Proteomic Analysis of Excretory-Secretory Products of Heligmosomoides polygyrus Assessed with Next-Generation Sequencing Transcriptomic Information

The murine parasite Heligmosomoides polygyrus is a convenient experimental model to study immune responses and pathology associated with gastrointestinal nematode infections. The excretory-secretory products (ESP) produced by this parasite have potent immunomodulatory activity, but the protein(s) responsible has not been defined. Identification of the protein composition of ESP derived from H. polygyrus and other relevant nematode species has been hampered by the lack of genomic sequence information required for proteomic analysis based on database searches. To overcome this, a transcriptome next generation sequencing (RNA-seq) de novo assembly containing 33,641 transcripts was generated, annotated, and used to interrogate mass spectrometry (MS) data derived from 1D-SDS PAGE and LC-MS/MS analysis of ESP. Using the database generated from the 6 open reading frames deduced from the RNA-seq assembly and conventional identification programs, 209 proteins were identified in ESP including homologues of vitellogenins, retinol- and fatty acid-binding proteins, globins, and the allergen V5/Tpx-1-related family of proteins. Several potential immunomodulators, such as macrophage migration inhibitory factor, cysteine protease inhibitors, galectins, C-type lectins, peroxiredoxin, and glutathione S-transferase, were also identified. Comparative analysis of protein annotations based on the RNA-seq assembly and proteomics revealed processes and proteins that may contribute to the functional specialization of ESP, including proteins involved in signalling pathways and in nutrient transport and/or uptake. Together, these findings provide important information that will help to illuminate molecular, biochemical, and in particular immunomodulatory aspects of host-H. polygyrus biology. In addition, the methods and analyses presented here are applicable to study biochemical and molecular aspects of the host-parasite relationship in species for which sequence information is not available

Effect of MDSC depletion on worm burdens in Hpb-infected C57BL/6 and <i>Irf8</i>^<i>-/-</i> mice.

<p>Numbers of F4/80^-CD11b^hiGr1^hi cells in MLN of infected C57BL/6 (B6) mice treated with PBS or 5-FU on (A) day 7 or (B) day 14 p.i. (C) Adult worm burdens on day 14 p.i. of individual B6 mice treated with PBS or 5-FU. Numbers of (D) F4/80^-CD11b^hiGr1^hi and (E) CD4⁺GATA3⁺ T cells in MLN of infected <i>Irf8</i>^<i>-/-</i> mice treated with PBS or 5-FU on day 14 p.i. (F) Adult worm burdens on day 14 p.i. and (G) fecal egg counts on day 13 p.i. of individual infected <i>Irf8</i>^<i>-/-</i> mice treated with PBS or 5-FU. Data are representative of two replicate experiments each with n = 5 mice per group. Data are presented as mean ± SEM. ns, not significant; *, <i>p</i> ≤ 0.05; **, <i>p</i> ≤ 0.01.</p

Immunophenotype of cells in MLN of C57BL/6 and IRF-8 deficient mice after primary Hpb infection.

<p>(A) Gating strategy used to analyze F4/80^-CD11b^hiGr1^hi (MDSC) and F4/80⁺CD11b⁺CD206⁺ (AAMØ) cells by flow cytometry. Total numbers of F4/80^-CD11b^hiGr1^hi cells in MLN of (B) C57BL/6 (B6) and <i>Irf8</i>^<i>-/-</i> mice and (C) B6 and BXH-2 mice on day 14 p.i. Total numbers of (D) CD4⁺GATA3⁺ T cells and (E) F4/80⁺CD11b⁺CD206⁺ cells in MLN of B6 and <i>Irf8</i>^<i>-/-</i> mice on day 14 p.i. n = 5 mice/group. Data are representative of three replicate experiments in B6 and <i>Irf8</i>^<i>-/-</i> mice and two replicate experiments in B6 and BXH-2 mice. Data are presented as mean ± SEM. ns, not significant; *, <i>p</i> ≤ 0.05; **, <i>p</i> ≤ 0.01.</p

<i>Irf8</i> expression is down-regulated in MDSC and contributes to higher worm burdens after Hpb infection.

<p>(A) <i>Irf8</i> expression in CD11b⁺Gr1⁺ cells purified from spleens of naïve and infected C57BL/6 (B6) mice on day 7 p.i. Each point represents cells pooled from 2 naïve mice or one infected B6 mouse. Representative results of one of two replicate experiments are shown. Data are presented as relative quantity of <i>Irf8</i> normalized against the endogenous control <i>Actb</i>. (B) Adult worm burden after primary Hpb infection in WT B6 and progenitor C3H/HeJ mice compared to <i>Irf8</i>^<i>-/-</i> and IRF-8 deficient BXH-2 mice. The worm burdens of individual mice pooled from independent experiments are shown. (C) Adult worm burden after challenge Hpb infection in WT B6, <i>Irf8</i>^<i>-/-</i> and BXH-2 mice. The worm burdens of individual mice pooled from independent experiments are shown. Data are presented as mean ± SEM. ns, not significant; **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001; ****, <i>p</i> ≤ 0.0001.</p

CD4⁺CD25⁺Foxp3⁺ T cell expansion is significantly greater in <i>Irf8</i>^<i>-/-</i> than C57BL/6 mice during Hpb infection.

<p>(A) Numbers of total CD4⁺ T cells in MLN of naïve and infected C57BL/6 (B6) or <i>Irf8</i>^<i>-/-</i> mice on days 7 and 14 p.i. (B) Numbers of CD4⁺CD25⁺Foxp3⁺ cells in MLN of infected B6 and <i>Irf8</i>^<i>-/-</i>.mice on day 14 p.i. (C) Numbers of CD4⁺GATA3⁺ and CD4⁺CD25⁺Foxp3⁺ T cells in MLN of naïve B6 and <i>Irf8</i>^<i>-/-</i>.mice and on days 7 and 14 p.i. (D) Numbers of CD4⁺CD25⁺Foxp3⁺ T cells in MLN of <i>Irf8</i>^<i>-/-</i>.mice treated with isotype control antibody or anti-CD25 mAb on day 14 p.i. Numbers of (E) CD4⁺GATA3⁺ T cells, (F) F4/80⁺CD11c⁺CD206⁺ cells, and (G) F4/80^-CD11b^hiGr1^hi cells in MLN of <i>Irf8</i>^<i>-/-</i>.mice treated with isotype control antibody or anti-CD25 mAb on day 14 p.i. (H) Adult worm burdens on day 14 p.i. and (I) fecal egg counts on day 13 p.i. of <i>Irf8</i>^<i>-/-</i> mice treated with isotype control antibody or anti-CD25 mAb. Data from individual mice (n = 5 mice per group) are presented in panels A and D-I. Data are presented as mean ± SEM. **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001; ****, <i>p</i> ≤ 0.0001.</p

Analysis of CD4⁺ Th2 cell effector function in Hpb-infected C57BL/6 and <i>Irf8</i>^<i>-/-</i> mice.

<p>(A) MLN and (B) spleen cells obtained from C57BL/6 (B6) and <i>Irf8</i>^<i>-/-</i> mice (n = 5 mice per group) on day 14 p.i. were stimulated ex vivo with 50 μg AWH. Supernatants were collected 48 h later and IL-4 levels determined by ELISA. Data representative of two to three replicate experiments are presented as mean ± SEM. **; <i>p</i> ≤ 0.01; ***, p≤0.001. (C) HES-specific IgG1 and IgE titers in the sera of naïve and infected B6 and <i>Irf8</i>^<i>-/-</i> mice on day 14 p.i. Each point represents one mouse. Mean ± SEM for each group is shown. *, <i>p</i> ≤ 0.05; ***, p≤0.001. (D) MLN cells were obtained from naïve and infected B6 or <i>Irf8</i>^<i>-/-</i> mice on day 14 p.i. (n = 3 mice per group). CD11a and CD49d expression were analyzed on gated CD4⁺ T cells by flow cytometry. CD11a^hiCD49d^hi cells were gated and the expression of CD44 and CD62L was analyzed. Representative contour plots of one mouse from each group are shown for co-expression of CD11a and CD49d and CD44 and CD62L. Data presented for B6 mice are from one of 2 replicate experiments performed. (E) Gated CD4⁺ T cells obtained from naïve and infected B6 or <i>Irf8</i>^<i>-/-</i> mice on day 14 p.i. (n = 3 mice/group) were analyzed for intracellular expression of GATA3, Foxp3, IL-4, ROR-γt, IFN-γ, and IL-17 by flow cytometry. The proportions of each cell type are shown as a percent of total CD4⁺ T cells. (F) The ratios of the proportions of CD4⁺Foxp3⁺ to CD4⁺GATA3⁺ T cells were determined for C57BL/6 and <i>Irf8</i>^<i>-/-</i> mice based on the data shown in panel E. Each point represents one mouse. Mean ± SEM for each group is shown. *, <i>p</i> ≤ 0.05.</p

Analysis of the Trichuris suis excretory/secretory proteins as a function of life cycle stage and their immunomodulatory properties

Abstract Parasitic worms have a remarkable ability to modulate host immune responses through several mechanisms including excreted/secreted proteins (ESP), yet the exact nature of these proteins and their targets often remains elusive. Here, we performed mass spectrometry analyses of ESP (TsESP) from larval and adult stages of the pig whipworm Trichuris suis (Ts) and identified ~350 proteins. Transcriptomic analyses revealed large subsets of differentially expressed genes in the various life cycle stages of the parasite. Exposure of bone marrow-derived macrophages and dendritic cells to TsESP markedly diminished secretion of the pro-inflammatory cytokines TNFα and IL-12p70. Conversely, TsESP exposure strongly induced release of the anti-inflammatory cytokine IL-10, and also induced high levels of nitric oxide (NO) and upregulated arginase activity in macrophages. Interestingly, TsESP failed to directly induce CD4+ CD25+ FoxP3+ regulatory T cells (Treg cells), while OVA-pulsed TsESP-treated dendritic cells suppressed antigen-specific OT-II CD4+ T cell proliferation. Fractionation of TsESP identified a subset of proteins that promoted anti-inflammatory functions, an activity that was recapitulated using recombinant T. suis triosephosphate isomerase (TPI) and nucleoside diphosphate kinase (NDK). Our study helps illuminate the intricate balance that is characteristic of parasite-host interactions at the immunological interface, and further establishes the principle that specific parasite-derived proteins can modulate immune cell functions

Antigen-specific cytokine secretion by MLN cells from infected <i>Irf8</i>^<i>-/-</i> mice treated with anti-CD25 mAb.

<p>Single cell suspensions of MLN were prepared from infected <i>Irf8</i>^<i>-/-</i> mice treated with isotype control antibody or anti-CD25 mAb on day 14 p.i. The cells were stimulated in vitro with 50 μg AWH, supernatants were harvested 48 h later, and cytokine levels were determined using a Bio-Plex assay. (A) IL-4, (B) IL-5, (C) IL-13, (D) IL-6, and (E) IL-10 levels. Data from individual mice are presented as pg/ml and were calculated based on internal standards for each cytokine with the mean ± SEM for each group shown. n = 5 mice per group. *, <i>p</i> ≤ 0.05; **, <i>p</i> ≤ 0.01.</p

IRF-8 regulates expansion of myeloid-derived suppressor cells and Foxp3⁺ regulatory T cells and modulates Th2 immune responses to gastrointestinal nematode infection

<div><p>Interferon regulatory factor-8 (IRF-8) is critical for Th1 cell differentiation and negatively regulates myeloid cell development including myeloid-derived suppressor cells (MDSC). MDSC expand during infection with various pathogens including the gastrointestinal (GI) nematode <i>Heligmosomoides polygyrus bakeri</i> (Hpb). We investigated if IRF-8 contributes to Th2 immunity to Hpb infection. <i>Irf8</i> expression was down-regulated in MDSC from Hpb-infected C57BL/6 (B6) mice. IRF-8 deficient <i>Irf8</i>^<i>-/-</i> and BXH-2 mice had significantly higher adult worm burdens than B6 mice after primary or challenge Hpb infection. During primary infection, MDSC expanded to a significantly greater extent in mesenteric lymph nodes (MLN) and spleens of <i>Irf8</i>^<i>-/-</i> and BXH-2 than B6 mice. CD4⁺GATA3⁺ T cells numbers were comparable in MLN of infected B6 and IRF-8 deficient mice, but MLN cells from infected IRF-8 deficient mice secreted significantly less parasite-specific IL-4 ex vivo. The numbers of alternatively activated macrophages in MLN and serum levels of Hpb-specific IgG1 and IgE were also significantly less in infected <i>Irf8</i>^<i>-/-</i> than B6 mice. The frequencies of antigen-experienced CD4⁺CD11a^hiCD49d^hi cells that were CD44^hiCD62L^- were similar in MLN of infected <i>Irf8</i>^<i>-/-</i> and B6 mice, but the proportions of CD4⁺GATA3⁺ and CD4⁺IL-4⁺ T cells were lower in infected <i>Irf8</i>^<i>-/-</i> mice. CD11b⁺Gr1⁺ cells from naïve or infected <i>Irf8</i>^<i>-/-</i> mice suppressed CD4⁺ T cell proliferation and parasite-specific IL-4 secretion in vitro albeit less efficiently than B6 mice. Surprisingly, there were significantly more CD4⁺ T cells in infected <i>Irf8</i>^<i>-/-</i> mice, with a higher frequency of CD4⁺CD25⁺Foxp3⁺ T (Tregs) cells and significantly higher numbers of Tregs than B6 mice. In vivo depletion of MDSC and/or Tregs in <i>Irf8</i>^<i>-/-</i> mice did not affect adult worm burdens, but Treg depletion resulted in higher egg production and enhanced parasite-specific IL-5, IL-13, and IL-6 secretion ex vivo. Our data thus provide a previously unrecognized role for IRF-8 in Th2 immunity to a GI nematode.</p></div