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. 2013 Aug 26;8(8):e71949.
doi: 10.1371/journal.pone.0071949. eCollection 2013.

IL-4 attenuates Th1-associated chemokine expression and Th1 trafficking to inflamed tissues and limits pathogen clearance

Affiliations

IL-4 attenuates Th1-associated chemokine expression and Th1 trafficking to inflamed tissues and limits pathogen clearance

Christopher A Lazarski et al. PLoS One. .

Abstract

Interleukin 4 (IL-4) plays a central role in the orchestration of Type 2 immunity. During T cell activation in the lymph node, IL-4 promotes Th2 differentiation and inhibits Th1 generation. In the inflamed tissue, IL-4 signals promote innate and adaptive Type-2 immune recruitment and effector function, positively amplifying the local Th2 response. In this study, we identify an additional negative regulatory role for IL-4 in limiting the recruitment of Th1 cells to inflamed tissues. To test IL-4 effects on inflammation subsequent to Th2 differentiation, we transiently blocked IL-4 during ongoing dermal inflammation (using anti-IL-4 mAb) and analyzed changes in gene expression. Neutralization of IL-4 led to the upregulation of a number of genes linked to Th1 trafficking, including CXCR3 chemokines, CCL5 and CCR5 and an associated increase in IFNγ, Tbet and TNFα genes. These gene expression changes correlated with increased numbers of IFNγ-producing CD4+ T cells in the inflamed dermis. Moreover, using an adoptive transfer approach to directly test the role of IL-4 in T cell trafficking to the inflamed tissues, we found IL-4 neutralization led to an early increase in Th1 cell recruitment to the inflamed dermis. These data support a model whereby IL-4 dampens Th1-chemokines at the site of inflammation limiting Th1 recruitment. To determine biological significance, we infected mice with Leishmania major, as pathogen clearance is highly dependent on IFNγ-producing CD4+ T cells at the infection site. Short-term IL-4 blockade in established L. major infection led to a significant increase in the number of IFNγ-producing CD4+ T cells in the infected ear dermis, with no change in the draining LN. Increased lymphocyte influx into the infected tissue correlated with a significant decrease in parasite number. Thus, independent of IL-4's role in the generation of immune effectors, IL-4 attenuates lymphocyte recruitment to the inflamed/infected dermis and limits pathogen clearance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Short-term IL-4 blockade modulates the inflamed tissue environment.
A) Antigen-specific IL-4 and IFNγ production by ELISPOT, day 7 following OVA/CFA immunization. B) Heat map of TLDA array genes (n = 94 genes examined, Table S1) expressed in the ear dermis of control and anti-IL-4 treated OVA/CFA immunized mice. Anti-IL-4 treatment (or PBS) was administered on day 7 and 10 post-immunization and RNA extracted on day 14. 29 genes (rows) were identified based on the treatment p-value (<0.05) after a 2-way ANOVA. The columns correspond to 18 samples. The colored circles beneath the heat map indicate which of the four experiments the sample was processed in. Color in the heat map corresponds to the z-score of relative expression data. For each gene, the z-score was computed separately in each of the four experiments rather than using all 18 samples together. Genes were clustered using correlation and average linkage.
Figure 2
Figure 2. Immune trafficking genes upregulated following short-term IL-4 blockade.
A–C) Genes expressed in the ear dermis of control and anti-IL-4 treated OVA/CFA immunized mice. Anti-IL-4 treatment (or PBS) was administered on day 7 and 10 post-OVA/CFA immunization and RNA extracted on day 14. Relative gene expression for those genes significantly different between control and anti-IL-4 treated groups. Statistics by two-way ANOVA: *p<0.05, **p<0.01, ***p<0.001. Data from 4 independent experiments.
Figure 3
Figure 3. Alternatively activated macrophage genes down-regulated following short-term IL-4 blockade.
A–C) Gene expression in the ear dermis of control and anti-IL-4 treated OVA/CFA immunized mice. Anti-IL-4 treatment was administered on day 7 and 10 post-immunization and RNA extracted on day 14. A) Relative gene expression for Th2-associated genes, all genes shown were not significantly differentially expressed between groups by two-way ANOVA. B–C) Genes significantly down-regulated between anti-IL-4 treated and control groups. Statistics by two-way ANOVA: *p<0.05, **p<0.01. Data from 4 independent experiments.
Figure 4
Figure 4. Increased T cell trafficking to inflamed dermis following short-term IL-4 blockade.
A) Increased immune cell accumulation in the immunized dermis: left panel, number of CD45+ cells from one of 4 representative experiments, each symbol represents an individual mouse; right panel, number of CD4+CD3+ T cells, data from 3 experiments, each symbol represents an individual mouse. Statistics by two-tailed T test: *p<0.05, **p<0.01. B) Increased number of OVA-specific IFNγ producers in the ear dermis after IL-4 blockade, by ELISPOT. Each symbol represents an individual experiment with data obtained from pooled tissue from 3–4 mice; control and anti-IL-4 treated groups from the same experiment are paired. Statistics by paired T test, *p<0.05. C) cytokine production in draining LN from same experiments as in B). Statistics by paired T test, ns = p>0.05. D) Adoptive transfer of in vitro generated Th1 DO11.10+ Thy1.2+ T cells into Thy1.1+ mice immunized with OVA/CFA in one ear (+OVA) and CFA in other ear (−OVA). Mice were treated with anti-IL-4 (11B11) on days 7 and 10 after immunization, and Th1 cells transferred on day 12 after immunization. Left panel, representative FACS profile of endogenous (Thy1.1+) and transferred cells (Thy1.2+) in ear dermis 24 h after cell transfer. Middle and right panels, number of transferred cells in dermis or draining LN, respectively, 24 hours after cell transfer. Statistics by two-way ANOVA.
Figure 5
Figure 5. Early Type 2 immunity in the Leishmania major infected dermis with broad down-regulation of Type 1 chemokines.
A) Number of CD4+CD3+GFP+ T cells in the L. major infected ear dermis 2 weeks post-infection compared to PBS-injected control and ears from naïve mice. B) Number of innate cell types in L. major infected ear 2 weeks post-infection compared to PBS-injected control and naïve mice. C) Basophil+Eosinophil/Neutrophil ratio of cells in the dermis 2 weeks post-infection compared to PBS or OVA/CFA immunization and naïve mice. A–C, Statistics by Mann Whitney: *p<0.05, **p<0.01, ***p<0.001. D) Fold change in gene expression in the ear dermis of L. major-infected versus OVA/CFA immunized. RNA at 2 weeks post-infection/immunization analyzed by TLDA gene array as in Table S1. Table shows all genes statistically (TTest) differentially expressed between groups: bold, those genes underrepresented in the L. major infected dermis; not bold, those genes enriched in L. major infected dermis compared to OVA/CFA. Data from 3 independent experiments.
Figure 6
Figure 6. Increased T cell infiltration and improved L. major parasite clearance following short-term IL-4 blockade.
A) Increased immune cell accumulation after anti-IL-4 treatment in the L. major infected dermis: number of CD4+CD3+ T cells, representative data from one of three experiments. Anti-IL-4 treatment was administered on day 7 and 10 post-infection and cells analyzed on day 14. B) Anti-Leishmania IFNγ and IL-4 producing T cells by ELISPOT in infected ear (top) and draining LN (bottom) after anti-IL-4 treatment or PBS as in A). Data from 4 independent experiments. Statistics comparing PBS and 11B11 by ANOVA. C) Parasite load in ear dermis after L. major infection and anti-IL-4 treatment on day 7 and day 10, infected tissue harvested on day 21. A) and C) Statistics by Mann Whitney: *p<0.05. ***p<0.001. D) Relative gene expression in the L. major-infected dermis after 11B11 or PBS treatment (as in A); 8 mice per group, from two independent experiments. Statistics by one-tailed T test, * <0.05.

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