30th Annual Meeting of the German Society for Parasitology

Prof. Dr.

Prof.

Identifying mechanisms of preventing immunopathology during Trypanosoma brucei infection

Parasite Immunology

Parasite-Host Interaction

Abstract text Introduction: Induction of a robust immune response is essential for hosts to fight microbial infections. However, excessive immune responses without the presence of appropriate regulations also damage tissues, leading to immunopathology and death during infections. We have previously shown that IL-27 signaling is required for survival during African trypanosome infection by preventing lethal effect of CD4+ T cells and IFN-γ. However, the cellular and molecular mechanisms involved in preventing immunopathology still remain incompletely understood. Objectives to determine the cells and molecules involved in regulating immunopathology during T. brucei infection. Materials &amp; methods: Wild-type and knockout mice were infected with T. brucei. Flow cytometry, ELISA, and adoptive transfer experiments were performed to analyze the immune response in the infected mice. Results We found that CXCR6-/- mice infected with T. brucei survived significantly longer than infected wild-type mice, associated with demilished liver immunopathology and reduced pro-inflammatory cytokines including IFN-γ and TNF-α. Adoptive transfer of wild-type CD4+ T cells, but not CXCR6-/- CD4+ T cells, significantly reduced the survival of infected CXCR6-/- mice, demonstrating that CXCR6+CD4+ T cells mediated the early mortality of infected mice. Interestingly, mice deficient in LFA-1, an integrin molecule that is continuously expressed on Foxp3+ Tregs, died significantly earlier than wild-type mice during T. brucei infection, associated with enhanced activation of CD4+ T cells and higher secretion of IFN-γ. Importantly, infected LFA-1-/- mice exhibited significantly lower frequency and absolute number of Foxp3+ Tregs. Conclusion CXCR6+CD4+ T cells promote early mortality of mice infected with T. brucei, while LFA-1 is essential for preventing the early mortality of infected mice, likely by maintaining Foxp3+ Tregs numerically and functionally.

Talk

Parasitic molecular triggers of coccidia-induced NETosis: a role for cell surface glycans

Veterinary Parasitology

Abstract text Introduction Coccidian parasites represent an important threat for animal and human health. Infection-driven innate immune reactions of the hosts depend on the adequate recognition of parasites. One major mechanism relies on the recognition of parasite-own pathogen associated molecular patterns (PAMPs). Among PAMPS, glycan residues on the parasite&quot;s surface are one of the most important drivers of host immune responses. Polymorphonuclear neutrophils (PMN) are one of the first immune cells to arrive at the site of injury or infection. PMN-derived effector mechanisms against coccidian parasites include reactive oxygen species (ROS) production, degranulation and neutrophil extracellular trap (NET) formation. In this study, the glycosylation patterns in Besnoitia besnoiti and Toxoplasma gondii tachyzoites in addition to Eimeria bovis sporozoites and the relevance of different glycans in B. besnoiti- and T. gondii-induced NETosis were studied. Material and Methods Glycan pattern in B. besnoiti and T. gondii tachyzoites as well as E. bovis-sporozoites were determined by lectin blots using the following lectins: GNA, SNA, MAA, DSA, PNA. Blockage and modification of B. besnoiti and T. gondii tachyzoite-related glycan motifs was achieved by chemical treatments (NaIO4 oxidation), enzymatic treatments with PNGase F, PNGase A and O-glycosidase or by parasite culture in human foreskin fibroblasts (HFF) treated with the N-glycosylation inhibitor tunicamycin. After treatments, B. besnoiti and T. gondii tachyzoites were confronted with bovine PMN and NET formation was analyzed by confocal microscopy. Percentages of NET formation were calculated by image analysis. Results and conclusions Depending on the lectin used, overlapping and differing glycan pattern were revealed in tachyzoite (B. besnoiti, T. gondii) and sporozoite (E. bovis) stages by lectin blots. In contrast to tachyzoites, GNA-detected N-glycans proved absent in E. bovis sporozoites. Glycan oxidation via NaIO4 and PNGase F treatments led to a decrease in NET formation, but also affected tachyzoite viability. In the case of tunicamycin-treated HFF, T. gondii but not B. besnoiti tachyzoites were able to develop F2 tachyzoite generation. F2-tachyzoites proved impaired in their glycosylation repertoire and induced less NETs than those originating from untreated control HFF layers, thereby indicating a potential role of N-glycans in coccidia-induced NETosis.

P2X1 receptor antagonist NF449 inhibits Besnoitia besnoiti-induced neutrophil clustering and anchored extracellular trap (NET) formation

Molecular Parasitology

Abstract text Introduction Bovine besnoitiosis is a re-emerging cattle disease caused by the apicomplexan parasite Besnoitia besnoiti. Neutrophil extracellular traps (NETs) formation represents an efficient innate immune mechanism of polymorphonuclear neutrophils (PMN) against parasites, including B. besnoiti. PMN-derived purinergic signaling was proposed as critical factor for NET formation. ATP is an important purinergic ligand, which regulates several PMN actions, such as chemotaxis, ROS production and NET formation, via the P2 nucleotide receptor family. However, little data are available on the role of ATP as key modulator of purinergic signaling in B. besnoiti-triggered NETosis. Material and methods Bovine PMN were isolated from the blood of healthy cows by density gradient and exposed to B. besnoiti tachyzoites in the presence of ATP and purinergic receptor antagonists. Total and extracellular ATP levels were quantified by luminometry. NET formation was estimated as extracellular DNA by picogreen-derived fluorescence intensities. PMN oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were quantified by Seahorse instrumentation. NET and cluster formation of PMN exposed to B. besnoiti was evaluated by epifluorescence microscopy. Results Current data showed that B. besnoiti tachyzoite exposure neither changed total ATP levels in PMN nor in the extracellular environment even though significantly triggering NET formation. Interestingly, exogenous supplementation of ATP failed to affect B. besnoiti-induced OCR but led to an increase of ECAR in PMN. Likewise, parasite exposure enhanced ECAR in PMN. However, neither the latter effect nor tachyzoite-triggered total NET formation was influenced by exogenous ATP levels. B. besnoiti-induced anchored NET formation was selectively blocked by the inhibition of the purinergic receptor P2X1 whilst antagonists of P2Y2, P2Y6, P2X4 and P2X7 receptors all failed to affect parasite-driven NETosis. [AT1] As an interesting finding, we additionally observed that tachyzoites exposure increased the percentage of PMN forming clusters and that this effect was P2X1-dependent. Conclusion B. besnoiti induces an increase in clustering, oxidative activity and NET formation in bovine PMN. We identified the P2X1 as a pivotal purinergic receptor to be involved in B. besnoiti-induced anchored NET formation therefore suggesting this receptor as an interesting target for future treatments of NET-mediated tissue damage.

PD Dr.

Interactions of human dendritic cells with Cryptosporidium parvum

Abstract text ntroduction: Little is known on Cryptosporidium parvum-driven responses of innate immune cells, such as PMN, monocytes, or macrophages, and even less is reported on parasite interactions with dendritic cells (DCs). DCs are sentinel cells residing in nearly all tissues including the small intestine. DCs detect pathogenic microorganisms with a broad range of pattern recognition receptors, take up, process and present antigens and migrate to the lymphoid organs to present them to naïve T cells. They decide, moreover, based on the information obtained, which kind of immune response like Th1-, Th2-, Treg-, or Th17- is elicited. Objectives: We here intended to analyse early responses of human DCs driven by C. parvum stages. Materials and methods: Immature human myeloid DCs were generated from monocytic blood precursors derived from buffy coats using GM-CSF and IL-4. Cells were harvested on day 6 of culture and exposed to medium alone (negative control), C. parvum oocysts/sporozoites and/or LPS (positive control) over night. Phagocytosis assays were conducted as well as scanning electron microscopy (SEM) analysis to unveil interactions between DCs and these two C. parvum parasite stages. Flow-cytometry analysis (FACS) demonstrated not only upregulation of classical DC activation markers and costimulatory molecules required for T cell-stimulation after C. parvum exposure but also phagocytosis. Cytokine IL-6 and chemokine IL-8 production in C. parvum-exposed human DCs was analysed by ELISAs. Finally, live cell 3D holotomography microscopy (Nanolive) was performed for visualization of highly motile DCs during first encounters with C. parvum. Results: SEM shows not only DCs entrapping oocysts/sporozoites with their dendrites but also phagocytizing them. C. parvum induced IL-6- and IL-8- secretion in stimulated human DCs. Human DCs seem capable to phagocyte efficiently both stages of C. parvum (i. e. oocysts and sporozoites). Positive control (LPS stimulation) and C. parvum both upregulated DC maturation markers, i. e. CD83, antigen-presentation molecules (HLA-DR, CD1a), costimulatory molecules (CD40, CD86) and adhesion molecules (CD11b, CD58) as required for eliciting a strong adaptive immune response. Conclusions: Considering that a strong adaptive immune response is required to protect against C. parvum infection, we investigated how DCs interact with C. parvum to open a new avenue in the search for an effective medication against this zoonotic parasite.

Mechanisms of aggravation of cutaneous leishmaniasis by iron overload

Abstract text Iron overload can cause increased susceptibility to certain intracellular infectious pathogens, but the exact underlying mechanisms remain poorly defined. Here, we investigated how iron loading affects the disease outcome in self-healing C57BL/6 mice infected with the intracellular protozoan parasite Leishmania (L.) major. Iron-loaded L. major-infected C57BL/6 mice showed disease exacerbation with aggravated skin swelling at the site of infection and higher parasite loads in the lesion. This was paralleled by enhanced cell death in the infected tissue, increased influx of CD11b+Ly6G+Ly6C+ neutrophil-like cells lacking suppressor activity and decreased percentages of T-cells, eosinophils, macrophages and dendritic cells. Furthermore, iron overload increased the expression of IFN-gamma, TNF, IL-1ß, IL-4, IL-17A, TGF-beta and type 2 nitric oxide synthase (NOS2) mRNA in skin lesions, whereas NOS2 protein expression and NO production in vivo was suppressed compared to control mice. In vitro, iron partially rescued L. major from NO-dependent, but ferroportin-independent killing by macrophages and favored the differentiation of Th2 cells, which was due to an oxidant-dependent increased nuclear translocation of STAT6. Arginase (Arg) 1, which competes with NOS2 for the substrate L-arginine, was upregulated on mRNA and protein level. In C57BL/6 mice lacking Arg1 in hematopoietic and endothelial cells, the aggravated course of disease following iron loading was abolished and NO production in the skin lesions was partially restored. Similar effects were observed, when iron-loaded C57BL/6 mice were treated with anti-IL-4-antibodies, indicating that iron-driven Arg1 induction is mainly due to enhanced IL-4 levels. Thus, iron overload differentially affects NOS2 and Arg1 expression. Disease exacerbation and impaired parasite control by iron excess in the infected tissues at least partially result from an oxidant-dependent expansion of Th2 cells and an IL-4-mediated Arg1 expression that decreases NO production in iron-loaded mice.

Arginase 1 causes chronicity of Leishmania mexicana infection via the generation of a replicative monocytic host cell niche

Abstract text Background and objective Control of Leishmania (L.) parasites require interferon (IFN)γ-dependent type 2 nitric oxide synthase (NOS2), an enzyme that converts L-arginine into citrulline and nitric oxide (NO). NOS2 activity is counteracted by arginase (ARG) 1 and 2, both of which are induced by Th2 cytokines and cleave L-arginine into urea and ornithine, a precursor of polyamines. Recently, we observed that the expression of ARG1 and ARG2 steadily increased in L. mexicana-infected BALB/c and C57BL/6 mice during disease manifestation. Here, we studied the role of host cell arginases during L. mexicana-induced chronic cutaneous leishmaniasis (CL). Methods C57BL/6 wild-type (WT), germ-line k.o. (Arg2-/-) or cell-specific conditional k.o. mice (Arg1ΔTek, Arg1ΔCx3cr1, Il10ΔCd4) were infected into the skin with 3×106 L. mexicana promastigotes. Infected skin tissue was processed for mRNA/protein expression, metabolomics and single-cell (sc) RNAseq analyses at different time-points post infection (p.i.). Results C57BL/6 WT mice developed non-healing chronic CL, whereas Arg2-/- mice showed progressive disease with a delayed onset. Deletion of Arg1 in hematopoietic and endothelial cells (Arg1ΔTek) or in monocytes and macrophages (Arg1ΔCx3cr1) exhibited a strongly reduced pathology and ultimately resolved their skin lesions despite parasite persistence, suggesting that myeloid Arg1 accounts for chronic CL. Mice lacking IL-10 in CD4+ T cells (Il10ΔCd4) defined IL-10 as a factor inducing ARG1 during infection. Metabolomics revealed the depletion of L-arginine by ARG1 and a significant rise in polyamines in the infected WT skin and draining lymph nodes. At day 40 p.i., when Arg1 mRNA was already upregulated in WT mice, but disease manifestation, parasite burden and the metabolic profile were still comparable between WT and Arg1-deficient mice, similar levels of NO were seen in WT and Arg1-deficient mice, although the expression of NOS2 protein was much higher in WT mice. ScRNAseq analysis of skin lesion cells identified distinct myeloid subpopulations that were enriched in WT mice, including a prominent Arg1+/NOS2+ and IFNγ-dependent cluster of inflammatory macrophages. Conclusion ARG1 serves as an unexpected signal for the recruitment and differentiation of monocytes in the skin of L. mexicana-infected mice, which leads to an enhanced immunopathology and the generation of a cellular niche that favors parasite replication due to ARG1/NOS2 substrate competition.

Session X
Parasite Immunology II – Protozoa

Appointment

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Session XParasite Immunology II – Protozoa

Appointment

Chairs

Programme

Session X
Parasite Immunology II – Protozoa