Immune evasion genes from Brugia malayi: functional analyses of Bm-SPN-2, the major secreted microfilarial product

Abstract

Many parasites have evolved to release products that inhibit host defence mechanisms such as enzymes in the mammalian host, in order to promote and sustain their survival within the host. The human filarial nematode Brugia malayi produces larval microfilariae, which circulate in the blood stream. Their most abundant secreted product is a serine protease inhibitor Bm-SPN-2. Serine protease inhibitors (Serpins) are reported to be involved in how the nematodes avoid host immune defences, and in the case of Bm-SPN-2, the protein was found to specifically inhibit the enzymatic activity of human neutrophil elastase and cathepsin G in a dose-dependent manner. More recently, these two enzymes have been linked to the activation of a major innate cytokine IL-33, which is stored as a full-length 270-aa protein in the cell nucleus, and released as an active C-terminal domain upon stimulation.

As full-length (FL) human and murine IL-33 are not commercially available, soluble murine and human FL-IL-33 were produced in transfected HEK 293T cells, following mutation of the nuclear binding motif. In this form, IL-33 is no longer retained in the nucleus and can be purified as a soluble protein. It was confirmed that once cleaved, recombinant human IL-33 was able to induce significant IL-6 secretion by mast cells. Bm-SPN-2 was then shown to block human full-length IL-33 cleavage by inhibiting human neutrophil cathepsin G in a dose dependent manner, supporting the hypothesis that Bm-SPN-2 may act in vivo to prevent IL-33 activation and the promotion of the TH2 immune response. However, in the in vivo setting, it was unexpectedly found that IL-33R (ST2) gene deficiency did not enhance the survival of B. pahangi microfilariae. Furthermore, in the absence of IL-33R, murine immune responses to microfilariae were not significantly altered compared to wild-type BALB/c mice, other than in a significant increase in IL-33 expression. Hence while Bm-SPN-2 can act in vitro to forestall one of the key events in TH2 induction, this has not yet been shown to be crucial to the immune response to the parasite in vivo.