of AD. In addition, it has demonstrated that pro-inflammatory cytokines IFNγ and TNFα promoted apoptosis of basophils indispensable in papain-elicited allergic immune responses. Based on these previous studies, in chapter II, we investigated the cellular mechanism of γPGA-mediated suppression of allergic immune responses by employing the papain-elicited basophil-dependent model. We found that in vivo γPGA treatment dramatically reduced basophils which are totally dependent on TLR4, DC, and IL12. Importantly, the cellular target of γPGA/TLR4/DC/IL12 axis proved to be iNKT cells since iNKT cell-deficient CD1d KO mice had no effect on basophil depletion post-γPGA treatment. Furthermore, iNKT cell-mediated basophil reduction was mainly attributed to Th1 cytokines such as IFNγ and TNF, which is consistent with aforementioned studies. Thus, these results demonstrated that γPGA-induced iNKT cell polarization toward the Th1 phenotype induces apoptotic basophil depletion, leading to the suppression of Th2 immune responses. In summary, my PhD thesis studies have demonstrated that NKDCs and iNKT cells activated by DC-derived IL12 upon stimulation with γPGA were critically important as the source of IFNγ for Th1 responses at early time point post-γPGA injection and increased levels of IFNγ from these cells might lead to either enhance anti-tumor immune responses or prevent allergic responses via controlling Th1/Th2 imbalance.
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