IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4⁺ T Cells

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production of IL-17 by CD4+ T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4+ T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents.ArtinM是一种从面包树（Artocarpus heterophyllus）种子中提取的D-甘露糖结合凝集素（D-mannose-binding lectin），可与Toll样受体2（TLR2）的N-聚糖结合并激活抗原呈递细胞（antigen-presenting cells, APCs），具体表现为白细胞介素12（IL-12）的产生。体内给予ArtinM可诱导Th1免疫，并对多种胞内病原体感染产生防护作用。在白色念珠菌（Candida albicans）感染的小鼠模型中，研究人员证实，除Th1免疫外，ArtinM还可诱导Th17免疫，表现为给药组动物体内白细胞介素17（IL-17）水平显著升高。本研究探讨了ArtinM诱导IL-17产生的潜在机制。我们发现，ArtinM可刺激BALB/c或C57BL/6小鼠的脾细胞产生IL-17，该反应在缺乏白细胞介素23（IL-23）、髓系分化因子88（MyD88）或白细胞介素1受体（IL-1R）时显著减弱。此外，我们证实ArtinM可直接诱导巨噬细胞表达IL-23 mRNA并产生白细胞介素1（IL-1）。与此一致的是，在去除巨噬细胞的细胞悬液中，ArtinM刺激产生的IL-17水平降低了53%；且外源性IL-23可与ArtinM协同促进脾细胞悬液产生IL-17。我们证实，缺乏IL-23、IL-1R或MyD88可抑制但并未完全阻断ArtinM刺激的脾细胞产生IL-17。因此，我们进一步探究了ArtinM是否可直接作用于CD4阳性T细胞（CD4+ T cells）以促进IL-17产生。事实上，去除CD4阳性T细胞的脾细胞悬液在ArtinM刺激下仅释放极低水平的IL-17。同样，分离的CD4阳性T细胞在ArtinM刺激下可增强转化生长因子β（TGF-β）mRNA的表达，并释放高水平的IL-17。考虑到IL-23与ArtinM之间的协同作用，我们使用IL-23基因敲除（IL-23 KO）小鼠的细胞来评估该凝集素对CD4阳性T细胞的直接作用。我们证实ArtinM可显著促进IL-17的产生，这一反应在CD4阳性T细胞预先用抗CD3抗体（anti-CD3 antibody）孵育后受到显著抑制。综上，ArtinM可通过两种主要途径促进CD4阳性T细胞产生IL-17：（I）通过诱导抗原呈递细胞产生IL-23和IL-1；（II）通过与CD4阳性T细胞表面的CD3直接相互作用。本研究有助于阐明ArtinM诱导Th17免疫的相关机制，并为利用糖类识别因子（carbohydrate recognition agents）调控免疫的策略设计提供了新的研究视角。