Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Sep 7;17(9):3153-3175.
doi: 10.1021/acs.jproteome.8b00263. Epub 2018 Aug 29.

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

Affiliations
Free PMC article

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

Nagihan Bostanci et al. J Proteome Res. .
Free PMC article

Abstract

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.

Keywords: Cp40; GCF; alternative complement pathway; complement; gingival crevicular fluid; gingival inflammation; label-free quantitation; periodontitis; primates; proteome.

Conflict of interest statement

Conflict of Interest Disclosure

J.D.L. is the founder of Amyndas Pharmaceuticals, which is developing complement inhibitors (including third-generation compstatin analogs such as AMY-101), and is the inventor of the compstatin technology licensed to Apellis Pharmaceuticals (4(1MeW)7W/POT-4/APL-1 and PEGylated derivatives). J.D.L. and G.H. are inventors of a joint patent that describes the use of complement inhibitors for therapeutic purposes in periodontitis.

Figures

Figure 1.
Figure 1.
A) Schematic representation of the protein discovery and quantification workflow by use of label-free quantitative proteomics (LFQ) in uncovering Macaca gingival crevicular fluid (GCF) proteome. The C3 inhibitor Cp40 was administered locally in the maxillary gingival tissue of adult systemically healthy cynomolgus monkeys (Macaca fascicularis) with established periodontitis, once a week ((‘1x treatment’; n=5 animals) or three times per week ((‘3x-treatment’; n=10 animals) for six weeks (end of treatment) followed by another six weeks of observation in the absence of treatment (end of monitoring). More details can be found under Materials and Methods. B) Qualitative protein survey in Macaque GCF in the ‘3 x treatment’ group. C) Qualitative protein survey in Macaque GCF in the ‘1 x treatment’ group.
Figure 2.
Figure 2.
Gene Ontology (GO) mapping of quantified GCF proteins. Gene ontology analysis of total 573 proteins involved in GCF from monkey natural periodontitis using PANTHER program. PANTHER Classification System was used to investigate the protein function based on their cellular component (A), biological process (B) and molecular function (C) and immune system process (D).
Figure 3.
Figure 3.
Gene ontology analysis of GCF proteome from monkey natural periodontitis using PANTHER program. Bar plot showing the enrichment of PANTHER biological pathways. Fold change of enrichment and p value are shown (cutoff: fold change >= 8, p <=10−7).
Figure 4.
Figure 4.
Scatter plot of GCF proteome in response to the ‘3x-treatment’ of Cp40 at A) Week 6 Vs. Week 0 B) Week 12 Vs. Week 6. Red dots: fold change ε 2 and p-value <0.05; blue dots: fold change δ 0.5 and p < 0.05) C) Venn diagram showing overlapping between significantly down-regulated proteins of Week 6 Vs. Week 0 (blue) and significantly up-regulated proteins in Week 12 Vs. Week 6 (red).
Figure 5.
Figure 5.
Scatter plot of GCF proteome in response to the ‘1x-treatment’ of CP40 at A) Week 6 Vs. Week 0 B) week12 Vs. Week 6. (red dots: fold change ε 2 and p - value <0.05; blue dots: fold change δ 0.5 and p < 0.05). C) Venn diagram showing overlapping between significant down-regulated proteins of Week 6 Vs. Week 0 (blue) and significantly up-regulated proteins in Week 12 Vs. Week 6 (red). Among them, 210 proteins down-regulated first and then up-regulated (the common changed proteins).
Figure 6.
Figure 6.
Heat map showing the fold change (log2(FC)) of complement activation related proteins in total 573 proteins involved in GCF from monkey natural periodontitis according to PANTHER GO database. Alternative activation pathway (A), classical activation pathway (B) and complement activation (C). The red color indicates up-regulation, the blue color indicates down-regulation.
Figure 7.
Figure 7.
The C3 inhibitor Cp40 and its targets in the setting of periodontitis. Complement is involved both in the dysbiotic transformation of the periodontal microbiota and in the inflammatory process that leads to the degradation of periodontal tissue which in turn exacerbates dysbiosis, leading to the generation of a vicious cycle of dysbiosis and destructive inflammation. Complement inhibition using the C3 inhibitor Cp40 (AMY-101) can block both dysbiosis and destructive inflammation, thereby reversing periodontitis.

Similar articles

See all similar articles

Cited by 1 article

Publication types

MeSH terms

Feedback