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

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.

Host‐microbe interactions: Profiles in the transcriptome, the proteome, and the metabolome

“…The investigations targeted many forms of the disease, ranging from gingivitis to mild, moderate, chronic, and aggressive periodontitis . Most studies have focused on analyses of samples from saliva, gingival crevicular fluid, and sulcular/gingival tissues …”

“…Many studies have been carried out in humans with the goal of discovering a proteomic signature for periodontal health 29,30 (Table 2).…”

“…[31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49] The investigations targeted many forms of the disease, ranging from gingivitis 44 to mild, moderate, chronic, 33,36,37,41,47 and aggressive periodontitis. 42,46,48 Most studies have focused on analyses of samples from saliva, 30,34,36,38,44,47,49 gingival crevicular fluid, 29,31,33,37,41,43,45,48 and sulcular/gingival tissues. 35,40 Samples of saliva and gingival crevicular fluid can be collected noninvasively.…”

“…Animal studies have also been carried out to discover a proteomic signature of periodontal disease 25,30,61 or of the response to the treatment of periodontitis. 30 Although the number of proteomic studies carried out in animals is limited, this type of study is useful for exploring novel therapies for periodontitis, which are challenging to carry out in humans. For example, proteomic-based animal studies, such as those conducted in primate and canine models, reported the effect of complement system-inhibiting proteins in the treatment of periodontitis in preclinical experimental periodontitis.…”

Proteomic Investigations of In Vitro and In Vivo Models of Periodontal Disease

“…The cynomolgus monkey ( Macaca fascicularis ) is an old world monkey highly related to humans, sharing 92.5% of the human genome . It is one of the most important non‐human primate animal models in biomedical research and has been used to understand the effect of complement 3 (C3) inhibitor Cp40 in reducing periodontal destruction, such as probing pocket depth and clinical attachment loss, in monkeys with established periodontitis . As with the canine model reported by Davies et al .…”