A138 DOES AN EXTRACT OF THE TAPEWORM HYMENOLEPIS DIMINUTA PROMOTE A REGULATORY NEUTROPHIL

Abstract

Background

Data are emerging that neutrophils may play an overlooked role in the response to infection with helminths, perhaps early in the infection and particularly those species that cause tissue damage (e.g. Nippostrongylus brasiliensis - a gut nematode (roundworm)). We have noted an early recruitment of neutrophils into the peritoneal cavity following injection of a crude extract of the tapeworm Hymenolepis diminuta (HdE), and this HdE suppresses dinitrobenzene sulfonic acid (DNBS)-induced colitis. Thus we hypothesized that HdE induced an anti-inflammatory phenotype in murine neutrophils.

Aims

(1) to determine if HdE directly converts neutrophils to an anti-inflammatory phenotype in vitro, and (2) to determine if intraperitoneal (ip.) injection of HdE induces an anti-inflammatory phenotype in recruited neutrophils.

Methods

Neutrophils were isolated by a Percoll gradient from the bone marrow of healthy BALB/c mice and were treated with HdE (100–1000 μg/mL) and the following measures of activity assessed: (a) viability, (b) Ca²⁺ signaling, (c) chemotaxis, (d) cytokine production. HdE (1 mg) was injected ip. into BALB/c mice and 4h later, peritoneal exudate cells (PECs) were collected. These cells were then co-cultured with anti-CD3/anti-CD28 stimulated splenic CD4⁺ T cells for 4 days, and IL-10 measured in the culture medium by ELISA.

Results

Direct application of HdE to neutrophils was not preferentially cytotoxic as assessed by trypan blue staining or LDH release, and elicited a slow increase in intracellular Ca²⁺ that was also observed in Ca²⁺-free medium, suggesting the increased Ca²⁺-detected was via release from intracellular stores. HdE did not induce neutrophil chemotaxis, nor did it directly stimulate the production of TNF-α, or IL-10 (n=3–9), but it did suppress LPS-driven neutrophil chemotaxis (n=4). The PECs retrieved 4h after injection of HdE (90–95% neutrophils based on nuclear morphology), but not LPS, evoked increased production of the anti-inflammatory cytokine IL-10 from co-cultures of activated murine splenic CD4⁺ T cells. Co-culturing HdE-treated bone marrow derived neutrophils with activated CD4⁺ T cells did not replicate this result.

Conclusions

Treatment with HdE induced a subtle Ca²⁺ mobilization in murine neutrophils and remarkably suppressed their movement in response to LPS. The PECs recruited in response to HdE promoted IL-10 production from T cells. If validated, this would represent a novel aspect of immunoregulation whereby neutrophils recruited in response to helminths can educate and enhance the ‘differentiation’ of a T cell into a regulatory phenotype characterized by IL-10 production (i.e. Tr1-like phenotype).

Funding Agencies

CCC, CIHRNSERC