30th Annual Meeting of the German Society for Parasitology

Prof. Dr.

Dr. rer nat

Prof

Kinetic analysis of IL-9 expressing cells during murine infection with the parasitic nematode Strongyloides ratti

Abstract text Introduction: During their migration helminths induce the release of tissue-derived alarmins such as IL-33 which promote the initiation of a protective type 2 immune response. We have previously demonstrated that mucosal mast cells are central for the timely ejection of Strongyloides ratti from the intestine of Infected mice. Application of recombinant IL-33 or enhancement of endogenous IL-33 accelerated the degranulation of mucosal mast cell in an IL-9 dependent manner, resulting in rapid expulsion of the S. ratti. Objective: Aim of the study was to identify the cellular sources of IL-9 in S. ratti infected mice. Material and Methods: BALB/c IL-9 GFP Reporter mice were used to analyze IL-9 expressing cells in 4 different groups: naïve, IL-33 treated, IL-33 treated + S. ratti infected and S. ratti infected mice. Lung, mesenteric lymph nodes (mLN) and lamina propria-derived cells were isolated at day 2, 6 and 10 p.i. Frequencies of granulocytes, mast cells, B cells, NK cells, various T cell (CD4, CD8, gd T cells) and innate lymphoid cell (ILC) subsets were analyzed in several organs and their expression of the pluripotent cytokine IL-9 was quantified by flow cytometry. Results: Kinetic analysis revealed a pronounced expansion and expression of IL-9 GFP by ILC subsets such as ILC2 and NK cells in lung and mLN cells of IL-33 treated mice, as expected. ILC2s were identified as the main IL-9 expressing cell type in the lung of all 3 groups compared to naïve mice. Strikingly, the expansion of these cell types was not visible in the lung and mLN of S. ratti infected mice and was not altered by additional IL-33 treatment. In the small intestine (lamina propria) we observed a late expression of IL-9 by GATA-3+ CD4+ T cells and mast cells at day 10 p.i. in S. ratti infected mice. Expression of IL-9 by these cells was already detectable at day 6 p.i. in IL-33 treated + S. ratti infected mice and was absent in IL-33 treated mice indicating a S. ratti-specific expression of IL-9 at the site of infection. Conclusion: Artificial activation of innate cells by application of the alarmin cytokine IL-33 induces a rapid and pronounced expression of IL-9 by ILC subsets. By contrast, simultaneous infection with S. ratti dampened the expansion of ILC subsets indicating counter regulation of IL-33 induced anti-helminth immune response.

Parasite Immunology

Parasite-Host Interaction

Talk

The role of CD160 in the control of helminth infection

Abstract text Introduction: Helminths dampen their host`s immune response. Among other things, helminths induce expansion of regulatory lymphocytes and checkpoint molecules with co-inhibitory functions such as B and T lymphocyte attenuator (BTLA), a ligand of Herpes Virus Entry Mediator (HVEM). Another HVEM ligand is CD160, which is described on various T cell subsets, NK cells, innate lymphoid cells (ILCs) and mast cells. Objective: Aim of the study was to investigate the role of the CD160 in helminth infected mice. Material and Methods: I used Strongyloides ratti was used as a model for helminth infection with a tissue migration and an intestinal phase. Taking advantage of CD160-deficient (CD160-/-), we performed a tight kinetic study of S. ratti infection. Furthermore, the impact of CD160 on the innate immune response using RAG-/-mice that lack T and B lymphocytes but express CD160, and CD160-/-RAG-/- mice that additionally lack CD160 was investigated. Worm burden was analyzed at different timepoints. Flow cytometry was used to analyze which cell types express CD160 in S. ratti-infected mice. Additionally the expansion/contraction of CD160-expressing cells during S. ratti infection was investigated. Results: Numbers of migrating larvae in the tissues were unchanged but numbers of adult worms in the intestine day 6 p.i. were reduced in CD160-/- mice compared to wildtype mice. CD160-/-RAG-/-mice also showed reduced intestinal parasite burden day 6 p.i. Although RAG-/- mice cannot completely eject Strongyloides parasites, they control the parasite burden from initially ca 100 parasites to 2-5 parasitic adults per mouse. In RAG-/- mice with additional absence of CD160the initially lower parasite burden day 6 p.i. remained stable for almost 100 days. This was associated with impaired mast cell activation in CD160-/-RAG-/-mice. Intestinal NK cells, ILC1, ILC2 and ILC3 express CD160 in naïve mice. During S. ratti infection sSpecifically, CD160+ ILC2 expand while CD160-deficiency interfered with ILC2 but not with NK cell expansion during infection. Conclusion: In combination, these results suggest that CD160-mediated signals have dual and opposing functions during intestinal helminth infection, antagonizing early innate immune responses but promoting late innate immune response.

Characterization of the regulative role of the C-type lectin receptor MINCLE in the initiation of anti-helminth immune responses

Abstract text Objectives One quarter of the human population is infected with helminths, large multicellular parasites, that are controlled in the context of a type 2 immune response. Using Strongyloides ratti infection in mice, we aim to analyse the role of C-type lectin receptors (CLR), an ancient family of innate pattern recognition receptors, in the initiation and modulation of protective type 2 immunity to helminths. Methods Screening a CLR-hFc library, we found yet unidentified S. ratti derived ligand(s), engaging Macrophage inhibitory C type lectin receptor (MINCLE) and further signalling into a MINCLE reporter cell line. We used a MINCLE-deficient mouse to analyse this interaction and its effects in vitro and in vivo. Results &amp; Conclusions Strikingly, the loss of this CLR in MINCLE-deficient mice resulted in reduced intestinal parasite burden when compared to wildtype mice, suggesting absence of the CLR did not impair but improved the host defence immune response. Parameters of Type 2 immunity like mucosal mast cell activation, S. ratti specific Th2 cell responses, protective memory and also the intestinal microbiota were unchanged in MINCLE-deficient mice. However, an increase in MINCLE+ cell populations, like eosinophils, neutrophils and dendritic cells, was observed in the lungs and intestine, offering a possible target for a MINCLE-ligand. Additionally, in vitro stimulation of MINCLE+, but not MINCLE-deficient, macrophages and dendritic cells with a baseline concentration of the MINCLE agonist TDB and increasing concentrations of S. ratti lysate, lead to a dose dependent reduction of TDB-induced TNF production. By contrast, production of TDB-induced IL-6 and IL-10 were synergistically increased by co-stimulation with S. ratti lysate. We are currently investigating the nature of the putative S. ratti -derived MINCLE ligand and its modulatory effects on the hosts immune response.

Dr. rer. nat.

Microfilariae-induced eosinophil ETosis is NADPH oxidase- and inflammasome-dependent

Abstract text Introduction Eosinophils as well as their cytotoxic granules are important for protective immunity against parasitic filarial nematodes, which can be mediated through extracellular DNA trap cell death (ETosis). Thereby, intracellular DNA is explosively released, entrapping and killing pathogens. Neutrophil ETosis (NETosis) can be induced in a NADPH oxidase (NOX) or Ca2+-dependent manner. Moreover, the activation of the inflammasome can lead to NETosis with gasdermin D (GSDMD) being an essential enzyme in forming pores and thus, releasing the DNA traps. However, eosinophil ETosis (EETosis) is understudied and the exact signaling cascade for filaria-induced ETosis is not known yet. Objectives The current study investigated the signaling pathway during EETosis in response to microfilariae (MF) using the rodent filaria Litomosoides sigmodontis. Materials &amp; Methods To study EETosis in response to MF, eosinophils were generated from the bone marrow of wild-type and different knockout mice (NOX, caspase-1, ASC, NLRP3 and AIM2) and cells were stimulated with MF. EETosis was investigated using fluorescence microscopy as well as DNA quantification methods. Results Our results demonstrate that MF induce EETosis in a NOX-dependent manner since eosinophils generated from NOX knockout mice did not release DNA traps in response to MF and PMA. In addition, similar to PMA, histone citrullination occured during a later state of MF-induced EETosis. Moreover, the canonical inflammasome pathway is involved during EETosis. The canonical inflammasome assembles after activation of a sensor molecule, which recruits pro-caspase-1 and the adaptor molecule ASC. As an executing caspase, caspase-1 is able to cleave GSDMD, which forms pores in the cellular membrane. During MF-induced EETosis, active caspase-1 was detected in DNA traps. Inhibition of caspase-1 as well as eosinophils generated from caspase-1 or ASC knockout mice failed to release DNA in response to MF. Using eosinophils from AIM2 and NLRP3 knockout mice, we identified AIM2 and not NLRP3 as the responsible inflammasome sensor, since AIM2 knockout eosinophils, in contrast to NLRP3 knockout eosinophils, were unable to form DNA traps in response to MF. Moreover, we visualized GSDMD during EETosis and observed pore formation in the membrane and the nucleus. Conclusion Our results indicate that EETosis in response to microfilariae is dependent on NOX and the AIM2-caspase 1 inflammasome.

Repeated sensitization of mice with microfilariae of Litomosoides sigmodontis induce pulmonary eosinophilia in an IL-33 dependent manner

Abstract text Background: Eosinophilia is a hallmark of helminth infection and eosinophils are essentially involved in the protective immune responses against helminths. The distinct role of eosinophils during parasitic filarial infection, allergy and autoimmune disease-derived pathology is still not sufficiently understood. In this study, we established a mouse model for tropical pulmonary eosinophilia (TPE), a clinical manifestation that is caused by eosinophil hyper-responsiveness within the lung of lymphatic filariasis patients to investigate involved mechanisms. Methods: Wild-type (WT) BALB/c mice were sensitized with dead microfilariae (MF) of the rodent filarial nematode Litomosoides sigmodontis in weekly intervals for a total of three times and subsequently challenged with viable microfilariae (MF) to induce TPE. The resulting immune response was compared to non-sensitized wild-type mice as well as sensitized eosinophil-deficient dblGATA mice using flow cytometry, lung histology and ELISA. Further, the role of IL-33 signaling during TPE development was investigated using the IL-33 signaling blocker HpARI. Results: Sensitized WT mice displayed increased eosinophil numbers in blood, BAL, spleen and lung. The eosinophilia was accompanied with enhanced MF clearance from the blood, increased immune cell numbers in the lung, increased IL-33 serum levels and activation of lung eosinophils in TPE-induced WT mice in comparison to non-sensitized WT mice and dblGATA TPE mice. Further, TPE-induced WT mice had an increased number of group 2 innate lymphoid cells (ILC2) and alternatively activated macrophages (AAM) within the lung when compared to non-sensitized WT mice. Lung retention of MF was drastically increased in sensitized mice. Interestingly, blocking of IL-33 signaling prevented a type–2 immune shift, reducing M2 macrophages, lung ILC2s and eosinophils. Conclusion: Repeated sensitization with L. sigmodontis MF induces pulmonary eosinophilia accompanied with a strong type 2 immune response mimicking human TPE. Using this newly established L. sigmodontis TPE mouse model, we demonstrate that IL-33 is a key alarmin, which is essentially involved in the induction of TPE-associated immune responses.

Lung immune responses during infection with the filarial nematode Litomosoides sigmodontis – crosstalk between lung and pleural cavity

Abstract text Introduction Recent advances in immunological studies have reinforced the existence of communication between tissues and organs, contributing to maintenance of host homeostasis but also to disease development. Helminth parasites are a great tool to investigate such inter-tissue communication because of their migration through different tissues and organs within their hosts. Objectives The filarial nematode Litomosoides sigmodontis (Ls) resides in the pleural cavity but has a transient lung stage between days 3-5 post infection and later during patency, microfilariae (MF) can accumulate in the lungs. However, pulmonary immune response during Ls infection are not well characterized so far. Here, we present a detailed overview of the ongoing immunological events within the lungs over the course of Ls infection. Further, we aimed to investigate whether immune responses within the lung can affect responses in the pleural cavity, the site of infection. Materials &amp; Methods We used multi-color flow cytometry to investigate cellular composition, T cell and macrophage phenotypes as well as neutrophil-eosinophil kinetics in the lung and compared them side-by-side with the responses within the pleural cavity. ELISA and qPCR were performed to investigate cytokine/chemokine milieu and type 2 associated genes to obtain an overview of ongoing immune responses. Results We analyzed the early events (d3-5pi) of the lung response and found that BALB/c mice, but not semi-susceptible C57BL/6 mice that clear the infection shortly after the molt into adult filariae, upregulate IL-17A production by γδ T cells. This was associated with increased early lung tissue damage only seen in BALB/c mice. Depleting this early lung IL-17A resulted in significantly lower CD4+ T cell and eosinophil numbers and decreased production of the type 2 cytokines IL-5 and IL-13 in the pleural cavity d15pi. Currently we are deciphering the mechanisms by which pulmonary IL-17A regulates the pleural cavity response. Furthermore, with onset of microfilaremia, the lung displays an immune reaction similar to the pleural cavity, even though L4 and adult stages solely live in the pleural cavity. Interestingly, MF+ mice have a higher eosinophil frequency in the lung compared to MF- mice, indicating MF-dependent immune responses. Conclusion Our data suggests a crosstalk between the lung and the pleural cavity that supports the induction and maintenance of protective immune responses against filariae.

Session VII
Parasite Immunology I – Helminths 1

Appointment

Chairs

Programme

Session VIIParasite Immunology I – Helminths 1

Appointment

Chairs

Programme

Session VII
Parasite Immunology I – Helminths 1