Liviane Maria Alves Rabelo¹, Tiago de Oliveira Batista², João Vitor Afonso de Carvalho², João Miguel Vieira Ripardo², Nylane Maria Nunes de Alencar¹, Felipe Domingos de Sousa³

Frutapin (FTP) is a glucose/mannose-binding lectin found in the seeds of Artocarpus altilis (breadfruit). It shares structural similarity with ArtinM (from Artocarpus heterophyllus, jackfruit), known for its immunomodulatory and wound-healing properties. However, due to its low yield in native form, our group standardized its heterologous expression in a prokaryotic system, significantly increasing production (~40 mg/L of culture) and enabling studies on its pharmacological potential. This study aimed to investigate the immunomodulatory potential of recombinant frutapin (rFTP) in murine macrophage RAW 264.7 cells. Cell viability assays (0.15 – 1 mg/mL, resazurin) were performed. Nitric oxide (NO, Griess assay) and cytokine production (TNF-α, IL-1β, IL-6, and IL-10; ELISA) were monitored with or without LPS stimulation. Macrophages exposed to rFTP for 24, 48, and 72 hours underwent proteomic profiling using label-free DIA LC-MS to assess differential expression compared to an untreated control group (CTRL). Data were filtered to show only statistically significant differences (p < 0.05, ANOVA) coupled with a ≥1.5-fold change in protein abundance (Maximum Fold Change—MFC ≥1.5). Label-free quantification used the HiN approach. Protein quantification data were imported into the Omicscope portal; significant changes between conditions were identified using thresholds of ≥1.5-fold change and p-value ≤ 0.05, followed by analysis via volcano plots and heatmaps. rFTP showed dual effects on RAW 264.7 macrophages: increased short-term viability (high doses) but long-term reduction, suggesting dose-dependent modulation. In LPS-activated cells, rFTP significantly reduced NO production (regulating inflammation) while maintaining basal levels. Cytokine analysis revealed: 1) Elevated basal TNF-α (immune priming), 2) Increased early IL-10 (anti-inflammatory), and 3) No effect on IL-1β/IL-6, demonstrating selective immunomodulation. These findings suggest rFTP balances inflammatory responses (via NO/TNF-α/IL-10 regulation), potentially benefiting chronic wound healing. Future studies should explore molecular mechanisms (e.g., NF-κB pathways). Regarding the proteomic profile, 1,555 proteins were identified, with 878 differentially regulated. Principal Component Analysis (PCA) showed expected consistency within biological conditions, while samples exhibited separation in the longitudinal analysis. The Dynamic Range Plot indicated most proteins were in high abundance. Enrichment Analysis visualizations (dot plot and heatmaps) revealed relevant information about FTP\'s influence on RNA binding and cadherin binding pathways—consistent with RAW cell activation leading to increased proliferation. Cadherins are a group of cell adhesion molecules mediating cell-cell adhesion and tissue organization; notably, we observed up-regulation of CD166 antigen (ALCAM) for up to 48 hours after FTP exposure. CD166 is a cell adhesion molecule mediating heterotypic cell-cell contacts that promote T-cell activation and proliferation. rFTP modulated the inflammatory response in activated macrophages, significantly increasing cell proliferation and production of the anti-inflammatory cytokine IL-10, while reducing levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β.

Agradecimentos: