Immunomodulatory effects of fractions from Aframomum melegueta K.Schum. and Xylopia aethiopica (Dunal) A.Rich. on cytokine responses in PBMCs from individuals with hyperreactive onchocerciasis

Hyperreactive onchocerciasis (HO) is characterized by a severe skin inflammation. We previously showed that hydro-ethanolic crude extracts of Aframomum melegueta K.Schum. (AM) and Xylopia aethiopica (Dunal) A.Rich. (XA) possess anti-helminthic activities and exhibit Th2 and Th17 anti-inflammatory responses on peripheral blood mononuclear cells (PBMCs) from HO individuals. However, the specific bioactive fraction of the plant extracts that carries this activity remain unknown. This study aims to assess the anti-inflammatory potential of bioactive fractions-derived from the crude hydro-ethanolic extracts (HEE) of these plants. The crude HEE underwent bioguided fractionation between ether fraction (EF), dichloromethane (DCMF), and aqueous fraction (AF), yielding three fractions per plant. PBMCs from HO individuals were co-cultured with DCMF, AF, and EF of XA and AM at a final concentration of 200 µg/mL, as well as 0.005% EF of AM in the presence or absence of microbeads conjugated with monoclonal anti-CD3/CD28 to evaluate anti-inflammatory activity. Cytokine responses was evaluated using snadwich ELISA and Interferon-gamma ( IFN-γ) , Interleukin-17 A (IL-17​A) , IL-5 and IL-10 gene expression was assessed using Real time qPCR (RT-qPCR). Data were analysed using SPSS software version 21 and GraphPad PRISM version 9.2.0. A p-value less than 0.05 was considered statistically significant. As expected HEE extracts and Ibumol reference drug​ inhibited Th1, Th2 and Th17 cytokine responses following TCR activation. The tested fractions differentially modulated Th1, Th17 and Th2 cytokines production. Among them, HEE-derived DCMF and EF of AM significantly inhibited IFN-γ and IL-17​A secretion (DCMF/AM: 1 206ug/mL to 0.2ug/mL and 437.7ug/mL to 0.001ug/mL respectively). A stronger and broader effect was observed on IL-​5. ​Hence, DCMF of AM significantly inhibited its secretion (DCMF/AM: 44.51 ug/mL to 0.001ug/mL) and notably, all XA fractions significantly lowered IL-5 levels (EF, AF and DCMF: 44.51 ug/mL to 4.5, 3.7 and 4.5 ug/mL respectively). IL-10 production was also significantly inhibited by HEE-derived ethereal fraction of AM (EF/AM: 395.7ug/mL to 0.04ug/mL) and by HEE-derived DCMF of XA (DCMF/XA: 395.7ug/mL to 84.9 ug/mL). Consistent with these functional findings, IL-5 and IL-17​A gene expression was also downregulated across the various fractions of both plants. Overall, dichloromethane fraction from HEE extracts of AM carried anti-inflammatory potential. The activity was regulated at transcriptional and protein levels.