Latex protein extracts from Calotropis procera with immunomodulatory properties protect against experimental infections with Listeria monocytogenes

Abstract

Background: The latex from the medicinal plant Calotropis procera is often used in folk medicine against infectious and inflammatory diseases.

Purpose: In this study, we investigate a protein fraction with immunomodulatory properties, named LPPI, against experimental infections, in vitro and in vivo, with a virulent strain of Listeria monocytogenes.

Study design: LPPI was exposed to cultured macrophages or Swiss mice and then challenged with L. monocytogenes.

Methods: Peritoneal macrophages were obtained from Swiss mice, and cultured in 96-well microplates. Soluble latex proteins (LP) were subjected to fractionation by ion-exchange chromatography. The major peak (LPPI) was added into wells at 10 or 100µg/ml. Albumin (100µg/ml) was used for comparison between protein treatments. After incubation for 1h at 5% CO2/ 37°C, the supernatant was discarded and 0.2ml of L. monocytogenes overnight culture was added in the wells. Following 4h and 24h infection, the cytokine mRNA expression was evaluated as well as the number of intracellular colony forming units. Swiss mice (n=16) were injected intraperitoneally (i.p.) with LPPI (5 and 10mg/kg) while the control mice received albumin (10mg/kg) or LP (10mg/kg). After 24h, all animal groups were challenged with L. monocytogenes (10(6) CFU/ ml), also by i.p. route.

Results: LPPI was not toxic to uninfected macrophages (pMØ) and significantly increased mRNA expression of TNF-α, IL-6, IL-1β and iNOS. Following infection, cell viability was reduced by 50% in albumin-treated pMØ (control); but only 17% in pMØ treated with LPPI at 100µg/ml. In this case, LPPI increased expression of TNF-α and IL-6 whereas the number of bacterial colony-forming units was reduced 100-fold in comparison to control groups. Swiss mice pretreated with LPPI showed dose-dependent survival rates that reached 80%, while mice that received albumin died 1-3 days after infection. After 24h infection, leukocyte migration to the infectious foci was high in LPPI-treated mice whereas the number of viable bacteria in the peritoneal fluid, liver and bloodstream were significantly reduced.

Conclusion: We conclude that LPPI present immunomodulatory properties that are beneficial for prevention of systemic bacterial infections caused by the intracellular bacteria L. monocytogenes.

Keywords: Cytokines; Experimental infections; Intracellular infection; Laticifer fluid.