Elsevier

Acta Tropica

Volume 120, Issue 3, December 2011, Pages 191-205
Acta Tropica

Sensitization with anti-inflammatory BmAFI of Brugia malayi allows L3 development in the hostile peritoneal cavity of Mastomys coucha

https://doi.org/10.1016/j.actatropica.2011.08.005Get rights and content

Abstract

Filarial parasites survive by inducing tolerance in host but the antigens and mechanisms involved are not clear. Recently we found that BmAFI, a Sephadex G-200 eluted fraction of Brugia malayi adult worm extract, stimulates IL-10 release from THP-1 cells. In the present study, we determined the SDS-PAGE profile of BmAFI and infective 3rd stage larva (L3), investigated the effect of pre-sensitization of host with BmAFI on the survival and development of L3 in the non-permissive peritoneal cavity (p.c.) of the permissive host Mastomys coucha and in the p.c. of non-permissive Swiss mice, and studied immunological correlates for the observed effects. The parasite development and burden in p.c., was determined in sensitized infected M. coucha and Swiss mice and the release of TGF-β, IL-4, IL-10, IL-13, IFN-γ and NO, cellular proliferative response to Con A and BmAFI and levels of IgG subclasses and IgE were determined in sensitized infected M. coucha. Cellular proliferative response to Con A and BmAFI, mRNA expression of GATA-3, CTLA-4 and T-bet were determined in sensitized Swiss mice. In addition, the parasitological parameter was also studied in BmAFI-sensitized M. coucha exposed to the infection by standard subcutaneous (s.c.) route to assess whether sensitization enhances the intensity of infection. BmAFI-sensitization permitted survival of L3 and their development to adult stage by day 60 p.i. in the p.c. of M. coucha; in non-sensitized animals L3 could molt to L4 only and no parasite could be recovered beyond day 30 p.i. In M. coucha that received infection by s.c. route, pre-sensitization with BmAFI enhanced the microfilaraemia and adult worm recovery. In sensitized Swiss mice L3 could successfully molt to L4 in p.c. with improved recovery of parasite. BmAFI sensitization upregulated TGF-β and IL-10 release, IgG1 and IgG2b levels, GATA-3 and CTLA-4 mRNA expression, suppressed the cellular proliferative response and downregulated Con A stimulated response, IgE, IL-13, IFN-γ and NO responses. Immunoblot analysis showed that the BmAFI antiserum also strongly reacts with some L3 molecules. The results show, for the first time, that sensitization with the anti-inflammatory BmAFI which shares some of its molecules with those in L3, facilitates parasite survival in the non-permissive p.c. of the permissive host M. coucha, render a non-permissive Swiss mouse partially permissive to infection and enhances parasite load in M. coucha receiving the infection through permissive s.c. route by evoking a modified Th2 type of response and anti-inflammatory milieu. In conclusion, the findings suggest that the anti-inflammatory BmAFI fraction facilitates survival of B. malayi infection even in non-permissive environment.

Graphical abstract

Sensitization with BmAFI which shares some molecules with L3, generates anti-inflammatory milieu and modified Th2 response and facilitates development of infection in hostile host environment.

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Highlights

► Facilitated survival and development of L3 to adult stage in hostile host environment. ► Upregulated TGF-β & IL-10 release, IgG1 & IgG2b, and CTLA-4 & GATA-3 mRNA expression. ► Downregulated cell proliferative response, IgE, IL-13, IFN-γ and NO responses.

Introduction

Lymphatic filariasis (LF), a longstanding chronic infection caused by Wuchereria bancrofti, Brugia malayi and Brugia timori, is prevalent in many parts of the tropics and sub-tropics of the world. Currently over 120 million people are affected by the infection with 40 million people showing chronic disease symptoms (Molyneux, 2003). Active modulation of the host's immune response is part of the parasites’ strategies for long-term survival which is characterized by a marked cellular hyporesponsiveness and a shift of the cytokine balance toward a Th2/Th3 response (Doetze et al., 2000, King et al., 1993, Plier et al., 1996). It is reported that the down-regulation of inflammatory reactions in the host is induced by secreted products of the parasites (Allen and MacDonald, 1998, Hewitson et al., 2009, Whelan et al., 2000) by manipulating the cytokine network (Hartmann et al., 1997), signal transduction pathways (Harnett et al., 1998) or inhibitors of essential enzymes (Hartmann and Lucius, 2003) which eventually contribute to cellular hypo-responsiveness (Harnett and Harnett, 2008) and a possible pathogenicity factor essential for the persistence of parasite within its host (Lawrence, 2001, Maizels et al., 2004, Schonemeyer et al., 2001). Also, strong Th2 responses induced by the parasite molecules, even regulatory T cells (Taylor et al., 2007) or alternatively activated macrophages (Allen and MacDonald, 1998, Kreider et al., 2007) lead to blocking of protective immune effector responses, allowing parasites to survive in a “modified Th2” environment muting effector Th1 and Th2 responses (Hewitson et al., 2009, Maizels and Yazdanbakhsh, 2003).

Our earlier study has shown that BmAFI, a Sephadex G-200 eluted fraction of B. malayi adult worm extract, stimulated the release predominantly anti-inflammatory cytokine (IL-10) and facilitated survival of B. malayi adult worms instilled in to the peritoneal cavity (p.c.) in Mastomys coucha (Dixit et al., 2004).

In the present study, we used two rodent species: Mastomys coucha and Swiss mouse for determining the effect of sensitization with BmAFI on the survival and development of subsequently introduced infection and the hosts’ immunological correlates for the observed fate of the infection. M. coucha is a highly susceptible host for B. malayi infection if the infection (L3) is introduced through subcutaneous route (a route through which humans get the infection) and the immunological profile of this rodent model of filarial infection is well characterized (Dixit et al., 2006, Sahoo et al., 2009, Tyagi et al., 1994, Tyagi et al., 1985). However, M. coucha does not support the infection if it is introduced in to the peritoneal cavity (Gupta et al., 2004, Murthy et al., 1997). This model is therefore unique in that it provides a non-permissive pocket (peritoneal cavity; p.c.) in an otherwise highly permissive host. As a result, by just choosing the appropriate route of initiation of infection, both parasite survival-facilitating (i.p. route) as well as survival-inhibiting or survival-enhancing potential (s.c. route) of sensitization could be studied in this model. The second rodent, Swiss mouse is a non-permissive host and the infection fails to develop whether it is introduced s.c. or i.p. Therefore, the non-permissive Swiss mouse is used for confirmation of the effects of sensitization on parasite load. An advantage of mouse is that it is a good model for studying short term immune response.

The parasite development and burden in p.c. was determined in sensitized infected M. coucha and Swiss mice and the release of TGF-β, IL-4, IL-10, IL-13, IFN-γ, NO and cellular proliferative response to Con A and BmAFI and levels of IgG subclasses and IgE were determined in sensitized infected M. coucha. Cellular proliferative response to Con A and BmAFI, mRNA expression of GATA-3, CTLA-4 and T-bet were determined in sensitized Swiss mice. In addition, the parasitological parameter was also studied in BmAFI-sensitized M. coucha exposed to the infection by standard s.c. route to assess whether sensitization enhances the intensity of infection. Further, in order to find out whether BmAFI shares some antigens with those of L3, we compared SDS-PAGE profile of L3 with that of BmAFI and then immunoblotted L3 extract with anti-BmAFI sera from sensitized M. coucha with or without L3 infection.

Section snippets

Animals

Healthy 8–10 week-old male M. coucha and Swiss mice (22–24 g) from the Institute's Animal Facility were used in the study in compliance with the Institutional Animal Ethics Committee guidelines. Throughout the study, they were housed in climatically controlled animal quarters (Temperature: 23 ± 2 °C; RH: 60% and photoperiod: 12 h light–dark cycles) and fed standard rodent chow supplemented with dried shrimps (M. coucha) and water ad libitum.

Isolation of parasites and preparation of parasite extract

Adult B. malayi parasites were recovered from p.c. of jirds

Effect of sensitization with BmAFI on the survival and development of L3 in the peritoneal cavity of M. coucha and Swiss mice

Sensitization of M. coucha with BmAFI and subsequent L3 infection by i.p. route showed survival and development of the L3 to adult worms (Fig. 1A). The sensitized animals showed around 54% and 30% recovery of developing worms (L4) on days 15 and 30 p.i., respectively (Fig. 1A); young adults were recoverable (11%) at day 45 p.i. The full-grown adult parasite (Fig. 2E) recovery on day 60 p.i. from these animals was 1.3%. The parasite recovery data in non-sensitized i.p.-infected animals indicate

Discussion

It is not clear which of the parasite stages has the ability to modulate host's immune system in a way that facilitates the parasite survival. We chose adult worms in preference to other parasite life stages for characterizing parasite proteins (fractions) for several reasons: (1) Among L3, L4 and adult stages, adults can be recovered in large numbers from rodent hosts, (2) adult worm represents the fully developed parasite, (3) adult worms are considered to be largely responsible for much of

Acknowledgements

Thanks are due to Dr. T.K. Chakraborty, Director, CDRI, Lucknow, for his encouragement and providing facilities. The authors thank Dr. Mario T. Philipp, Ph.D. Professor of Microbiology and Immunology Chair, Division of Bacteriology and Parasitology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Lousiania, for critical review of the manuscript and Dr. P.S.R. Murthy for helpful suggestions. Thanks are also due to Mr. V.K. Bose for technical assistance and Mr.

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