Nippostrongylus brasiliensis: identification of intelectin-1 and -2 as Stat6-dependent genes expressed in lung and intestine during infection

Abstract

Elimination of the helminth parasite Nippostrongylus brasiliensis from infected mice is mediated by IL-4 or IL-13 and dependent on the IL-4Ralpha chain and the transcription factor Stat6 in non-hematopoietic cells. However, it is not clear which Stat6-dependent effector molecules mediate worm expulsion. We identified intelectin-1 and -2 as Stat6-dependent genes that are induced during infection. Intelectins can bind galactofuranose, a sugar present only in microorganisms and might therefore serve as microbial pattern element. To analyze whether constitutive expression of intelectin-1 or -2 leads to accelerated pathogen clearance, transgenic mice were generated which express high levels of these genes selectively in the lung. Infection with N. brasiliensis or Mycobacterium tuberculosis did not result in accelerated pathogen clearance in transgenic as compared to wild-type mice. Further, no significant modulation of the immune response in lung or lymph nodes was observed. Thus, under these conditions, intelectins did not enhance pathogen clearance.

Figure 1

Microarray analysis of Stat6-dependent gene expression in lungs and small intestine after N. brasiliensis infection. A) Microarray analysis was performed 9 days after N. brasiliensis infection of BALB/c (WT) or Stat6-deficient mice (Stat6^-/-) as described in Materials and Methods. Each dot corresponds to a single gene on the microarray. A-values on the x-axes indicate the expression levels on a log₂ scale and M-values on the y-axes indicate the difference in expression levels between WT and Stat6^-/- mice on a log₂ scale. Horizontal lines indicate the cut-off for genes considered significantly up- or down-regulated in a Stat6-dependent fashion (M>1.5 or <-1.5, which corresponds to more than 2.8 fold up- or down-regulation, respectively). B) Venn diagram of genes that were up- or down-regulated by Stat6 in lung and/or intestine.

Figure 2

Quantitative RT-PCR for intelectin-1 and intelectin-2. A) Expression levels of intelectin-1 and intelectin-2 in lung and small intestine was analyzed on day 0, 3, 6 and 9 after N. brasiliensis infection of BALB/c mice by quantitative RT-PCR. B) Expression levels of intelectin-1 and intelectin-2 in lung and small intestine of naïve wild-type mice (WT naive) or day 9 N. brasiliensis-infected WT, Stat6^-/- or Rag^-/- mice were determined by quantitative RT-PCR. Data were normalized to HPRT. Error bars indicate standard deviations.

Figure 3

Expression of intelectin-1 and intelectin-2 in the lung of transgenic mice. A) Schematic representation of the expression vector used to generate intelectin-1 (SPINT-1) and intelectin-2 (SPINT-2) transgenic mice. B) RT-PCR from lung, spleen and liver of SPINT-1 and SPINT-2 transgenic mice with transgene-specific primers. C) Quantitative RT-PCR from lung of SPINT-1 or SPINT-2 mice and their negative littermates (neg. litt.) normalized to HPRT expression. Error bars indicate standard deviations.

Figure 4

Administration of galactofuranose (Galf) or galactopyranose (Galp)containing–glycoconjugates. A) Chemical structures of the β-D-Galf glycosides and the β-D-Galp-(1→6)-D-Galp disacharide used for intranasal administration. B) Recruitment of eosinophils and basophils to the lung was determined by flow cytometry 4 days after administration of pooled Galf-glycosides or the Galp-disacharide in SPINT-1 or SPINT-2 transgenic mice or their negative littermates. Untreated transgenic or wild-type mice served as control.

Figure 5

Analysis of the immune response of SPINT-1 and SPINT-2 transgenic mice after N. brasiliensis infection. A) Flow-cytometric analysis of lung and paratracheal lymph nodes of SPINT-1, SPINT-2 and 4get mice on day 9 after N. brasiliensis infection. SPINT mice were crossed to 4get mice to visualize IL-4-expressing cells ex vivo, as described in Materials and Methods. Dot-plots in the second row are gated on R1 (CD4-GFP+) from the dot-plots above and show the frequency of basophils (IgE+SiglecF-) and eosinophils (IgE-SiglecF+) within R1. B) Total number of eosinophils, basophils and Th2 cells in the lung on day 9 after infection. Uninfected wild-type mice were used as controls. C) Total serum IgE levels on day 9 after N. brasiliensis infection. D) The kinetics of worm expulsion was determined in SPINT-1, SPINT-2 and negative littermate control mice. Five mice per group were analyzed. Error bars indicate standard deviations.

Figure 6

Infection of mice with aerosolized Mycobacterium tuberculosis.A) Intelectin transgenic mice (closed bars) or wild-type mice (open bars) were infected with M. tuberculosis as described in Materials and Methods. Colony forming units (cfu) in the lung were determined on day 0, 14 and 42 after infection. B) 42 days after infection cfu were determined in spleen and liver. 3-4 mice per group were analyzed. Error bars indicate standard deviations.