Extracts of the rat tapeworm, Hymenolepis diminuta, suppress macrophage activation in vitro and alleviate chemically induced colitis in mice

Abstract

Analysis of parasite-host interactions can reveal the intricacies of immunity and identify ways to modulate immunopathological reactions. We assessed the ability of a phosphate-buffered saline-soluble extract of adult Hymenolepis diminuta to suppress macrophage (human THP-1 cell line, murine peritoneal macrophages) activity in vitro and the impact of treating mice with this extract on colitis induced by dinitrobenzene sulfonic acid (DNBS). A high-molecular-mass fraction of adult H. diminuta (HdHMW) or excretory/secretory products reduced macrophage activation: lipopolysaccharide (LPS)-induced interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) and poly(I:C)-induced TNF-alpha and IL-6 were suppressed by HdHMW. The active component in the HdHMW extract was minimally sensitive to boiling and trypsin digestion, whereas the use of sodium metaperiodate, as a general deglycosylation strategy, indicated that the immunosuppressive effect of HdHMW was at least partially dependent on a glycan: treating the HdHMW with neuraminidase and alpha-mannosidase failed to inhibit its blockade of LPS-induced TNF-alpha production by THP-1 macrophages. Mice treated with DNBS developed colitis, as typified by wasting, shortening of the colon, macroscopic and microscopic tissue damage, and an inflammatory infiltrate. Mice cotreated with HdHMW (three intraperitoneal injections) displayed significantly less inflammatory disease, and this was accompanied by reduced TNF-alpha production and increased IL-10 and IL-4 production by mitogen-stimulated spleen cells. However, cotreatment of mice with neutralizing anti-IL-10 antibodies had only a minor impact on the anticolitic effect of the HdHMW. We speculate that purification of the immunosuppressive factor(s) from H. diminuta has the potential to lead to the development of novel immunomodulatory drugs to treat inflammatory disease.

FIG. 1.

(A) The crude and high-molecular-mass (HdHMW, >50 kDa) extracts of H. diminuta significantly reduce LPS (1 μg/ml, 48 h)-induced TNF-α production by THP-1 macrophages. The low-molecular-mass (HdLMW, i.e., <50 kDa) preparation was ineffective. (B) Dose dependency of HdHMW in reducing LPS-induced TNF-α (n = 9 THP-1 preparations from three experiments). (C and D) LPS-induced IL-1β and IL-6 production is reduced by concomitant treatment with HdHMW (100 μg/ml) or excretory/secretory products from H. diminuta (HdES, 20 μg/ml) (n = 9 to 18 THP-1 cell preparations/group from three to six experiments; mean ± the SEM; * and #, P < 0.05 compared to the control and LPS, respectively).

FIG. 2.

Bar graph showing that THP-1 cells exposed to LPS (1 μg/ml) and cotreated with either HdHMW (100 μg/ml) or HdES products (20 μg/ml) produced significantly less TNF-α by 4 to 24 h poststimulation (n = 6 to 9 THP-1 cell preparations/group from two to three experiments; mean ± the SEM [*, P < 0.05 compared to the time-matched LPS group]).

FIG. 3.

Bar graph showing the dose dependency of HdES inhibition of LPS-induced TNF-α production by THP-1 cells (n = 3 macrophage preparations; mean ± the SEM [* and #, P < 0.05 compared to the control and LPS, respectively]).

FIG. 4.

(A) A 48-h exposure to HdHMW (>50 kDa; 100 μg/ml) or excretory/secretory products from H. diminuta (HdES, 20 μg/ml) does not significantly alter expression of the Toll-like receptor 4 (TLR4) mRNA. β-Actin was used as a loading control (n = 3; six to nine preparations per group; mm, molecular marker). (B) Representative gels of semiquantitative RT-PCR products demonstrating that 3 h of treatment with LPS (1 μg/ml) or HdHMW (100 μg/ml) does not appreciably alter the expression of markers indicative of an alternatively activated macrophage (i.e., the mannose receptor (MMR or CD206) in humans, YM1 in mice, or Arg-1. Indeed, untreated THP-1 macrophages showed constitutive expression of mRNA for these molecules. (C) iNOS mRNA was induced in peritoneal macrophages by a 48-h treatment with LPS, but not HdHMW, which also does not affect Ym1 or Arg-1 levels (n = 3 macrophage preparations).

FIG. 5.

(A) Treating the high-molecular-mass (>50 kDa; 100 μg/ml) extract of H. diminuta (HdHMW) or excretory/secretory products from H. diminuta (HdES, 20 μg/ml) with proteinase K (PK, 1 mg/ml; 3 h), followed by boiling (20 min), results in a small reduction in the ability of either preparation to inhibit LPS (1 μg/ml, 48 h)-induced TNF-α production by THP-1 macrophages (n = 15 to 27 THP-1 cell preparations/group from five to eight experiments). (B) Bar graph showing that deglycosylation of HdHMW with sodium metaperiodate (SMP) significantly reduces its ability to inhibit TNF-α production by THP-1 cells stimulated with LPS (mean ± the SEM; n = 3 to 4 THP-1 cell preparations [* and #, P < 0.05 compared to the control and LPS, respectively]; the superscript “a” indicates that the active component of HdHMW was recovered from G-25 Sephadex desalting columns that were used to remove the SMP from the HdHMW + SMP preparation).

FIG. 6.

(A) LPS (1 μg/ml)-induced TNF-α production by human (THP-1) and murine (J774) macrophage cell lines is inhibited by treatment with the MAPKK inhibitor, U0126 (10 μM) (mean ± the SEM; n = 20 THP-1 preparations from six experiments and n = 12 J774 preparations from four experiments [*, P < 0.05 compared to the control “con”]). (B) Representative immunoblot (n = 3) showing that LPS (30 min) evokes Erk activation (i.e., increased levels of phosphorylated Erk [pErk]) in THP-1 cells and that this is not affected by HdHMW (100 μg/ml) cotreatment (total Erk expression is shown as a loading control; two separate THP-1 preparations are shown per condition).

FIG. 7.

Bar graphs illustrating reduced severity of colitis in male BALB/c mice induced by intrarectal instillation of DNBS (3 mg in 50% ethanol) with three i.p. injections of the high-molecular-mass (>50 kDa; 1 mg of HdHMW in 200 μl of PBS) extract of H. diminuta (HdHMW) as gauged by changes in body weight (A), representative images of colon length (arrowheads indicate a 7-cm length) (B), and colon length measurements (mean ± the SEM; n = 5 to 11 mice from three experiments; *, P < 0.05 compared to the control) (C).

FIG. 8.

Bar graph illustrating reduced severity of colitis in male BALB/c mice induced by intrarectal instillation of DNBS (3 mg in 50% ethanol) with three i.p. injections of the high-molecular-mass (>50 kDa; 1 mg of HdHMW in 200 μl of PBS) extract of H. diminuta, as gauged by disease activity score (mean ± the SEM; n = 5 to 11 mice from three experiments [*, P < 0.05 compared to the control]).

FIG. 9.

Treatment with the high-molecular-mass (>50 kDa; 1 mg of HdHMW in 200 μl of PBS) extract of H. diminuta reduced DNBS-induced colonic histopathology, as shown by representative hematoxylin-and-eosin-stained images (m, muscle; e, edema; l, lumen of the colon; *, inflammatory infiltrate; arrowhead, epithelium [original magnification, ×400]) (A) and the calculation of histological damage scores (B) and is corroborated by reductions in MPO activity (C) (mean ± the SEM; n = 5 to 11 mice from three experiments [*, P < 0.05 compared to the control]).