Regulation of T cell Differentiation

在本研究中，我们在两种小鼠关节炎模型中测试了与IL-12/IL-23 p40反应并因此对IL-12 p70和IL-23以及抗IL-23 p19和TNFR-Fc具有活性的中和mAbs的功效，一种是推定的Th 17介导的疾病CIA，另一种是抗体/免疫复合物介导的疾病CAIA。令我们有些惊讶的是，结果表明p40或p19的抗体中和对这两种关节炎小鼠模型具有完全相反的作用。 在CIA模型中，抗p40和p19抗体以及TNFR-Fc均下调Th 1相关细胞因子（IFN？，IL-12（p40，p35））和Th 17相关细胞因子（IL-17 A，IL-17 F，IL-23（p40，p19））以及下游促炎细胞因子（IL-6，TNF-？和IL-1？）和伴随炎症的蛋白酶，并改善关节炎。 有趣的是，抗p40抗体具有最深刻的临床效果，但其对细胞因子产生的作用与抗p19之间的唯一差异是对p40和p35产生的作用更大。 这表明其更大的作用是由于IL-12 p70相关的下游细胞因子，该细胞因子在本研究中未测量。 这些研究与先前报告山羊多克隆抗p19抗体可减弱大鼠CIA的研究一致，但该研究未详细分析抗体对细胞因子的影响，也未比较抗p19抗体与抗p40抗体的影响（ART 2007 Yago）。 此外，这些研究与已经提到的显示携带p19缺失的小鼠对CIA具有抗性的研究一致。 因此，这些研究为CIA关节炎主要由依赖于IL-23 p19的Th 17反应驱动的结论提供了额外的支持。 然而，由于抗p40比抗p19具有更深刻的临床作用，因此很明显，该模型中的关节炎也由不受IL-23控制的IL-12 p70细胞因子介导。 在用各种抗体治疗CAIA后获得了非常不同的结果。 在这种情况下，抗p40和抗p19都伴随着疾病的严重加剧，只有TNFR-Fc继续具有改善作用。 该临床结果反映在爪细胞因子产生的分析中，其显示首先CAIA伴随低水平的Th 1或Th 17细胞因子mRNA表达，其不被抗p40和抗p19治疗进一步降低，其次这种抗体治疗伴随下游促炎细胞因子（IL-6和IL-1？）以及基质金属蛋白酶升高。此外，抗p40和抗p19治疗CAIA（但不是CIA）导致一种潜在的诱导性细胞因子IL-27（p28）的产生增加，提高了这种细胞因子导致疾病严重程度增加的可能性。 为了了解抗p40或抗p19治疗CAIA后IL-27产生增加的起源，我们进行了体外研究以确定p40或p19阻断是否影响IL-27分泌。 特别地，我们用单独的LPS或与胶原抗原-抗体复合物组合刺激RAW细胞（来自RAW 264.7巨噬细胞系的细胞），并且发现当在中和性抗p40或抗p19存在下培养时，细胞产生增加量的IL-27和IL-6。这表明，巨噬细胞产生IL-12和/或IL-23对IL-27产生发挥自分泌负调节作用，因此，增加的IL-27产生在抗p40和抗p19治疗的CAIA中观察到的IL-12或IL-6的产生（以及IL-6的产生）可能是由于阻断了IL-12或IL-19的基线水平的负面作用。23对在LPS存在下通过抗原-抗体复合物在体内刺激的关节巨噬细胞的作用。类似的炎症机制不适用于抗体治疗的CIA的事实可以归因于在该模型中局部巨噬细胞不被免疫复合物或引起高IL-27应答的其他因子刺激的可能性，并且在用抗p40和/或抗p19有效治疗的CIA中，炎症不像在未治疗的CIA中报道的那样继发产生抗胶原抗体。需要进一步研究，其中抗p40或抗p19处理的CAIA小鼠也用抗IL-27（抗p28）处理，以进一步确定IL-27数据的可能解释。

In the present study, we tested the efficacy of neutralizing mAbs reacting with IL-12/IL-23p40 and thus active against both IL-12p70 and IL-23, as well as anti IL-23p19 and TNFR-Fc in two mouse arthritis models, one a putative Th17-mediated disease, CIA, and the other an antibody/immune complex-mediated disease, CAIA. Somewhat to our surprise, the results demonstrated that antibody neutralization of either p40 or p19 had quite an opposite effect on these two arthritis mouse models. In the CIA model, both anti-p40 and p19 antibody as well as TNFR-Fc down-regulated the increase in Th1-related cytokines (IFN?, IL-12 (p40, p35)) and Th17-related cytokines (IL-17A, IL-17F, IL-23 (p40, p19)) as well as down-stream proinflammatory cytokines (IL-6, TNF-? and IL-1?) and proteinases accompanying the inflammation in paw tissues and ameliorated the arthritis. Of interest, the anti-p40 antibody had the most profound clinical effect, but the only difference between its effect on cytokine production and that of anti-p19 was a greater effect on p40 and p35 production. This suggests that its greater effect was due to an IL-12p70-related downstream cytokine that was not measured in this study. These studies are consistent with a previous study reporting that a goat polyclonal anti-p19 attenuates rat CIA, but this study did not contain a detailed analysis of the effect of the antibody on cytokines and did not compare the effect of anti-p19 with anti-p40 (ART 2007 Yago). In addition, these studies are consistent with a study already mentioned showing that mice bearing a p19 deletion are resistant to CIA. Thus, these studies provide additional support for the conclusion that the arthritis in CIA are primarily driven by Th17 response dependent on IL-23p19. However, since anti-p40 had a more profound clinical effect than anti-p19, it is clear that the arthritis in this model is also mediated by an IL-12p70 cytokine(s) that is not under IL-23 control. A very different result was obtained upon treatment of CAIA with the various antibodies. In this case both anti-p40 and ant-p19 was accompanied by a major intensification of disease and only the TNFR-Fc continued to have an ameliorative effect. This clinical result was reflected in the analysis of paw cytokine production which showed first that CAIA was accompanied by low levels of Th1 or Th17 cytokines mRNA expression which were not further lowered by anti-p40 and anti-p19 treatment and second that such antibody treatment was accompanied by elevations of downstream proinflammatory cytokines (IL-6 and IL-1?) as well as elevations in matrix metalloproteinases. In addition, anti-p40 and anti-p19 treatment of CAIA (but not CIA) led to increased production of one potential inductive cytokine, IL-27 (p28), raising the possibility that this cytokine was responsible for the increased disease severity. To understand the origin of the increased IL-27 production following anti-p40- or anti-p19-treated CAIA we performed in vitro studies to determine if p40 or p19 blockade affects IL-27 secretion. In particular we stimulated RAW cells (cells from the RAW264.7 macrophage line) with LPS alone or in combination with collagen antigen-antibody complexes and found that the cells produced increased amounts of IL-27 and IL-6 when cultured in the presence of neutralizing anti-p40 or anti-p19. This suggested that macrophage production of IL-12 and/or IL-23 exerts an autocrine negative regulatory effect on IL-27 production and therefore that the increased IL-27 production (as well as IL-6 production) noted in anti-p40- and anti-p19-treated CAIA could arise from blockade of the negative effect of baseline levels of IL-12 or IL-23 on joint macrophages being stimulated in vivo by antigen-antibody complexes in the presence of LPS. The fact that a similar mechanism of inflammation does not apply to antibody-treated CIA can be ascribed to the likelihood that in this model regional macrophages are not being stimulated by immune complexes or other factors that evoke high IL-27 responses and that in CIA effectively treated with anti-p40 and/or anti-p19 the inflammation does not secondarily generated anti-collagen antibodies as reported in untreated CIA. Further studies in which anti-p40 or anti-p19 treated mice with CAIA are also treated with anti-IL-27 (anti-p28) will be necessary to further establish this possible interpretation of the IL-27 data.