Abstract:A flavonoid Astragalin (kaempferol-3-O-β-d-glucopyranoside, Ast) has several biological activities including anti-oxidant, anti-HIV, and anti-allergic effects. Nonetheless, its insolubility in hydrophilic solvents imposes restrictions on its therapeutic applications. In this study, we investigated the effects of water-soluble astragalin-galactoside (kaempferol-3-O-β-d-isomaltotrioside, Ast-Gal) on murine bone marrow-derived dendritic cell (DC) maturation and T helper (Th) cell-mediated immune responses. Ast-Gal significantly increased maturation and activation of DCs through the upregulation of surface markers, such as cluster of differentiation (CD)80, CD86, and Major histocompatibility complex (MHC) II in a dose-dependent manner, while Ast had little effects. Additionally, Ast-Gal-treated DCs markedly secreted immune-stimulating cytokines such as interleukin (IL)-1β, IL-6, and IL-12. Importantly, Ast-Gal strongly increased expression of IL-12, a polarizing cytokine of Th1 cells. In a co-culture system of DCs and CD4+ T cells, Ast-Gal-treated DCs preferentially differentiates naïve CD4+ T cells into Th1 cells. The addition of neutralizing IL-12 monoclonal antibody (mAb) to cultures of Ast-Gal-treated DCs and CD4+ T cells significantly decreased interferon (IFN)-γ production, thereby indicating that Ast-Gal-stimulated DCs enhance the Th1 response through IL-12 production by DCs. Injection with Ast-Gal-treated DCs in mice increased IFN-γ-secreting Th1 cell population. Collectively, these findings indicate that hydrophilically modified astragalin can enhance Th1-mediated immune responses via DCs and point to a possible application of water-soluble astragalin-galactoside as an immune adjuvant.Keywords: astragalin galactoside; hydrophilic modification; Th1 cell; dendritic cell; adjuvant
Autoimmune inflammation of the CNS in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), is mediated by antigen specific Th1 and Th17 cells . For many years integrin targeted blocking of T helper cell trafficking into the CNS has appeared to be an attractive approach to treat immunopathology in MS . In particular, monoclonal antibodies (natalizumab) to the α4 subunit of the integrin VLA-4 (α4β1 heterodimer) were successfully used to prevent the influx of immune cells into the CNS and to treat CNS autoimmunity . However, in experimental models it has been shown that distinct encephalitogenic T cell subsets vary in their equipment with VLA-4 . While Th1 cells maintain high amounts of VLA-4 expression, Th17 cells are low in VLA-4. As a consequence, blockade of VLA-4 is more efficient in preventing the recruitment of Th1 cells than of Th17 cells into the CNS parenchyma.
Similar to the murine system, neutralization of α4-integrins by natalizumab in humans mainly blocks CD4+ T cells from entering into the CNS compartment but spares CD8+ T cells [23, 24]. Yet, intracerebral reconstitution of CD4+ effector T helper cell responses has been proposed to be necessary for efficient clearance of various viruses from the CNS including JC virus . We wanted to dissect the essential features of an intracerebral T helper cell response that would be able to control viral infection. Thus, we combined a system of T cell conditional ablation of α4-integrin expression (CD4 Cre+x Itga4flox/flox mice, α4 CKO mice) with antibody mediated depletion of CD8+ T cells in order to investigate the differential contribution of Th1 cells vs Th17 cells to host protection in CNS infection in the absence of CTLs. To exclude possible alterations in priming of antigen specific T helper cell responses in the peripheral immune compartment of α4 CKO mice, we compared the fractions of antigen specific T cells on day 10 after subcutaneous immunization with MVA/CFA in the spleens of CD4 Cre-x Itga4flox/flox (wild type control) versus CD4 Cre+x Itga4flox/flox (α4 CKO) mice (Additional file 1: Figure S1). Upon ex vivo restimulation with I-Ab restricted VV epitopes, the fractions of antigen specific (CD40L+) CD4+ T cells and their cytokine profile were similar in wild type vs α4 CKO mice. Moreover, the anti-VV neutralizing serum response was equally effective in both groups on day 10 after immunization (Additional file 1: Figure S1). Thus, sensitization for adaptive cellular immune responses against VV in draining lymph nodes and spleen was not impaired by the lack of α4-integrins on T cells.
Next, we challenged MVA immune and CD8+ depleted wild type and α4 CKO mice with i.th. VV. In contrast to control littermates, α4 CKO mice rapidly lost weight and succumbed to infection (Figure 5A). Even in the complete absence of CD8+ T cells (Figure 5B), CD4+ effector T helper cells were protective in wild type mice but failed to control virus replication in the CNS of α4 CKO mice (Figure 5C). Recapitulating our observations with anti-α4 integrin (PS/2) administration, α4-integrin deficient T helper cells re-isolated from the CNS of VV challenged mice exhibited a Th17 like phenotype while the number of Th1 like cells was significantly reduced in α4 CKO animals as compared with controls (Figure 5D, E). Consistent with their cytokine production upon ex vivo stimulation, CNS derived α4-integrin deficient T helper cells expressed higher amounts of Th17 signature markers such as Rorc, Il17, Il22, Il6, Ccr6, or Il1r1 whereas Th1 associated genes like Cxcr3, Ccr5, or Ccr2 prevailed in wild type T helper cells (Figure 5F). In conclusion, while Th1 cells appeared to be sufficient to provide host protection in intrathecal VV infection, Th17 cells failed to control cerebral virus replication in the absence of CD8+ T cells resulting in lethal encephalitis.
It was possible that the reduced capacity of Th17 cells to provide host protection in intrathecal VV infection was simply due to reduced availability of IFN-γ within the CNS compartment. In order to test this possibility, we neutralized IFN-γ by monoclonal antibodies in MVA immune and CD8+ T cell depleted wild type mice (Additional file 2: Figure S2). In this purely T helper cell dependent scenario, VV specific Th1 and Th17 cells were generated and had access to the VV challenged CNS. Notably, the mice recovered from VV encephalitis despite ablation of IFN-γ. Efficiency of IFN-γ blockade was documented by reduced levels of MHC class II expression on microglial cells in the CNS compartment (Additional file 2: Figure S2). These data suggested that IFN-γ was redundant as an effector molecule in the clearance of intrathecal VV infection and refuted the idea that diminished IFN-γ expression by Th17 cells was responsible for the failure to clear intracerebral VV infection.
In order to define potential molecular mechanisms of T helper cell mediated host protection in intracerebral viral infection, we screened the expression profile of highly purified CD4+ effector T cells isolated from the CNS of VV challenged wild type vs α4 CKO mice for molecules directly involved in virus defense (Figure 7A). CD4+ effector T cells expressed Tnf, Grzmb, and Fasl irrespective of whether they were derived from the Th1 biased inflammatory infiltrate of wild type mice or from the Th17 biased inflammatory milieu of α4 CKO mice. In contrast, the expression of perforin-1 (Prf1) was markedly reduced in α4-integrin deficient as compared with wild type T helper cells. In order to correlate lack of Prf1 expression with the Th17 transcriptional program, we purified CD3+CD4+CD44-Foxp3- naïve T cells from Foxp3gfp.KI mice and stimulated them without exogenous cytokines (Th0) or differentiated them into Th1 cells or Th17 cells (Figure 7B). Consistent with our in vivo data, we found a significant reduction in Prf1 mRNA and protein in Th17 as compared with Th1 and Th0 cells. Notably, protein expression of Prf1 was only seen at late time points (Figure 7B). Since expression of Prf1 has been reported to depend on the expression of the transcription factor eomesodermin (Eomes) , we measured RNA levels of Eomes in CD4+ T cells isolated from brains of VV infected wild type and α4 CKO mice. Consistent with the low expression of Prf1 in Th17 cells, Eomes mRNA levels were reduced in CD4+ T cells isolated from α4 CKO mice as compared with wild type controls (Figure 7C). To formally validate whether reduced Eomes expression in Th17 cells accounted for diminished Prf1 expression as compared with Th0 and Th1 cells, we polarized Th17 cells in vitro and overexpressed Eomes by retroviral transduction (Figure 7D). FACS sorted transduced (GFP+) Th17 cells expressed abundant levels of Eomes as compared with control vector proving effective transduction; concomitantly, Prf1 RNA was significantly increased in Eomes transduced Th17 cells. These data suggested that the failure of Th17 cells to express Prf1 was due to reduced Eomes expression.
In order to explore the importance of CD4+ T cell derived Prf1 for successful host defense in CNS virus infection in vivo, we immunized Prf1 deficient mice (Prf1-/-) with MVA/CFA and established viral encephalitis in CD8+ T cell depleted animals by intrathecal infection with VV (Figure 8). While wild type mice recovered from i.th. VV challenge, Prf1 deficient mice succumbed to viral encephalitis. Numbers and fractions of CNS infiltrating immune cells were comparable between groups (data not shown) indicating that antigen specific priming in the peripheral immune compartment and establishment of inflammatory infiltrates within the CNS were not impaired in Prf1-/- mice. Moreover, wild type mice that were vaccinated with MVA and then depleted of CD4+ T cells in addition to CD8+ T cells succumbed to intrathecal challenge with VV although they had similar fractions of NK cells and NK T cells in the CNS as their CD4+ T cell replete counterparts (Additional file 3: Figure S3) suggesting that alternative sources of Prf1 other than CD4+ T cells were insufficient to provide protection in this model. Taken together, these data demonstrated that Prf1 expression was indispensable for effector T helper cell mediated control of intrathecal VV infection. 153554b96e