(1993) Immunological relationships during primary infection with Heligmosomoides polygyrus (Nematospiroides dubius): downregulation of specific cytokine secretion (IL-9 and IL-10) correlates with poor mastocytosis and chronic survival of adult worms. The Nottingham ePrints service makes this work by researchers of the University of Nottingham available open access under the following conditions. A note on versions: The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the repository url above for details on accessing the published version and note that access may require a subscription. Immunological relationships during primary infection with Heligmosomoides polygyrus (Nernatospiroides dubius): downregulation of specific cytokine secretion (IL-9 and IL-10) correlates with poor mastocytosis and chronic survival of adult worms SUMMARY Mice were infected either with Trichinella spiralis (day U) , Heligmosomoides polygyrus (day-14) or concurrently with both species and were killed in groups, together with nai've control mice, on 2 occasions (day 8 and 15 post injection with T. spiralis, corresponding to days 22 and 29 p.i. with H. polygyrus). The expulsion of T. spiralis was slowed significantly in concurrently infected mice and this was ussociated with a reduced mastocytosis and lower serum mucosal must cell protease levels. Mesenteric lymph node (M L N) lymphocytes from all three experimentul groups secreted IL-3 and IL-4 in copious amounts when stimulated in vitro by Concanavalin A (Con-A), hut the secretion o f high levels of IL-9 und IL-10 was essentially conjned to mice infected with T. spiralis alone. It is suggested that adult H. polygyrus selectively modulate cytokine secretion by Th2 cells within the M L N during infection and that this is brought about as a direct consequence of the mechanism employed by H. polygyrus to depress mucosal inflammatory responses in order to facilitate its own survival.

Specific serum and intestinal immunoglobulin (Ig)G1 and IgA responses to Heligmosomoides polygyrus were measured in a panel of seven inbred mouse strains which exhibit ‘rapid’ (< 6 weeks (SWR × SJL)F1), ‘fast’ (< 8 weeks, SJL and SWR), ‘intermediate’ (10–20 weeks, NIH and BALB/c) or ‘slow’ (> 25 weeks, C57BL/10 and CBA) resolution of primary infections. Mice with ‘rapid’, ‘fast’ or ‘intermediate’ response phenotypes produced greater serum and intestinal antibody responses than those with ‘slow’ phenotypes. The F1 hybrids ((SWR × SJL)F1) of two ‘fast’ responder strains showed the earliest antibody response with maximum titres evident within 6 weeks of infection. There was a negative correlation between the serum IgG1 responses and worm burdens in individual mice within a number of mouse strains, and also between serum IgG1 and IgA responses and worm burdens in the ‘rapid’ ((SWR × SJL)F1) responder strain. The presence of IgG1 in the gut was found to be due to local secretion rather than plasma leakage. Using Western immunoblotting, serum IgG1 from ‘rapid’ and ‘fast’ responder but not ‘slow’ responder mice was found to react with low molecular weight antigens (16–18 kDa) in adult worm excretory/secretory products.

Abstract The induction of acquired resistance to H. polygyrus, following treatment of mice by a 6 day immunizing infection abbreviated with the anthelmintic drug ivermectin (6d I-AI), was investigated. Four worms were sufficient to elicit >80% protection against challenge and immunizing infections >50 worms generated >95% protection in female NIH mice. A few worms were recovered during the second week from immunized challenged mice but these were rapidly expelled from the gut lumen. Treatment with hydrocortisone from day 10 postinfection, permitted worm burdens to accumulate over the following 2 weeks. The 6d I-AI protocol enabled females of strains previously designated as weak responders to develop potent acquired resistance to challenge (CBA mice showed >90% protection), although weak responder strain male mice were not significantly protected. Delaying treatment with ivermectin by as little as 24 h resulted in poorer expression of acquired resistance. A positive correlation between the increasing interval from infection to treatment with ivermectin and worm burdens after challenge, and the negative correlation with IgGl antibody responses after challenge indicated that the immunodepressive activities of 7 day and older worms down-regulated local intestinal immune responses. Mice characterized by weak responder phenotype were significantly more sensitive to downregulation than mouse strains showing strong responder phenotype. In consequence, optimal timing of treatment with anthelmintics during exposure to the immunizing infection, intending to minimize exposure to the immunodepressive stages of the parasite, is sufficient to overcome reported genetic constraints on the development of resistance in this system.

SUMMARY The primary immune response to infection with Heligmosomoides polygyrus was studied in mice differing in response phenotype (fast-SWR, intermediate-NIH, slow-CBA). Marked IgGl and IgE but not IgG2a antibody responses were detected in infected mice and the former were more intense in fast compared with slow responder strains. Mastocytosis, MMCP-1, and the secretion of cytokines by mesenteric lymph node cells, following stimulation in vitro by Con A, were also more intense initially in SWR mice. Secretion of IL-4 declined in all strains by the 4th week of infection, irrespective of response phenotype. IL-10 was only produced briefly by SWR mice. However, the temporal patterns of secretion of IL-3 and IL-9 clearly distinguished fast from slow responder phenotypes. Following initial intense secretion of IL-3, production declined in all strains but in the 5–6th weeks enhanced secretion was evident in SWR and NIH mice and was sustained until week 10 p.i. In contrast, CBA mice never recovered from the initial down-regulation in weeks 3–4 and secretion declined to background levels by week 6 p.i. despite the continued presence of adult worms. Temporal changes in the secretion of IL-9 were very similar: secretion declined in CBA mice by week 6 p.i., whilst SWR and NIH mice continued to secrete high amounts. We suggest that fast and slow responder mice differ not only in their initial responsiveness to parasite antigens but also in their ability to sustain a Th2 response to the parasite and we propose that the latter is in part determined by their different susceptibilities to parasite-mediated immunomodulation. Only the fast responder strains can sustain a Th2 response of sufficient intensity to facilitate expulsion of adult worms.

The inheritance of response phenotype to Heligmosomoides polygyrus was investigated in F1 hybrid progeny of fast and slow responder mouse strains. The fast responses of SJL(H-2S) and SWR(H-2q) mice were mediated by dominant genes complementing each other in F1 hybrids which lost worms earlier and produced faster parasite-specific IgG1 antibody responses than either parent. However, the response of F1 hybrids from crosses of C57BL/10 (B10, H-2b) mice with either SJL or SWR differed from that of the parental strains and from each other: (B10 x SWR)F1 lost worms earlier than SWR whilst (B10 x SJL)F1 lost worms later than SJL mice. The F1 progeny of SJL mice with congenic strains B10.G (H-2q) and B10.S (H-2S) lost worms as quickly as SJL. Therefore, the response phenotype mediated by the genome of SJL mice was unaffected by H-2 heterozygosity (with H-2q) or homozygosity (H-2S) despite heterozygosity with B10 background genes, but was slowed significantly by heterozygosity with H-2b. All hybrids involving heterozygosity with B10, irrespective of MHC haplotype or background, failed to clear worms completely, in each case a proportion of mice harbouring residual worm burdens after loss of worms from parental strains.

SummaryMice were infected either with Trichinella spiralis (day 0). Heligmosomoides polygyrus (day – 14) or concurrently with both species and were killed in groups, together with naïve control mice, on 2 occasions (day 8 and 15 post infection with T. spiralis, corresponding to days 22 and 29 p.i. with H. polygyrus). The expulsion of T. spiralis was slowed significantly in concurrently infected mice and this was associated with a reduced mastocytosis and lower serum mucosal mast cell protease levels. Mesenteric lymph node (MLN) lymphocytes from all three experimental groups secreted IL‐3 and IL‐4 in copious amounts when stimulated in vitro by Concanavalin A (Con‐A), but the secretion of high levels of IL‐9 and IL‐10 was essentially confined to mice infected with T. spiralis alone. It is suggested that adult H. polygyrus selectively modulate cytokine secretion by Th2 cells within the MLN during infection and that this is brought about as a direct consequence of the mechanism employed by H. polygyrus to depress mucosal inflammatory responses in order to facilitate its own survival.

IgG1 antibody responses to Heligmosomoides polygyrus were measured in eight mouse strains supporting acute (< 8 weeks, SJL, SWR), intermediate (10-20 weeks, NIH, BALB/c) or chronic (> 25 weeks, C57BL/0, CBA, C3H, AKR) primary infections. Mice supporting acute or intermediate infections produced more intense antibody responses and total serum IgG1 concentrations were higher than in mice tolerating chronic infections. Positive correlations across mouse strains between the intensity of the antibody response and the percentage loss of worms in weeks 6 and 10 were established. No correlation was found between the response within mouse strains and loss of worms by individual mice. Heavy infections gave marginally higher antibody titres than low intensity infections, but few significant differences were detected and it was concluded that infection intensity did not markedly influence the magnitude of the antibody response. Male and female mice responded similarly despite the earlier loss of worms from females. No association was found between the primary response phenotype and recognition of particular antigens in Western blot analysis, nor did intensity of infection or host gender affect recognition. The possibility that immunomodulatory properties of adult worms may have had a differential influence on ability of strains of contrasting response phenotype to mount IgG1 responses was discussed.

L3 and L4 stages of H. polygyrus were prevented from developing further and were probably killed within 24 h of treatment with ivermectin although total parasite burdens, particularly when treatment was given 4-6 days after infection, declined over a longer period lasting several days. Strong resistance to challenge infection was expressed by infected mice dosed with ivermectin during the tissue phase of larval development. Even immunizing infections as brief as 12-36 h (when only L3 larvae would have been present in the mucosa) elicited strong acquired immunity. When infections were terminated 4-6 days after infection, acquired resistance was 95-100%. The stronger resistance of mice exposed to both L3 and L4 stages was associated with the recognition of low molecular weight polypeptides in adult worm homogenate and there was a highly significant correlation between percentage protection and anti-L4/anti-adult worm serum IgG1 antibodies.

The course of primary infection was studied in BALB and B10 H-2 congenic mouse strains. The duration of infection, as assessed with regular faecal egg counts and worm burdens, was shorter in mice carrying the H-2 s , H-2 d or H-2 q haplotypes when compared to mice with H-2 b . Strains with H-2 k were intermediate. An experiment was carried out to test the hypothesis proposed by Wassom, Krco & David (1987) predicting that the progeny of I–E +ve mouse strains crossed with I–E -ve strains, would show susceptibility rather than resistance to infection. This hypothesis was not substantiated by our data and we conclude that it does not apply to primary infections with Heligmosomoides polygyrus . It is proposed that the gene products of at least two loci within the H-2 (associated with the H-2 b and H-2 k haplotypes) are crucial in determining the response phenotype of mice to primary infection with H. polygyrus . One allele, (associated with the H-2 b haplotype) may be preferentially affected by parasite-mediated immunosuppression.

SUMMARY The time-course of low and high intensity primary infections with Heligmosomoides polygyrus was monitored in SJL and SWR mice, both of which usually expel worms within 7 weeks of larval administration. Worm expulsion in these strains was not dependent on the intensity of infection, with low and high intensity worm burdens being lost within the same period of time. The ability to expel worms rapidly was inherited in a dominant manner in F 1 offspring of SJL or SWR mice mated with C 57 Bl 10 mice; the latter being a strain in which no loss of worms was evident within 10 weeks of infection. However, neither (SJL × C 57 Bl 10 )F 1 nor (SWR × C 57 Bl 10 )F 1 mice expelled worms as rapidly as the parental SJL and SWR strains. (SWR × B10G)F 1 [H-2 q ] mice eliminated worms faster than (SWR × C 57 Bl 10 )F 1 [H-2 bq ], suggesting that the b haplotype had a moderating influence on the expulsion process. In fact (SWR × B10G)F 1 mice showed a significant reduction in worm burdens by week 4 but by weeks 6–8 the rate of worm loss had slowed considerably. In contrast, SJL and SWR mice, whilst initiating rejection slightly later, (after week 4) expelled all worms within the following 2 weeks. Thus two distinct patterns of response were observed among the fast responder strains as exemplified by SWR and SJL mice on the one hand and (SWR × B10G)F 1 on the other. Our results support the hypothesis that the course of a primary infection with H. polygyrus is influenced by multiple host gene loci, some of which are encoded within the MHC. SJL and SWR mice probably have similar if not identical gene combinations at loci which determine a fast responder phenotype, distinguishing them from the other mouse strains which have been studied.

The efficacy of ivermectin against Heligmosomoides polygyus was investigated in 5 mouse strains (CFLP, NIH, C57Bl10, BALB/c and CBA) using a variety of dose levels, and subcutaneous and oral administration. Heligmosomoides polygyrus were not completely eliminated when 5 mg kg−1 of ivermectin was given 6 days after infection, but 10 mg kg−1 was totally effective. Significant mouse-strain variation in drug efficacy was detected, NIH mice requiring treatment with higher doses than CFLP mice in order to bring about a comparable level of larvicidal activity. Invermectin was more effective when given subcutaneously than when given orally, regardless of the dose administered or the strain of mouse tested. The anthelmintic effect of the treatment was more persistent in CFLP mice given 20 mg kg−1 subcutaneously than in NIH mice or in mice treated orally, and a 20-day interval between administration and infection was insufficient to prevent inhibition of parasite survival. Ivermectin was shown to be totally effective at 20 mg kg−1 given subcutaneously in killing immune arrested larvae of H. polygyrus.