IL-4 Attenuates Th1-Associated Chemokine Expression and Th1 Trafficking to Inflamed Tissues and Limits Pathogen Clearance

<div><p>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 <i>Leishmania major</i>, as pathogen clearance is highly dependent on IFNγ-producing CD4+ T cells at the infection site. Short-term IL-4 blockade in established <i>L. major</i> 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.</p></div

Immune trafficking genes upregulated following short-term IL-4 blockade.

<p><b>A–C</b>) 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.</p

Increased T cell infiltration and improved <i>L. major</i> parasite clearance following short-term IL-4 blockade.

<p><b>A</b>) Increased immune cell accumulation after anti-IL-4 treatment in the <i>L. major</i> 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>B</b>) Anti-<i>Leishmania</i> 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. <b>C</b>) Parasite load in ear dermis after <i>L. major</i> 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. <b>D</b>) Relative gene expression in the <i>L. major</i>-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.</p

Short-term IL-4 blockade modulates the inflamed tissue environment.

<p><b>A</b>) Antigen-specific IL-4 and IFNγ production by ELISPOT, day 7 following OVA/CFA immunization. <b>B</b>) Heat map of TLDA array genes (n = 94 genes examined, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071949#pone.0071949.s001" target="_blank">Table S1</a>) 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.</p

Alternatively activated macrophage genes down-regulated following short-term IL-4 blockade.

<p><b>A–C</b>) 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. <b>A</b>) Relative gene expression for Th2-associated genes, all genes shown were not significantly differentially expressed between groups by two-way ANOVA. <b>B–C</b>) 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.</p

Increased T cell trafficking to inflamed dermis following short-term IL-4 blockade.

<p><b>A</b>) 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>B</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. <b>C</b>) cytokine production in draining LN from same experiments as in B). Statistics by paired T test, ns = p>0.05. <b>D</b>) 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.</p

Early Type 2 immunity in the <i>Leishmania major</i> infected dermis with broad down-regulation of Type 1 chemokines.

<p><b>A</b>) Number of CD4+CD3+GFP+ T cells in the <i>L. major</i> infected ear dermis 2 weeks post-infection compared to PBS-injected control and ears from naïve mice. <b>B</b>) Number of innate cell types in <i>L. major</i> infected ear 2 weeks post-infection compared to PBS-injected control and naïve mice. <b>C</b>) Basophil+Eosinophil/Neutrophil ratio of cells in the dermis 2 weeks post-infection compared to PBS or OVA/CFA immunization and naïve mice. <b>A–C</b>, Statistics by Mann Whitney: *p<0.05, **p<0.01, ***p<0.001. <b>D</b>) Fold change in gene expression in the ear dermis of <i>L. major</i>-infected versus OVA/CFA immunized. RNA at 2 weeks post-infection/immunization analyzed by TLDA gene array as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071949#pone.0071949.s001" target="_blank">Table S1</a>. Table shows all genes statistically (TTest) differentially expressed between groups: bold, those genes underrepresented in the <i>L. major</i> infected dermis; not bold, those genes enriched in <i>L. major</i> infected dermis compared to OVA/CFA. Data from 3 independent experiments.</p

Pre-existing neutralizing antibody mitigates B cell dysregulation and enhances the Env-specific antibody response in SHIV-infected rhesus macaques

<div><p>Our central hypothesis is that protection against HIV infection will be powerfully influenced by the magnitude and quality of the B cell response. Although sterilizing immunity, mediated by pre-formed abundant and potent antibodies is the ultimate goal for B cell-targeted HIV vaccine strategies, scenarios that fall short of this may still confer beneficial defenses against viremia and disease progression. We evaluated the impact of sub-sterilizing pre-existing neutralizing antibody on the B cell response to SHIV infection. Adult male rhesus macaques received passive transfer of a sub-sterilizing amount of polyclonal neutralizing immunoglobulin (Ig) purified from previously infected animals (SHIVIG) or control Ig prior to intra-rectal challenge with SHIV_SF162P4 and extensive longitudinal sampling was performed. SHIVIG treated animals exhibited significantly reduced viral load and increased <i>de novo</i> Env-specific plasma antibody. Dysregulation of the B cell profile was grossly apparent soon after infection in untreated animals; exemplified by a ≈50% decrease in total B cells in the blood evident 2–3 weeks post-infection which was not apparent in SHIVIG treated animals. IgD+CD5+CD21+ B cells phenotypically similar to marginal zone-like B cells were highly sensitive to SHIV infection, becoming significantly decreased as early as 3 days post-infection in control animals, while being maintained in SHIVIG treated animals, and were highly correlated with the induction of Env-specific plasma antibody. These results suggest that B cell dysregulation during the early stages of infection likely contributes to suboptimal Env-specific B cell and antibody responses, and strategies that limit this dysregulation may enhance the host’s ability to eliminate HIV.</p></div

Auto-antigen microarray profiles of 9G4+ IgG isolated from HIV-infected patients.

<p>Plasma, 9G4+ and 9G4- antibodies were screened for IgG binding to 85 auto-antigens. Columns represent HIV samples, rows represent antigens and color maps to log₁₀ signal (gray indicates lower limit of detection, LLOD). Columns are organized by sample type (unfractionated plasma, 9G4+, 9G4-) and subject order within groups is the same for each type. Rows are clustered based on Euclidean distance to facilitate visualization. Antigens that resulted in no signal are listed to the right of the heat map. Antigens in red indicate higher 9G4+ binding relative to 9G4- based on a Wilcoxon signed-rank test at p<0.05. Average signal data where either the signal intensity was greater than 100 or the signal-to-noise ratio was greater than 2 were considered to be above the LLOD; these data were background-subtracted (based on PBS control per antigen) and then log₁₀ transformed.</p

9G4+ antibodies from HIV-1 infected patients bind apoptotic lymphocytes.

<p>Unfractionated serum from normal donors (n=16), SLE (n=21) and HIV- infected (n=57) were used in apoptotic binding assays. (<b>A</b>-<b>C</b>) The Jurkat Human T cell line was treated with camptothecin to induce apoptosis then incubated with patient serum. Serum binding to cells was detected using FITC- labeled 9G4 rat anti-human monoclonal antibody. Apoptotic Cell Gate was drawn around cells stained positive for viability dye, representing the dying cell population. (<b>C</b>) The shaded histogram represents cells incubated with PBS in the absence of patient serum. The thin-line represents the antibody binding from a normal serum donor and the Bold-line is representative of 9G4+ antibody binding from an HIV infected patient. (<b>D</b>) Data from all samples are plotted. The dashed-line represents the normal donor mean plus 2 standard deviations. Significance determined by Mann-Whitney test.</p