Helminth regulation of host IL-4Ralpha/Stat6 signaling: mechanism underlying NOS-2 inhibition by Trichinella spiralis

Abstract

Gastrointestinal nematode infection is known to alter host T cell activation and has been used to study immune and inflammatory reactions in which nitric oxide (NO) is a versatile player. We previously demonstrated that Trichinella spiralis infection inhibits host inducible NO synthase (NOS-2) expression. We now demonstrate that (i) an IL-4 receptor alpha-subunit (IL-4Ralpha)/Stat6-dependent but T cell-independent pathway is the key for the nematode-induced host NOS-2 inhibition; (ii) endogenous IL-4 and IL-13, the only known IL-4Ralpha ligands, are not required for activating the pathway; and (iii) treatment of RAW264.7 cells with parasite-cultured medium inhibits NOS-2 expression but not cyclooxygenase 2 expression. We propose that a yet-unidentified substance is released by the nematode during the host-parasite interaction.

Fig. 6.

Inhibition of NOS-2 expression in RAW264.7 cells by a T. spiralis-secreted substance(s). (A) Immunoblotting for NOS-2 and Cox-2 in RAW264.7 cells cultured with NBL medium and control medium. LPS was used for induction of NOS-2 and Cox-2. Treatment with NBL medium inhibited LPS (50 ng/ml)-stimulated NOS-2 expression by 100%. Increasing the concentration of LPS to 100 ng/ml only partially overcame the inhibition by worm-cultured medium [expression in response to 100 ng/ml LPS was still <50% of the expression in response to LPS (50 ng/ml) in control medium]. In contrast, the expression of COX-2 was not significantly altered. (B) Real-time quantitative RT-PCR analysis of NOS-2 mRNA in RAW264.7 cells cultured with NBL medium and control medium. Changes in RAW264.7 cell NOS-2 mRNA were quantified after normalizing to NOS-2 standard. Data are expressed as means ± SEM (n = 3–6). *, P < 0.01 compared with control. (C) In vitro culture of T. spiralis adult worm and NBL.

Fig. 1.

Suppression of NOS-2 by T. spiralis (T.s.) infection depends on an IL-4Rα/Stat6-mediated signaling pathway (scheme in Left). Immunoblotting for NOS-2 in ileum from IL-4Rα^–/– and WT control mice (A); tyrosine kinase inhibitor herbimycin A treated and control mice (B); and Stat6^–/– and WT control mice (C). Each indicated lane number represents an individual animal used in the experiment. Ileal NOS-2 expression was abolished after 9 days of T. spiralis infection in control groups, whereas no change was noticed in IL-4Rα^–/– (A, lanes 4 and 5), herbimycin A-treated (B, lanes 5 and 6), and Stat6^–/– (C, lanes 4–6) mice.

Fig. 2.

A T cell-independent IL-4Rα/Stat6 pathway is activated during T. spiralis-induced inhibition of NOS-2. Immunoblotting for NOS-2 in ileum from athymic mice (A); γδ TCR^–/– and WT control mice (B); and αβγδ TCR^–/– and WT control mice (C). Each indicated lane number represents an individual animal used in the experiment. Ileal NOS-2 expression was abolished after 9 days of T. spiralis infection in both control and T cell-deficient mice.

Fig. 3.

Endogenous IL-4 deficiency failed to affect T. spiralis-induced NOS-2 inhibition. (A) Tissue levels of IL-4 within the jejunal and ileal mucosa from T. spiralis-infected CF-1 mice were measured (uninfected animal was labeled as day 0). A marked increase of IL-4 after 5 days of infection in jejunum and after 9 days of infection in ileum was observed (*, P < 0.01). Data are the mean of four animals in each group. (B) Immunoblotting for NOS-2 in ileum from IL-4^–/– and WT control mice. Each indicated lane number represents an individual animal used in the experiment. Ileal NOS-2 expression was abolished after 9 days of T. spiralis infection in both control and IL-4^–/– mice.

Fig. 4.

The impact of endogenous IL-4 and T cells on serum concentration of IL-13. (A) Despite a lack of endogenous IL-4 production, a 9-day T. spiralis infection resulted in a marked increase in serum IL-13 (85-fold). The elevations of IL-13 were even higher (431 pg/ml) in WT control mice. Data are the mean of nine animals in each group (*, P < 0.01). (B) T. spiralis-infection-stimulated IL-13 levels were markedly decreased in α/β/γ/δ TCR^–/– mice, whereas they remained high in WT control mice. Data are the mean of three animals in each group (*, T.s. vs. control, P < 0.05; †, T cell KO vs. T cell WT, P < 0.01).

Fig. 5.

Deficiency of both endogenous IL-4 and IL-13 failed to affect T. spiralis-induced inhibition of NOS-2. (A) Immunoblotting for NOS-2 in ileum from IL-4^–/–/IL-13^–/– and WT control mice. (B) Immunoblotting for NOS-2 and β-actin in ileum from IL-4^–/–/IL-13^–/– mice. Each indicated lane number represents an individual animal used in the experiment. Ileal NOS-2 expression was abolished after 9 days of T. spiralis infection in both IL-4^–/–/IL-13^–/– (A, lanes 6 and 7) and WT control (A, lanes 8–10) mice. The same sample was blotted with anti-β-actin (45 kDa) antibody, and no inhibition was observed (B).