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

Regulation of the host immune system by helminth parasites

Helminth parasite infections are associated with a battery of immunomodulatory mechanisms, which impact all facets of the host immune response to ensure their persistence within the host. This broad-spectrum modulation of host immunity has intended and unintended consequences, both advantageous and disadvantageous. Thus the host may benefit from suppression of collateral damage during parasite infection, and from reduced allergic, autoimmune and inflammatory reactions. However, helminth infection can also be detrimental in reducing vaccine responses, increasing susceptibility to co-infection, and potentially reducing tumor immunosurveillance. In this review we will summarize the panoply of immunomodulatory mechanisms used by helminths, their potential utility in human disease, and prospective areas of future research

Cellular and molecular mechanisms of helminth-induced immunomodulation

About a third of the world’s population is infected with gastrointestinal (GI) nematodes that cause high morbidity in the developing world. As a result of host-parasite co-evolution, helminths evolved strategies to modulate host immunity to prevent their elimination and to induce minimal pathology in the host. Heligmosomoides polygyrus bakeri (Hpb), a murine GI nematode, is one of the most widely used models to study host-parasite interactions. Primary Hpb infection is chronic in some inbred mouse strains, but the mechanism(s) is unclear. Adult worms secrete excretory-secretory molecules (HES) that induce regulatory T cells (Treg) and tolerogenic dendritic cells (Tol DC), which in turn, are thought to suppress immune responses to unrelated antigens, including pathogens. Challenge infection after anthelminthic treatment results in the clearance of adult worms due to a highly polarized Th2 immune response characterized by IL-4-producing CD4+GATA3+ Th2 cells, B cell production of parasite-specific IgG1, and alternatively activated macrophages (AAM).The overall aim of this Ph.D. project was to use Hpb as an experimental model to understand the cellular and molecular mechanisms by which helminths induce immunomodulation. To understand the evasion strategies employed by GI nematodes to establish chronic infections, we first investigated the mechanism(s) involved in the establishment of chronic Hpb infection in B6 mice. We observed significant increases in F4/80-CD11b+Gr1hiLy6G+Ly6C+ myeloid-derived suppressor cells (MDSC) in mesenteric lymph nodes and spleen early after primary but not after challenge infection. MDSC from infected B6 mice suppressed antigen-specific CD4+ T cell proliferation via a nitric oxide-dependent mechanism as well as IL-4 secretion. Adoptive transfer of MDSC from infected B6 mice resulted in high adult worm burdens and increased egg production in naïve recipients. These findings indicate that primary Hpb infection induces MDSC that suppress Th2 responses and promote chronic infection. To further understand the role of MDSC during Hpb infection, we next investigated the role of the transcription factor, interferon regulatory factor 8 (IRF-8), which plays an important role in myeloid cell development. Irf8-/- mice and BXH-2 mice, a recombinant inbred strain that carries a point mutation in the Irf8 gene, have uncontrolled expansion of CD11b+Gr1+ MDSC. Irf8-/- and BXH-2 mice had significantly lower numbers of Th2 cells and AAM in lymphoid tissues and higher worm burdens compared to B6 mice during primary and challenge Hpb infections. In vitro co-culture of MDSC from Irf8-/- mice suppressed CD4+ T cell proliferation and antigen-specific IL-4 secretion. In vivo depletion of MDSC in Irf8-/- mice resulted in significant increases in Th2 cells and IL-4 production. These data highlight the importance of IRF-8 in the development of an adequate Th2 immune response to Hpb infection.Finally, we characterized HES to identify the immunosuppressive component(s) that modulate the antigen presenting function of DC. The immunosuppressive activity was found to be heat stable and partially resistant to proteinase K. We fractionated HES by high-performance liquid chromatography (HPLC) and evaluated the capacity of each fraction collected to suppress DC function. Mass spectrometry analyses of suppressive fractions derived from two independent HPLC rounds identified 21 and 8 proteins, 3 of which were common. Expression and purification of the proteins identified will be used to validate their immunosuppressive activity. In summary, this Ph.D. project identified novel cellular and molecular mechanisms by which Hpb modulates host immune responses. Identification of these mechanisms may lead to the development of better approaches to alleviate GI nematode infections as well as to the development of new therapeutics for use in autoimmune and inflammatory diseases in humans.Environ la moitié de la population mondiale est infectée par des nématodes gastro-intestinaux (GI), une catégorie d'helminthes, qui causent une morbidité sévère dans les pays en développement. Étant donné la co-évolution de proximité de l'hôte et des parasites, ces helminthes ont élaboré des stratégies pour réguler l'immunité de l'hôte et minimiser la pathologie pour ainsi éviter d'être éliminés. Heligmosomoides polygyrus bakeri (Hpb), un nématode GI de la souris, est un des modèles les plus reconnus pour étudier les interactions hôte-parasite. Une infection primaire avec Hpb devient chronique dans certaines lignées de souris consanguines, mais les mécanismes demeurent incertains. Une infection secondaire suivant un traitement avec des anthelminthiques mène à l'élimination de l'infection au stade larvaire en raison d'une forte réponse immunitaire Th2 caractérisée par la présence de cellules CD4+GATA3+ Th2 qui produisent de l'IL-4, de cellules B produisant des IgG1 et de macrophages activé alternativement (AAM).La visée globale de ce projet de PhD est d'utiliser Hpb comme modèle pour identifier et comprendre les mécanismes par lesquels helminthes modulent l'immunité au niveau cellulaire et moléculaire. Nous avons observés une augmentation significative du nombre de cellules myéloïdes suppressives (MDSC) F4/80-CD11b+Gr1hiLy6G+Ly6C+ dans les ganglions mésentériques et la rate peu après l'infection primaire, mais pas après l'infection secondaire. Les MDSC de souris infectées abrogèrent la prolifération de cellules T CD4+ antigène-spécifiques via un mécanisme dépendent à l'oxyde nitrique et la sécrétion d'IL-4. Le transfert adoptif de MDSC de souris infectées mena à un taux de vers adultes et une production d'œufs accrus dans de récipiendaires naïfs. Collectivement, ces résultats indiquent qu'une infection primaire de Hpb augmente les MDSC qui abrogent les réponses Th2 ce qui promeut une infection chronique.Pour comprendre d'avantage le rôle des MDSC pendant une infection par Hpb, nous avons étudié l'implication du facteur de transcription interferon regulatory factor 8 (IRF-8), qui joue un rôle important dans le dévéloppement des cellules myéloïdes. Les souris Irf8-/- et BXH-2, une lignée consanguine recombinante porteuse d'une forme mutée du gène Irf8, ont une expansion incontrôlée de MDSC CD11b+Gr1+. Nous avons observé que les souris Irf8-/- et BXH-2 avaient des nombres diminués de lymphocytes Th2 et d'AAM et des taux de vers accrus par rapport au souris B6 suite aux infections primaire et secondaire par Hpb. La déplétion in vivo des MDSC dans les souris Irf8-/- causa une augmentation des cellules Th2 et la production d'IL-4. Nos données relèvent l'importance d'IRF-8 dans le développement d'une réponse immunitaire de type Th2 en réponse à une infection par Hpb.Finalement, nous avons caractérisé les HES dans le but d'identifier les molécules immunosuppressives relâchées par Hpb qui modulent la capacité de présentation d'antigène des cellules dendritiques (DC). Nous avons fractionné les HES par chromatographie en phase liquide à haute performance (HPLC) et évalué la capacité de chaque fraction collectée à diminuer les fonctions des DC. Des analyses par spectroscopie par masse provenant de deux fractionnements indépendants par HPLC ont identifé 21 et 8 protéines (dont 3 en commun). L'expression et la purification de ces protéines identifiées pourra valider l'activité immunosuppressive de ces dernières.En conclusion, ce projet de PhD a pu identifer de nouveaux mécanismes cellulaires et moléculaires par lesquels Hpb modulent les réponses immunitaires de l'hôte. L'identification de ces mécanismes a le potentiel de mener à l'élaboration d'approches alternatives pour soulager les infections par les nématodes GI et au développement de nouvelles thérapies pour le traitement de maladies auto-immunitaires et inflammatoires chez l'humain

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

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

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