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Keywords:

  • DC;
  • Escherichia coli;
  • excretory/secretory;
  • helminth;
  • immunomodulation;
  • LPS;
  • mouse;
  • Neisseria meningitides;
  • toll-like receptor;
  • Trichinella spiralis

Summary

  1. Top of page
  2. Summary
  3. Research note
  4. References

Evidence from experimental studies indicates that during chronic infections with certain helminth species a regulatory network is induced that can down-modulate not only parasite-induced inflammation but also reduce other immunopathologies such as allergies and autoimmune diseases. The mechanisms however, and the molecules involved in this immunomodulation are unknown. Here, we focus on the effect of Trichinella spiralis excretory/secretory antigens (TspES) on the innate immune response by studying the effect of TspES on DC maturation in vitro. Bone marrow-derived DC from BALB/c mice were incubated with TspES either alone or in combination with LPS derived from two different bacteria. As indicators of DC maturation, the cytokine production (IL-1α, IL-6, IL-10, IL-12p70 and TNF-α) and the expression of various surface molecules (MHC-II, CD40, CD80 and CD86) were measured. Results indicate that while TspES alone did not change the expression of the different surface molecules or the cytokine production, it completely inhibited DC maturation induced by Escherichia coli LPS (E. coli LPS). In contrast, DC maturation induced by LPS from another bacterium, Neisseria meningitidis, was not affected by TspES. These results were confirmed using TLR4/MD2/CD14 transfected HEK 293 cells. In conclusion, T. spiralis ES antigens lead to suppression of DC maturation but this effect depends on the type of LPS used to activate these cells.


Research note

  1. Top of page
  2. Summary
  3. Research note
  4. References

Trichinella spiralis is a zoonotic helminth with worldwide distribution. Humans and other mammals become infected after ingestion of the larval stage present in raw or undercooked meat. The larvae migrate to the intestine of the infected host and mature to the adult reproductive stage where the parasites mate and newborn larvae (NBL) are released within 6 days after infection. These NBL migrate to striated muscle, where they encyst and can remain in a dormant stage for years. Infection can result in clinical disease characterized by an acute, followed by a convalescent and eventually chronic phase in which the larvae are still present (1).

During infection, pathogens and/or their products may interact with DCs via various families of pattern recognition receptors such as toll-like receptors (TLRs) (2,3), giving rise to DC maturation which is fundamental for the generation and polarization of the adaptive immune response.

Several studies using experimental models have shown that infection with certain helminth species, including T. spiralis, leads to suppression not only of parasite-induced inflammation but also of other immunopathologies (4–7). The precise mechanisms and molecules involved in this process are unknown. Furthermore, few studies on the interaction between helminths and/or their products and the innate immune system have been carried out (8). In this study, we aim at determining the effect of T. spiralis excretory/secretory (ES) antigens (TspES) on DC maturation in vitro.

Bone marrow-derived DC from Balb/c mice, were prepared using a modified procedure previously described by Lutz et al. (9). Briefly, cells were incubated in six-well plates (BD Falcon™; BD Europe, Erembodegem, Belgium) and at day 2 and 4, 20 ng/mL rGM-CSF (Cytocen, Utrecht, The Netherlands) was added. At day 7, cells were incubated with the ES antigens from T. spiralis (TspES) and/or the following TLR ligands: Ultra Pure E. coli K12 LPS (LPSEco) (Invivogen, Toulouse, France) or LPS of Neisseria meningitidis (LPSNeiss), purified as described previously (10). Trichinella spiralis ES antigens were prepared from muscle larvae from infected rats, recovered by acid-pepsin digestion and washed and incubated in RPMI medium for 19 h as described by Gamble (11). After centrifugation, the supernatant containing TspES was dialyzed, concentrated and the protein concentration was determined. Endotoxin determination was performed using the QCL-1000 chromogenic LAL Endpoint Assay (Lonza, Basel, Switzerland). The endotoxin value in TspES was below the detection limit of the assay (0·2 EU/mL).

After O/N incubation of DCs with TspES alone, TspEs in combination with LPSEco or LPSNeiss or each LPS alone, supernatants were harvested for cytokine determination and phenotypes of the cells were analysed by flow cytometry (FACScan; BD Biosciences, Erembodegem, Belgium) analysis, following standard FACS protocols using monoclonal antibodies directed against the following surface molecules: CD40, CD80, CD86 and MHC class II, labelled with phycoerythrin and CD11c labelled with allophycocyanin. All fluorochrome-conjugated antibodies were purchased from BD Biosciences. Propidium iodide (Sigma-Aldrich, Zwijndrecht, Netherlands) was used to determine the viability of the cells. Viable cells were 70–95% positive for the DC marker CD11c. For analysis, CD11c-positive cells were gated. Production of cytokines: IL-1, IL-6, IL-10, IL-12p70 and TNF-α was measured using ELISA kits (BD OptEIA; BD Biosciences), according to the manufacturer’s instructions. Data are represented as mean ± SD. An unpaired, two-tailed Student’s t-test was used to determine significant differences. P < 0·05 was considered statistically significant.

In addition to bone marrow-derived DC, we used HEK293 cells, transfected with mouse TLR4/MD2/CD14 (Invivogen). Cells (2 × 105/well) were incubated O/N with TspES alone, TspEs in combination with LPSEco or LPSNeiss or each LPS alone. The production of IL-8 in response to the different antigens (or combinations) was measured to analyse the effect of TspES on LPS-induced activation of the cells.

Incubation of DC with 1 ng/mL LPSEco or LPSNeiss, resulted as expected in increased expression of the surface molecules MHC II, CD40, CD80 and CD86. Incubation of the cells with 5 μg/mL TspES alone did not result in changes in the expression of any of these surface molecules as compared to cells incubated in medium only. However, when cells were incubated with LPSEco in combination with TspES, the surface molecule expression was reduced to background levels (Table 1). The suppression of LPSEco induced surface molecule expression by TspES was dose dependent. At TspES concentrations of 5 and of 0·5 μg/mL, the expression of surface molecules induced by 1 ng/mL of LPSEco was almost completely suppressed, whereas at 0·05 μg/mL the suppressive effect ranged between 32% and 57% and at a concentration of 0·005 μg/mL suppression was no longer observed (data not shown).

Table 1.   Expression of surface molecules on mouse bone marrow-derived DC
Incubation of DC with TspES and LPS
Surface moleculesMHC IICD40CD80CD86
MFI (SD)% pos (SD)MFI (SD)% pos (SD)MFI (SD)% pos (SD)MFI (SD)% pos (SD)
  1. Median fluorescence intensity (MFI) and percentage of cells positive (% pos), for the different surface molecules after O/N incubation of the cells in medium only or with T. spiralis ES antigens (TspES), E. coli LPS (LPSEco), LPSEco together with TspES, N. meningitidis LPS (LPSNeiss) or LPSNeiss together with TspES. Significant differences between MFI and % pos cells incubated with LPS only and cells incubated with LPS together with TspES are indicated with asterisk (*P < 0·05). Values in the table are from one experiment performed in triplicate (±SD) that has been repeated at least 10 times with similar results.

medium106 (79)49 (12)40 (1)21 (1)83 (9)41 (3)41 (19)22 (7)
TspES563 (100)72 (1)40 (4)20 (3)97 (4)46 (1)74 (3)38 (1)
LPSEco2036 (326)90 (1)119 (7)68 (2)261 (23)71 (3)357 (86)75 (4)
LPSEco + TspES534 (97)*72 (3)*42 (4)*23 (3)*103 (13)*48 (3)*73 (4)*38 (2)*
LPSNeiss952 (345)87 (2)142 (11)74 (1)323 (165)68 (6)378 (129)76 (3)
LPSNeiss + TspES1086 (429)87 (2)147 (13)74 (2)304 (78)70 (2)421 (108)77 (3)

In contrast, LPSNeiss induced expression of the surface molecules was not affected by TspES (Table 1), even when different concentrations of LPSNeiss were used to activate DC (data not shown).

Similar to the findings regarding the expression of surface molecules, TspES alone did not induce DC to produce cytokines. However, the production of all cytokines induced by LPSEco was reduced to background levels when it was combined with TspES. This reduction was statistically significant (P < 0·05) in all cases. In contrast, no differences could be observed between cytokine production of cells incubated with LPSNeiss alone and LPSNeiss in combination with TspES (Figure 1a–e).

image

Figure 1.  Cytokine production, determined by ELISA, after O/N incubation of cells in medium (Med) only or with T. spiralis ES antigens (TspES), E. coli LPS (LPSEco), LPSEco together with TspES, N. meningitidis LPS (LPSNeiss) or LPSNeiss together with TspES. The different cytokines are indicated at the top of every figure. Error bars represent SD of one experiment carried out in triplicate, representative of three independent experiments. Significant differences in cytokine production between cells incubated with LPS only and cells incubated with LPS together with TspES are indicated with asterisk *P < 0·05. (a–e) Cytokine concentrations in supernatant of DC, in pg/ml. (f) Cytokine concentration in supernatant of HEK cells transfected with TLR4/MD2/CD14, in ng/mL.

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Cell viability was measured by flow cytometry analysis of cells stained with propidium iodide and annexin-V. The percentage of stained cells did not exceed 5% of the total number of cells (data not shown). As cells incubated with this TspES were highly viable, cell death by apoptosis by TspES could not explain the observed effects.

Using TLR4/MD2/CD14 transfected HEK cells incubated with either E. coli or N. meningitidis LPS alone, or together with TspES, resulted in findings comparable to those observed using DC. In this system, TspES suppressed IL-8 production induced by LPSEco but no significant inhibition of the IL-8 production was observed when TspES was combined with LPSNeiss (Figure 1f).

Little is known about the mechanisms involved in the modulatory effect of infection with T. spiralis on the immune response to other, unrelated, antigens. From murine models there is some evidence indicating that during the early phase of infection with T. spiralis the systemic immune response against heterologous antigens is suppressed (6), and severity of EAE in T. spiralis infected rats is reduced (7). Studies carried out in vitro have shown that ES components of T. spiralis have a suppressive effect on the function of human neutrophils and murine macrophages (12,13). Unlike the findings from our study, Ilic et al. (14) reported on up-regulation of co-stimulatory molecules (CD54 and CD86) and production of IL-10 by DC incubated with different types of T. spiralis antigens. The differences between these two studies may rely on the nature and concentration of the antigens used. Ilic et al. used 100 μg/mL of crude larval antigen; mannose-rich glycoproteins, ES products from adult parasites and soluble extract of NBL whereas we use 5 μg/mL of ES products from muscle larvae.

The suppressive effect of TspES on DC maturation by E. coli but not by N. meningitidis LPS were confirmed using HEK293 cells transfected with mouse TLR4/MD2/CD14. These results indicate that TspES affect the interaction of E. coli LPS with TLR4. Involvement of TLR4 in modulation of DC maturation by other helminth antigens has been previously described by Goodridge et al. (15) using ES62, a phosphorylcholine-containing glycoprotein secreted by the filarial nematode Acanthocheilonema viteae and by others using soluble egg antigens derived from Schistosoma mansoni (16,17). Recently, Segura et al. (18) reported on the inhibition of TLR-induced cytokine production of DC with Heligmosomoides polygyrus ES for several TLR ligands.

Results from our study do not only indicate a suppressive effect of TspES in TLR4 activation, but in addition show that this effect is dependent on the TLR4 ligand that is being used. The differential effect of TspES on DC maturation by LPS from the two different bacteria could be explained by the difference in sugar composition of these molecules. The sugar molecules present in TspES may compete with the carbohydrates present in the E. coli but not in the N. meningitidis LPS for the same binding site of the TLR4 complex. Another possible explanation is the involvement of the CD14 molecule within the TLR4/MD2/CD14 complex. LPS in the smooth (S) form, as synthesized by most wild type gram-negative bacteria, such as E. coli, requires the CD14 molecule as a co-receptor for TLR4 to activate the signalling pathway in DC (19,20). The R-form LPS synthesized by rough (R) mutants of gram-negative bacteria, which lacks the O-polysaccharide chain that is present in the S-LPS, is capable of DC activation through TLR4 independently of CD14. Neisseria meningitidis synthesizes LPS with a highly reduced number of sugar residues, thus resembling R-form LPS (21). The differential effect that we observed for TspES suppression on DC maturation induced by E. coli and N. meningitidis LPS could therefore suggest a role for CD14 in addition to TLR4. The role of these molecules and the mechanisms involved in the suppressive effect of Tsp ES on DC maturation are currently under investigation.

The contrasting effect of TspES on DC activated with two different bacterial LPS, both acting via TLR4, indicate a very specific mechanism of action for this helminth antigen. These results suggest that the immunosuppressive effect of helminth antigens on immune responses to unrelated antigens and other pathogens may depend not only on the helminth species involved (22) but also on the nature of the unrelated antigen. Future investigations including in vivo studies may provide a better insight into the mechanisms involved in immunomodulation by these helminth molecules. Finally, identification and purification of the molecules in TspES that exert the immunosuppressive effect is essential as they could be used as therapeutic agents for treatment of different immunopathological conditions.

References

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  4. References
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