Gingival Exudatome Dynamics Implicate Inhibition of the Alternative Complement Pathway in the Protective Action of the C3 Inhibitor Cp40 in Nonhuman Primate Periodontitis

Periodontitis
is a prevalent chronic inflammatory disease associated
with dysbiosis. Although complement inhibition has been successfully
used to treat periodontitis in animal models, studies globally analyzing
inflamed tissue proteins to glean insight into possible mechanisms
of action are missing. Using quantitative shotgun proteomics, we aimed
to investigate differences in composition of inflammatory gingival
tissue exudate (“gingival crevicular fluid”; GCF), before
and after local administration of an inhibitor of the central complement
component, C3, in nonhuman primates. The C3 inhibitor, Cp40 (also
known as AMY-101) was administered locally in the maxillary gingival
tissue of cynomolgus monkeys with established periodontitis, either
once a week (1×-treatment; n = 5 animals) or
three times per week (3×-treatment; n = 10 animals),
for 6 weeks followed by another 6 weeks of observation in the absence
of treatment. 45 GCF samples were processed for FASP digestion and
liquid chromatography–tandem mass spectrometry (LC–MS/MS)
analysis. Data were processed using the ProgenesisQI software. The
statistical significance of differences between the groups was determined
by RM-ANOVA, and a protein expression change was considered as a true
regulation at >2-fold and p < 0.05. The human
orthologues were subjected to Gene Ontology analyses using PANTHER.
Data are available via ProteomeXchange with identifier PXD009502.
573 proteins with >2 peptides were longitudinally quantified. Both
3× and 1× administration of Cp40 resulted in significant
down-regulation of dozens of proteins during the 6-week course of
treatment as compared to baseline. Following drug withdrawal at 6
weeks, more than 50% of the down-regulated proteins showed increased
levels at week 12. The top scored pathway was “complement activation,
alternative pathway”, and several proteins involved in this
pathway were down-regulated at 6 weeks. We mapped the proteomic fingerprint
changes in local tissue exudate of cynomolgus monkey periodontitis
in response to C3 inhibition and identified the alternative pathway
of complement activation and leukocyte degranulation as main targets,
which are thus likely to play significant roles in periodontal disease
pathogenesis. Label-free quantitative proteomics strategies utilizing
GCF are powerful tools for the identification of treatment targets
and providing insights into disease mechanisms.

牙周炎（Periodontitis）是一种与菌群失调相关的高发慢性炎症性疾病。尽管补体抑制已在动物模型中成功用于牙周炎的治疗，但目前仍缺乏针对炎症组织蛋白质组开展全局分析以阐明其潜在作用机制的相关研究。
本研究采用定量鸟枪法蛋白质组学技术，旨在探究非人灵长类动物局部给予中枢补体成分C3抑制剂前后，炎症性牙龈组织渗出液（即龈沟液（gingival crevicular fluid，GCF））的组成差异。研究对象为已构建牙周炎模型的食蟹猴，研究者在其上颌牙龈组织局部给予C3抑制剂Cp40（又称AMY-101），给药方案分为每周1次（1次给药组，n=5）与每周3次（3次给药组，n=10），持续给药6周，随后停药观察6周。
共收集45份GCF样本，经FASP酶解后进行液相色谱-串联质谱（liquid chromatography–tandem mass spectrometry，LC–MS/MS）分析。数据处理采用ProgenesisQI软件完成。组间差异的统计学显著性通过重复测量方差分析（RM-ANOVA）确定，当蛋白表达变化倍数>2且p<0.05时，认定为具有统计学意义的真实调控变化。通过PANTHER数据库对人类同源蛋白进行基因本体（Gene Ontology）富集分析。
本数据集已通过ProteomeXchange数据库公开，标识符为PXD009502。
本研究共对573种至少包含2条特征肽段的蛋白进行了纵向定量分析。与基线水平相比，3次给药组与1次给药组在6周给药期间均出现数十种蛋白的显著下调。停药至第12周时，超过50%的下调蛋白表达水平出现回升。
得分最高的通路为“补体激活旁路途径”，该通路中的多种蛋白在给药6周时均出现下调。
本研究绘制了食蟹猴牙周炎局部组织渗出液在C3抑制治疗后的蛋白质组指纹变化，鉴定出补体激活旁路途径与白细胞脱颗粒为主要作用靶点，提示二者可能在牙周炎发病机制中发挥关键作用。
基于龈沟液的无标记定量蛋白质组学策略，可有效识别治疗靶点并为疾病机制研究提供新的见解。