Immunity in Experimental Murine Filariasis: Roles of T and B Cells Revisited

Abstract

We have reevaluated the contributions of T and B cells in Brugia malayi infection by utilizing knockout mice on a uniform background (C57BL/6J). We find that B-cell-deficient mice are more permissive to infection than T-cell-deficient mice.

The nematode Brugia malayi is one of the causative agents of human lymphatic filariasis, a chronic disease associated with high morbidity and social dysfunction (1). Following entry into the mammalian host, L3 larvae develop into L4 larvae (7 to 10 days) and subsequently into adult worms (4 to 6 weeks). Normal, immunocompetent mice are resistant to the development of patent B. malayi infection. Infection of BALB/cByJ or C57BL/6J mice results in sterile immunity with complete elimination of B. malayi larvae and absence of microfilaremia (3, 4). The first major insight into the mechanism of resistance came with the demonstration that congenitally athymic nude (Hfh11^nu) mice lacking T cells were permissive to the development of adult worms and microfilariae (7, 8). More recently it was shown that T- and B-cell-deficient C.B-17-Prkdc^scid (SCID) mice were also susceptible to infection (5).

However, further studies of SCID mice revealed that the mere absence of the adaptive immune system does not confer susceptibility to B. malayi infection. Experimental infection of SCID mice on different strain backgrounds indicated that whereas C.B-17-Prkdc^scid, BALB/c-Prkdc^scid, and C57BL/6J-Prkdc^scid mice are highly permissive, NOD/LtSz-Prkdc^scid and NOD/LtSz-Prkdc^scidB2m^null mice are poorly permissive (2). These data highlight the importance of the background of the mouse strain used in studying host-parasite interactions. Therefore, direct comparison of the studies of nude mice (C3H background) and SCID mice (C.B-17 and C57BL/6J backgrounds) could lead to potentially erroneous conclusions about the roles of specific mutations in affecting the susceptibility to B. malayi infection.

We therefore decided to investigate the fate of B. malayi L3 larvae in T-cell-deficient, B-cell-deficient, and combined T- and B-cell-deficient mice on a C57BL/6J background. Our data reveal a more significant role for B cells in resistance to the early phases of experimental murine filariasis than previously envisaged. The data also call for caution in the interpretation and comparison of data obtained from mutant alleles in the absence of strain background uniformity.

C57BL/6J, C57BL/6J-Prkdc^scid (hereafter SCID), C57BL/6J-Rag1^null (hereafter RAG1), C57BL/6J-Igh6^null (hereafter μMT), and C57BL/6J-Hfh11^nu (hereafter nude) mice were obtained from the colony of L. D. Schultz at The Jackson Laboratory. Breeding pairs of C57BL/6J-TCRβδ^null (hereafter TCRβδ) mice were obtained from Adrian Hayday at Yale University, New Haven, Conn., and bred at the AAALAC-accredited animal facility at the University of Connecticut Health Center. All mice were housed in microisolator cages to reduce the incidence of intercurrent infections. To control for the confounding variables of age and sex, all mice used in the study were males between 4 and 8 weeks of age.

B. malayi L3 larvae were generated at the insectarium of T. R. Klei at Louisiana State University and shipped to the University of Connecticut Health Center under optimal shipping conditions (9). The L3 larvae were injected intraperitoneally at a dose of 40 to 50 L3 larvae per mouse. The mice were necropsied at 6 weeks following infection, and the peritoneal cavities were washed with RPMI 1640 (GIBCO BRL, Gaithersburg, Md.) to recover viable adult worms. In addition, mice were soaked in Tris-buffered saline, with their peritoneal cavities open, to allow the remaining worms to crawl out. Worm counts were performed under a dissecting microscope. The mean adult worm burdens (and the standard deviations) of mice in two different experiments are represented in Table 1 as percentages of input L3 larvae. The mouse mutants can be divided into three groups based on the worm yields following infection. (i) Mice lacking both T and B cells (SCID and RAG1) and mice lacking B cells (μMT) are highly permissive for infection and exhibit worm burdens significantly greater than those in (ii) mice lacking T cells alone (nude and TCRβδ), which are poorly to moderately permissive, but still not as resistant as (iii) mice with an intact immune system (C57BL/6J), which are nonpermissive to infection. In order to determine the time point at which B-cell-mediated resistance is important, we infected mice and necropsied them 2 weeks later to examine L4 larval yields (Table 2). We conclude that B cells are important for early resistance to infection, since μMT mice exhibit significantly greater larval burdens than nude and TCRβδ mice even at this early time point.

TABLE 1.

B. malayi infection in nude, RAG1, SCID, TCRβδ, μMT, and C57BL/6J mice

Mouse genotype (n = 5/group)	Worm burden (%)a
	Expt 1	Expt 2
RAG1	35.6 ± 13	19.6 ± 6
SCID	31.6 ± 7	15.2 ± 7
μMT	28 ± 9	11.1 ± 8
Nude	15.8 ± 12	3.3 ± 5
TCRβδ	10.8 ± 13	2.6 ± 4
+/+	0 ± 0	0 ± 0

^aValues represent the mean worm burdens at 6 weeks as percentages of L3 larvae injected ± the standard deviation for the groups in two different experiments. The yields of adult worms in RAG1, SCID, and μMT mice were significantly different from those in nude, TCRβδ, and C57BL/6J mice (P < 0.05). 

TABLE 2.

B. malayi L4 larval development in nude, RAG1, SCID, TCRβδ, μMT, and C57BL/6J mice

Mouse genotype (n = 5/group)	Yield of adult worms (%)a
μMT	48.8 ± 13
SCID	33.2 ± 15
RAG1	30.4 ± 10
+/+	23.6 ± 10
Nude	12.2 ± 11
TCRβδ	5.5 ± 4

^aValues represent mean larval yields at 2 weeks as percentages of L3 larvae injected ± the standard deviation for the groups. The yields of adult worms in the μMT, SCID, and RAG1 mice were significantly different from those in the nude and TCRβδ mice (P < 0.05). 

Our data are different from those of our previously published report that mice homozygous for the Igh6^null mutation on the segregating C57BL/6J × 129 background have few or no worms 12 weeks postinfection (6). This emphasizes the importance of genetic background in the study of immune resistance to the parasite. We are currently evaluating the following possibilities: (i) the role of antibodies by transfer of hyperimmune serum, (ii) the role of cellular and cytokine mechanisms by adoptive transfer of B cells into μMT and SCID mice, and (iii) the functional status of T cells in the B-cell-deficient environment. The elucidation of the mechanism by which B cells mediate clearance of B. malayi infection becomes of paramount importance, since it would provide us with valuable clues in developing vaccine strategies. Whatever the mechanism by which B cells mediate their effects, the understanding of the role of B cells as important effectors of resistance to B. malayi constitutes a significant insight into the immunity to complex, extracellular, metazoan parasites.