A Novel Non-invasive Method to Detect RELM Beta Transcript in Gut Barrier Related Changes During a Gastrointestinal Nematode Infection

Currently, methods for monitoring changes of gut barrier integrity and the associated immune response via non-invasive means are limited. Therefore, we aimed to develop a novel non-invasive technique to investigate immunological host responses representing gut barrier changes in response to infection. We identified the mucous layer on feces from mice to be mainly composed of exfoliated intestinal epithelial cells. Expression of RELM-β, a gene prominently expressed in intestinal nematode infections, was used as an indicator of intestinal cellular barrier changes to infection. RELM-β was detected as early as 6 days post-infection (dpi) in exfoliated epithelial cells. Interestingly, RELM-β expression also mirrored the quality of the immune response, with higher amounts being detectable in a secondary infection and in high dose nematode infection in laboratory mice. This technique was also applicable to captured worm-infected wild house mice. We have therefore developed a novel non-invasive method reflecting gut barrier changes associated with alterations in cellular responses to a gastrointestinal nematode infection

Age-dependent rise in IFN-γ competence undermines effective type 2 responses to nematode infection

The efficient induction of type 2 immune responses is central to the control of helminth infections. Previous studies demonstrated that strong Th1 responses driven by intracellular pathogens as well as a bias for type 1 activity in senescent mice impedes the generation of Th2 responses and the control of intestinal nematode infections. Here, we show that the spontaneous differentiation of Th1 cells and their expansion with age restrains type 2 immunity to infection with the small intestinal nematode H. polygyrus much earlier in life than previously anticipated. This includes the more extensive induction of IFN-γ competent, nematode-specific Th2/1 hybrid cells in BALB/c mice older than three months compared to younger animals. In C57BL/6 mice, Th1 cells accumulate more rapidly at steady state, translating to elevated Th2/1 differentiation and poor control of parasite fitness in primary infections experienced at a young age. Blocking of early IFN-γ and IL-12 signals during the first week of nematode infection leads to sharply decreased Th2/1 differentiation and promotes resistance in both mouse lines. Together, these data suggest that IFN-γ competent, type 1 like effector cells spontaneously accumulating in the vertebrate host progressively curtail the effectiveness of anti-nematode type 2 responses with rising host age

Parasitic Nematodes Exert Antimicrobial Activity and Benefit From Microbiota-Driven Support for Host Immune Regulation

Intestinal parasitic nematodes live in intimate contact with the host microbiota. Changes in the microbiome composition during nematode infection affect immune control of the parasites and shifts in the abundance of bacterial groups have been linked to the immunoregulatory potential of nematodes. Here we asked if the small intestinal parasite Heligmosomoides polygyrus produces factors with antimicrobial activity, senses its microbial environment and if the anti-nematode immune and regulatory responses are altered in mice devoid of gut microbes. We found that H. polygyrus excretory/secretory products exhibited antimicrobial activity against gram+/− bacteria. Parasites from germ-free mice displayed alterations in gene expression, comprising factors with putative antimicrobial functions such as chitinase and lysozyme. Infected germ-free mice developed increased small intestinal Th2 responses coinciding with a reduction in local Foxp3+RORγt+ regulatory T cells and decreased parasite fecundity. Our data suggest that nematodes sense their microbial surrounding and have evolved factors that limit the outgrowth of certain microbes. Moreover, the parasites benefit from microbiota-driven immune regulatory circuits, as an increased ratio of intestinal Th2 effector to regulatory T cells coincides with reduced parasite fitness in germ-free mice.Peer Reviewe

Parasitic Nematodes Exert Antimicrobial Activity and Benefit From Microbiota-Driven Support for Host Immune Regulation

Intestinal parasitic nematodes live in intimate contact with the host microbiota. Changes in the microbiome composition during nematode infection affect immune control of the parasites and shifts in the abundance of bacterial groups have been linked to the immunoregulatory potential of nematodes. Here we asked if the small intestinal parasite Heligmosomoides polygyrus produces factors with antimicrobial activity, senses its microbial environment and if the anti-nematode immune and regulatory responses are altered in mice devoid of gut microbes. We found that H. polygyrus excretory/secretory products exhibited antimicrobial activity against gram+/− bacteria. Parasites from germ-free mice displayed alterations in gene expression, comprising factors with putative antimicrobial functions such as chitinase and lysozyme. Infected germ-free mice developed increased small intestinal Th2 responses coinciding with a reduction in local Foxp3+RORγt+ regulatory T cells and decreased parasite fecundity. Our data suggest that nematodes sense their microbial surrounding and have evolved factors that limit the outgrowth of certain microbes. Moreover, the parasites benefit from microbiota-driven immune regulatory circuits, as an increased ratio of intestinal Th2 effector to regulatory T cells coincides with reduced parasite fitness in germ-free mice

Functional characterization of Th2/1 hybrid cells in the murine infection model Heligmosomoides polygyrus

Schätzungen der Weltgesundheitsorganisation zufolge sind zurzeit etwa 1,5 Milliarden Menschen weltweit mit Helminthen infiziert, wobei die Prävalenz vor allem in tropischen und subtropischen Regionen hoch ist. Helminthen sind in der Lage die Immunantwort des Wirts zu modulieren und damit die Wirksamkeit von Impfstoffen zu beeinträchtigen. In dieser Arbeit wurde die Differenzierung und Funktion von Th2/1-Hybridzellen im murinen gastrointestinalen Infektionsmodell Heligmosomoides polygyrus untersucht. H. polygyrus wird unter anderem als Modellorganismus für die humanpathogenen Hakenwürmern Necator americanus sowie Ancylostoma duodenale zur Erforschung der Immunantwort und der Entwicklung von Vakzinen gegen diese Wurmarten genutzt. In Helmintheninfektionen nachgewiesene Th2/1-Hybridzellen sind durch die Koexpression der Schlüsseltanskriptionsfaktoren und Zytokine von Th2- (GATA-3, IL-4, IL-5, IL-13) und Th1-Zellen (T-bet, IFN-g) gekennzeichnet. Die zentrale Fragestellung dieser Arbeit war, ob die Ausbildung von Th2/1-Hybridzellen ein regulatives Element der protektiven Th2-Immunantwort gegen Helminthen darstellt. Ein Hauptaugenmerk lag dabei auf der Rolle von Th2/1-Hybridzellen als IFN-g-Produzenten, da bereits früher in verschiedenen Infektionsmodellen mit Helminthen gezeigt werden konnte, dass eine erhöhte IFN-g Expression mit einer verminderten Kontrolle von Wurminfektionen einhergehen. In dieser Arbeit konnten folgende Charakteristika der Th2/1-Hybridzellantwort in Parasiteninfektionen gezeigt werden: Th2/1-Hybridzellen werden verstärkt in der Milz vorgefunden und stellen die dominante parasiten-spezifische IFN-g-Quelle in einer H. polygyrus Infektion dar. Es konnte gezeigt werden, dass Th2/1-Hybridzellen in vivo aus naiven CD4+ T-Zellen differenzieren können und die Differenzierung unabhängig von der Mikrobiota ist. Darüber hinaus konnte gezeigt werden, dass IFN-g wichtig für die Differenzierung von Th2/1-Hybridzellen ist. Die Applikation von IFN-g früh während der Infektion führt zu einer verstärkten Th2/1-Hybridzellantwort sowie Parasitenfitness. Zudem werden Th2/1-Hybridzellen verstärkt in dem hochempfängliche Mausstamm C57BL/6 als im eher resistente Mausstamm BALB/c induziert. Weiterhin konnten Hinweise gefunden werden, dass die starken Th2/1-Hybridzellantworten die Polarisierung von alternativ aktivierten Makrophagen limitieren und damit die Fitness von H. polygyrus positiv beeinflussen können. Darüber hinaus konnte zum ersten Mal gezeigt werden, dass BALB/c deutlich mehr parasiten-spezifische CD4+ T-Zellen im Vergleich zu C57BL/6 in einer H. polygyrus Infektion induzieren und dass beide Mausstämme verstärkter auf Antigene vom Larvenstadium 4 reagieren. Zusätzlich führen von Helminthen gewonnene Antigene ebenfalls zur Induktion von Th2/1-Hybridzellen, was bei der Entwicklung von Impfstoffen beachtet werden sollte.Infections with helminths affect approximately 1.5 billion people worldwide and are most prevalent in tropical and sub-tropical regions of the developing world. Helminths can modulate the immune response of the host and can thereby inhibit the efficiency of any applied vaccine. The differentiation and function of Th2/1 hybrid cells in murine nematode infection was analyzed in this work. The intestinal nematode Heligmosomoides polygyrus is closely related to the human hookworms Necator americanus and Ancylostoma duodenale. Therefore, it is widely used as a model to study the immune response to helminth infections and for vaccine development. Th2/1 hybrid cells occurring in helminth infections co-express the lineage-defining transcription factors and key cytokines of Th2 (GATA-3, IL-4, IL-5, IL-13) and Th1 (T-bet, IFN-g) cells. The aim of this work was to analyze the potential of Th2/1 hybrid cells as a regulatory element of the protective Th2 immune response to helminth infections. In several studies of helminth infections, it could be shown that an increase in IFN-g expression leads to suppressed control of infection. Therefore, the main focus of this work was to assess the ability of Th2/1 hybrid cells to express IFN-g. The studies performed here showed that the spleen in mice infected with the intestinal nematode H. polygyrus comprise the highest numbers of Th2/1 hybrid cells at all stages of infection and that Th2/1 hybrid cells were the major source of parasite-specific IFN-g. Furthermore, it could be shown that Th2/1 hybrid cells can differentiate from naive CD4+ T cells in vivo and that this differentiation is independent of the microbiota. Additionally, IFN-g is important for Th2/1 hybrid cell induction, as IFN-g supplementation during early nematode infection led to elevated Th2/1 differentiation, more parasite-specific cells expressing IFN-g, and increased parasite egg production. Susceptible C57BL/6 mice induce a more pronounced Th2/1 hybrid cell response compared to the more resistant BALB/c line. This work also provides evidence that Th2/1 hybrid cells inhibit the activation of alternatively activated macrophages, which are important for resistance against helminth infections. It was also shown for the first time that BALB/c mice induce significantly more parasite-specific CD4+ T helper cells compared to C57BL/6 mice. Independent of the mouse strain used, T cells reacted strongest to antigens obtained from H. polygyrus fourth-stage larvae. Interestingly, parasite antigens applied subcutaneously also led to the induction of Th2/1 hybrid cells, which should be considered during vaccine development

Table_4_A Novel Non-invasive Method to Detect RELM Beta Transcript in Gut Barrier Related Changes During a Gastrointestinal Nematode Infection.XLSX

Currently, methods for monitoring changes of gut barrier integrity and the associated immune response via non-invasive means are limited. Therefore, we aimed to develop a novel non-invasive technique to investigate immunological host responses representing gut barrier changes in response to infection. We identified the mucous layer on feces from mice to be mainly composed of exfoliated intestinal epithelial cells. Expression of RELM-β, a gene prominently expressed in intestinal nematode infections, was used as an indicator of intestinal cellular barrier changes to infection. RELM-β was detected as early as 6 days post-infection (dpi) in exfoliated epithelial cells. Interestingly, RELM-β expression also mirrored the quality of the immune response, with higher amounts being detectable in a secondary infection and in high dose nematode infection in laboratory mice. This technique was also applicable to captured worm-infected wild house mice. We have therefore developed a novel non-invasive method reflecting gut barrier changes associated with alterations in cellular responses to a gastrointestinal nematode infection.</p

Video_1_A Novel Non-invasive Method to Detect RELM Beta Transcript in Gut Barrier Related Changes During a Gastrointestinal Nematode Infection.MP4

Currently, methods for monitoring changes of gut barrier integrity and the associated immune response via non-invasive means are limited. Therefore, we aimed to develop a novel non-invasive technique to investigate immunological host responses representing gut barrier changes in response to infection. We identified the mucous layer on feces from mice to be mainly composed of exfoliated intestinal epithelial cells. Expression of RELM-β, a gene prominently expressed in intestinal nematode infections, was used as an indicator of intestinal cellular barrier changes to infection. RELM-β was detected as early as 6 days post-infection (dpi) in exfoliated epithelial cells. Interestingly, RELM-β expression also mirrored the quality of the immune response, with higher amounts being detectable in a secondary infection and in high dose nematode infection in laboratory mice. This technique was also applicable to captured worm-infected wild house mice. We have therefore developed a novel non-invasive method reflecting gut barrier changes associated with alterations in cellular responses to a gastrointestinal nematode infection.</p

Table_1_A Novel Non-invasive Method to Detect RELM Beta Transcript in Gut Barrier Related Changes During a Gastrointestinal Nematode Infection.XLSX

Currently, methods for monitoring changes of gut barrier integrity and the associated immune response via non-invasive means are limited. Therefore, we aimed to develop a novel non-invasive technique to investigate immunological host responses representing gut barrier changes in response to infection. We identified the mucous layer on feces from mice to be mainly composed of exfoliated intestinal epithelial cells. Expression of RELM-β, a gene prominently expressed in intestinal nematode infections, was used as an indicator of intestinal cellular barrier changes to infection. RELM-β was detected as early as 6 days post-infection (dpi) in exfoliated epithelial cells. Interestingly, RELM-β expression also mirrored the quality of the immune response, with higher amounts being detectable in a secondary infection and in high dose nematode infection in laboratory mice. This technique was also applicable to captured worm-infected wild house mice. We have therefore developed a novel non-invasive method reflecting gut barrier changes associated with alterations in cellular responses to a gastrointestinal nematode infection.</p