Browsing by Author "Finney, Constance A. M."

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    Cathelicidin contributes to prompt protective inflammation against Toxoplasma gondii
    (2019-07-10) Tan, Yi Lin; Cobo, Eduardo R.; Kastelic, John P.; Gilleard, John Stuart; Finney, Constance A. M.
    Toxoplasma gondii is an intracellular parasite infecting all warm-blooded animals, including humans. Although infected immunocompetent individuals are usually asymptomatic, in immunocompromised individuals, T. gondii can affect the central nervous system and may cause congenital toxoplasmosis and death. Cathelicidins are short cationic peptides secreted by leukocytes and epithelial cells with antimicrobial and immunomodulatory activities. It has been proposed that cathelicidin might be a key defense against intracellular pathogens. For instance, human cathelicidin (LL-37), either endogenous or synthetic exogenous, reduced survival of intracellular Mycobacterium tuberculosis in macrophages. However, the role of cathelicidin in toxoplasmosis has been barely investigated. The objective was to elucidate the contributions of cathelicidin during acute systemic and long-term infection with T. gondii. In an acute generalized model of toxoplasmosis, C57BL/6 cathelicidin-deficient (Camp-/-) and wild type (Camp+/+) mice were challenged with luciferase-green fluorescence protein tagged T. gondii (high virulence Type 1 RH, 1 x 105, intra-peritoneal). Although all mice had succumbed by day 5 post infection, Camp-/- mice failed to initiate pro-inflammatory responses in vital organs (ileum, colon, liver, spleen and brain; p<0.05) at early infection (1 d). Consequently, more parasite load was detected in those vital organs (p<0.05). In long-term toxoplasmosis, we determined that Camp-/- mice were more susceptible to oral challenge with T. gondii cysts (low-virulence Type 2 ME 49); all Camp-/- infected mice died by day 12 post infection (p<0.05), whereas Camp+/+ infected and PBS treated mice survived throughout the 14 d study. These Camp-/- mice had more severe hepatitis, with evident liver necrosis and increased inflammatory cytokines, Ifn-γ and Tnf-α gene expression, than Camp+/+ mice (p<0.05). In Camp-/- infected mice, there was pronounced inflammation and Ifn-γ gene synthesis in their spleen (p<0.05) and in their ileum, more severe epithelial disruption, with fewer goblet cells. In in vitro studies, human macrophages (THP-1) infected by T. gondii (ME 49) expressed elevated endogenous gene transcriptional cathelicidin (p<0.05) together with induced gene transcriptional expression of IFN-γ and TNF-α (p<0.05). Camp-/- bone marrow derived macrophages (BMMs) challenged with T. gondii (RH or ME 49) secreted more Tnf-α (p<0.05) compared to infected Camp+/+ BMMs. The T. gondii burdens (both RH and ME 49 strains) were similar between Camp+/+ and Camp-/- BMMs. However, when THP-1 cells macrophages were pre-stimulated with recombinant human LL-37 cathelicidin (2 uM), T. gondii burden was reduced (p<0.05). In summary, this study identified the critical role of cathelicidin in initiating host immune responses, promptly during toxoplasmosis. In the absence of cathelicidin, inflammatory responses in vital organs (liver and spleen) were exaggerated at later infection (3 and 14 d) and they were detrimental to the host. Immunomodulatory roles of cathelicidin were manifested in infected macrophages, where the peptide supressed exaggerated inflammatory responses and aided macrophage killing capabilities. Cathelicidin secreted by macrophages during T. gondii infection has a vital role in host-pathogen balance, controlling parasite burden and preventing overwhelming inflammatory responses.
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    Characterizing Host Protective Immune Responses to the Parasitic Worm Heligmosomoides polygyrus
    (2020-05-05) Ariyaratne, Anupama; Finney, Constance A. M.; Hirota, Simon Andrew; Storey, Douglas G.
    Intestinal roundworms cause chronic debilitating disease in humans and livestock. The lack of vaccines and the emerging drug resistance only increase the need to better understand worm-host interactions. H. polygyrus larvae induce an intestinal granuloma response whereby immune cells, mostly alternatively activated macrophages and eosinophils, accumulate around the tissue encysted parasites. In vitro, these cells and antibodies immobilize, damage/kill worms within the granuloma. This is yet to be confirmed in vivo. We focused on understanding: the mechanisms underlying effective granulomas (chapter 1), the role of resistin like molecule (RELM) alpha, a protein shown to downregulate Th2 immunity (chapter 2), and eosinophils, an enigmatic cell type whose role in anti-worm immunity remains controversial (chapter 3). C57Bl/6 mice are susceptible to H. polygyrus infection. However, mice that are infected using a more natural approach (multiple low doses – (trickle) - as opposed to one large dose (bolus)), had fewer adult worms with more granulomas. Their granulomas had decreased RELM- expression, increased anti-parasitic RELM- and fibrotic gene expression as well as a stronger IgG1 accumulation. In the absence of RELM-, granulomas from trickle-infected mice remained more protective, with upregulated genes involved in eosinophil chemotaxis. Despite this increased eosinophil recruitment, the granulomas from RELM--/- mice were not as protective as those from wild type animals highlighting the need for RELM- for maximal granuloma efficiency. In WT mice, we found eosinophils accumulated around tissue encysted H. polygyrus during acute and chronic infections. The spleen, mesenteric lymph nodes and Peyer’s patches (PP) had increased eosinophilia, with resident and recruited eosinophil surface markers. In addition, eosinophils accumulated around the protozoan parasite Toxoplasma gondii indicating that intestinal eosinophils may represent a key cell type in immunity to different types of incoming parasites. T. gondii infection did not increase eosinophil numbers in the intestine or PP. However, eosinophils showed an inflammatory phenotype marked by increased MHC II and CD101 expression. These findings highlight that the granuloma is a key, complex site of anti-worm immunity and that trickle infection models provide us with a novel tool to study the mechanisms at play within granulomas.
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    Transcriptional patterns of sexual dimorphism and in host developmental programs in the model parasitic nematode Heligmosomoides bakeri
    (2023-05-28) Pollo, Stephen M. J.; Leon-Coria, Aralia; Liu, Hongrui; Cruces-Gonzalez, David; Finney, Constance A. M.; Wasmuth, James D.
    Abstract Background Heligmosomoides bakeri (often mistaken for Heligmosomoides polygyrus) is a promising model for parasitic nematodes with the key advantage of being amenable to study and manipulation within a controlled laboratory environment. While draft genome sequences are available for this worm, which allow for comparative genomic analyses between nematodes, there is a notable lack of information on its gene expression. Methods We generated biologically replicated RNA-seq datasets from samples taken throughout the parasitic life of H. bakeri. RNA from tissue-dwelling and lumen-dwelling worms, collected under a dissection microscope, was sequenced on an Illumina platform. Results We find extensive transcriptional sexual dimorphism throughout the fourth larval and adult stages of this parasite and identify alternative splicing, glycosylation, and ubiquitination as particularly important processes for establishing and/or maintaining sex-specific gene expression in this species. We find sex-linked differences in transcription related to aging and oxidative and osmotic stress responses. We observe a starvation-like signature among transcripts whose expression is consistently upregulated in males, which may reflect a higher energy expenditure by male worms. We detect evidence of increased importance for anaerobic respiration among the adult worms, which coincides with the parasite’s migration into the physiologically hypoxic environment of the intestinal lumen. Furthermore, we hypothesize that oxygen concentration may be an important driver of the worms encysting in the intestinal mucosa as larvae, which not only fully exposes the worms to their host’s immune system but also shapes many of the interactions between the host and parasite. We find stage- and sex-specific variation in the expression of immunomodulatory genes and in anthelmintic targets. Conclusions We examine how different the male and female worms are at the molecular level and describe major developmental events that occur in the worm, which extend our understanding of the interactions between this parasite and its host. In addition to generating new hypotheses for follow-up experiments into the worm’s behavior, physiology, and metabolism, our datasets enable future more in-depth comparisons between nematodes to better define the utility of H. bakeri as a model for parasitic nematodes in general. Graphical Abstract