Eosinophils and IL-4 Support Nematode Growth Coincident with an Innate Response to Tissue Injury.

It has become increasingly clear that the functions of eosinophils extend beyond host defense and allergy to metabolism and tissue regeneration. These influences have strong potential to be relevant in worm infections in which eosinophils are prominent and parasites rely on the host for nutrients to support growth or reproduction. The aim of this study was to investigate the mechanism underlying the observation that eosinophils promote growth of Trichinella spiralis larvae in skeletal muscle. Our results indicate that IL-4 and eosinophils are necessary for normal larval growth and that eosinophils from IL-4 competent mice are sufficient to support growth. The eosinophil-mediated effect operates in the absence of adaptive immunity. Following invasion by newborn larvae, host gene expression in skeletal muscle was compatible with a regenerative response and a shift in the source of energy in infected tissue. The presence of eosinophils suppressed local inflammation while also influencing nutrient homeostasis in muscle. Redistribution of glucose transporter 4 (GLUT4) and phosphorylation of Akt were observed in nurse cells, consistent with enhancement of glucose uptake and glycogen storage by larvae that is known to occur. The data are consistent with a mechanism in which eosinophils promote larval growth by an IL-4 dependent mechanism that limits local interferon-driven responses that otherwise alter nutrient metabolism in infected muscle. Our findings document a novel interaction between parasite and host in which worms have evolved a strategy to co-opt an innate host cell response in a way that facilitates their growth

Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen

Eosinophils promote larval growth in an innate context.

<p>(A)–(C), C57BL/6, PHIL, Rag1^-/-, PHIL/Rag1^-/- and Rag2^-/-γc^-/- mice were injected IV with 25,000 NBL. (A) Body size (area) of larvae recovered from indicated strains 13 days post injection. (B) Body size (area) of larvae recovered from Rag1^-/- and Rag2^-/-γc^-/- mice, 13 days post injection. (C) PHIL/Rag1^-/- mice received PBS or 5 × 10⁶ eosinophils from infected IL-5Tg⁺ or IL-5Tg⁺/IL-4^-/- mice every 48 h from 0–5 days post IV infection. Body size (area) of larvae, 13 days post injection. Each data set was collected from two experiments with similar results. Values represent mean ± SD; n = 3–4 mice. Significant differences were determined by Student’s <i>t</i> test or ANOVA and Tukey’s test. ***p < 0.0001.</p

STAT6 signaling in bone marrow-derived cells is essential for parasite growth.

<p>B6.SJL and STAT6^-/- mice were reconstituted with bone marrow cells from B6.SJL or STAT6^-/- mice for eight weeks, then infected by IV injection of 25,000 NBL. Areas of larvae were estimated, 13 days post injection. Each data set was collected from two experiments with similar results. n = 4 mice. Significant differences were determined by ANOVA and Tukey’s test. ***p < 0.0001.</p

GLUT4 and phosphorylation of Akt are enhanced in nurse cells.

<p>Detection of (A) Akt, phospho-Akt^Ser473 and (B) GLUT4 in tongues collected from Rag1^-/- mice infected by IV injection with NBL, 13 days post injection. Arrows indicate nurse cells. Solid arrowheads indicate muscle larvae. Opened arrowheads indicate uninfected muscle cells. Scale bar = 100μm. n = 3 mice.</p

IL-4/STAT6 signaling in eosinophils is required for larval growth.

<p>(A) Body size (area) of larvae, 17dpi. ΔdblGATA mice received PBS or 5 × 10⁶ eosinophils every 48 h between 5 and 9 dpi or 11 and 15 dpi (oral infection). (B)–(C), ΔdblGATA mice received PBS or 5 × 10⁶ eosinophils from infected IL-5Tg⁺ or IL-5Tg/STAT6^-/- mice every 48 h from 5–9 dpi. (B) Total body larval burdens in muscle, 28 dpi. (C) Body size (area) of larvae, 17 dpi. (D) ΔdblGATA mice received PBS or 5 × 10⁶ eosinophils from infected IL-5Tg⁺ or IL-5Tg⁺/IL-4^-/- mice every 48 h from 5–9 dpi. Body size (area) of larvae, 17dpi. (E) ΔdblGATA mice received PBS or 5 × 10⁶ eosinophils from uninfected or infected IL-5Tg⁺ every 48 h from 5–9 dpi. Body size (area) of larvae, 17dpi. (F) Body size (area) of larvae recovered from C57BL/6 and IL-5^-/- mice injected 25,000 NBL IV, 13 days post injection. (G) IL-4^-/- mice received PBS or 5 × 10⁶ eosinophils from infected IL-5Tg⁺ or IL-5Tg⁺/IL-4^-/- mice every 48 h from 0–5 days post IV infection. Body size (area) of larvae, 13 days post injection. Each data set was collected from two experiments with similar results. Values represent mean ± SD; n = 4 mice. Significant differences were determined by ANOVA and Tukey’s test. *p < 0.05, **p < 0.001, ***p < 0.0001.</p

Eosinophils suppress STAT1 signaling and modulate metabolism in infected muscle.

<p>Microarray-based gene expression profiling of diaphragms of uninfected and infected WT and PHIL mice on 0, 2 and 7 days post IV infection. Pathways detected by GSEA to be enriched in (A) PHIL or (B) WT mice at 7 days post IV infection. (C) Selected genes from interferon signaling, TCA cycle and fatty acid metabolism pathways. Maximum mean fold change (FC) between PHIL and WT at 7 days post IV infection is shown. Color pattern represents row Z-score.</p

IL-4/STAT6 signaling pathway is essential for larval growth.

<p>Area of larvae recovered from (A)–(C), C57BL/6, STAT6^-/-, IL-4^-/- and IL-13^-/- mice injected 25,000 NBL IV, 13 days post injection. Total body larval burdens in muscles of (D) WT and STAT6^-/- and (E) WT and IL-4^-/-, 24 days post injection. (F) IL-10 detected in CLN cultures from WT and STAT6^-/- mice, 13 days post injection. (G) Number of CD4⁺IL-10⁺ cells per diaphragm of WT and IL-4^-/- mice, 13 days post injection. Each data set was collected from two experiments with similar results. Values represent mean ± SD; n = 4 mice. Significant differences were determined by Student’s <i>t</i> test. ***p < 0.0001.</p