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.