Search

The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in C57BL/6 mice. Here, using both C57BL/6 and BALB/c mice, we analyze immune cells in the pleural cavity. Unlike C57BL/6 mice, naive tissue-resident large-cavity macrophages (LCMs) of BALB/c mice failed to fully implement the tissue-residency program. Following infection with a pleural-dwelling nematode, these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6, but not in BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte-to-macrophage conversion required both T cells and interleukin-4 receptor alpha (IL-4Rα) signaling. The transition to tissue residency altered macrophage function, and GATA6+ tissue-resident macrophages were required for host resistance to nematode infection. Therefore, during tissue nematode infection, T helper 2 (Th2) cells control the differentiation pathway of resident macrophages, which determines infection outcome.

The early migratory phase of pulmonary helminth infections is characterized by tissue injury leading to the release of the alarmin IL-33 and subsequent induction of type 2 immune responses. We recently described a role for IL-17A, through regulation of IFNγ, as an important inducer of type 2 responses during infection with the lung-migrating rodent nematodeNippostrongylus brasiliensis. Here, we aimed to investigate the interaction between IL-17A and IL-33 during the early lung migratory stages ofN. brasiliensisinfection. In this brief report, we demonstrate that deficiency of IL-17A leads to impaired IL-33 expression and secretion early in infection, independent of IL-17A suppression of IFNγ. Impaired IL-33 production was evident in lung epithelial cells, but not innate immune cells. Therefore, our results demonstrate that IL-17A can drive IL-33 during helminth infection, highlighting an additional mechanism through which IL-17A can regulate pulmonary type 2 immunity.

Infections with helminths affect more than one billion people worldwide. Despite an urgent need there is no vaccine available that would confer long lasting protection against helminth infections. Previous studies indicated that a vaccination with irradiated infective L3 reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as adjuvant improves the efficacy of a vaccination with irradiated L3 larvae of the rodent filaria Litomosoides sigmodontis. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in increased neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-β RNA levels at the site of injection. To investigate the in vivo impact on parasite clearance, BALB/c mice received 3 subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Serum analysis before the challenge infection confirmed the induction of L. sigmodontis-specific antibodies in response to the immunization and serum from immunized mice significantly reduced larval motility in vitro with naïve cells. 63 days after the challenge infection, vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a significantly greater reduction in adult worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). Further, the treatment of L. sigmodontis infection with 3pRNA alone, but not poly(I:C), resulted in a reduced worm burden, supporting the therapeutic potential for the activation of RIG-I with 3pRNA. In conclusion, our data demonstrate that the additional activation of nucleic acid sensing immune receptors boosts the immune response and provides better protection against L. sigmodontis. Thus, the use of nucleic acid receptor agonists as vaccine adjuvants represents a promising novel strategy to improve the efficacy of vaccines against filariae and potentially of other helminths.Author SummaryFilarial nematodes can cause debilitating diseases such as onchocerciasis and lymphatic filariasis that present a major public health burden in the tropics and subtropics, putting more than a billion people at risk of infection. Filarial diseases are transmitted to humans by insect vectors as they take a blood meal. The WHO classifies both filarial infections as neglected tropical diseases and aims to eliminate the transmission of onchocerciasis and eliminate lymphatic filariasis as public health problem by 2030. However, up to date there is no vaccination available that could support the efforts to eliminate filarial diseases and potentially helminth infections in general. Here, we used the well-established murine model for filarial infection, Litomosoides sigmodontis, to test the use of nucleic acid receptor agonists as vaccine adjuvants to enhance local immune responses. We found that infection with L. sigmodontis induces type I IFN and our vaccine strategy enhances the production of type I IFN resulting in increased parasite-specific immune responses and enhanced worm clearance. In summary, our study provides a promising novel approach for a vaccination strategy using cytosolic RNA receptor agonists.

Infection with the filarial nematodes that cause diseases such as lymphatic filariasis and onchocerciasis represent major public health challenges. With millions of people at risk of infection, new strategies for treatment or prevention are urgently needed. More complete understanding of the host immune system's ability to control and eliminate the infection is an important step towards fighting these debilitating infectious diseases. Neutrophils are innate immune cells that are rapidly recruited to inflamed or infected tissues and while considered primarily anti-microbial, there is increasing recognition of their role in helminth infections. Filarial nematodes present a unique situation, as many species harbour the bacterial endosymbiont, Wolbachia. The unexpected involvement of neutrophils during filarial infections has been revealed both in human diseases and animal studies, with strong evidence for recruitment by Wolbachia. This present review will introduce the different human filarial diseases and discuss neutrophil involvement in both protective immune responses, but also in the exacerbation of pathology. Additionally, we will highlight the contributions of the murine model of filariasis, Litomosoides sigmodontis. While several studies have revealed the importance of neutrophils in these parasite infections, we will also draw attention to many questions that remain to be answered.

Filarial nematodes are parasitic worms transmitted by blood-feeding insects. Mainly found in tropical and subtropical areas of the developing world, diseases such as lymphatic filariasis and onchocerciasis represent major public health issues. With millions of people infected and billions at risk of infection, these diseases can stun economic growth and impair the life quality, hence the WHO classified both lymphatic filariasis and onchocerciasis as Neglected Tropical Diseases. The lesser known filarial disease loiasis is not only affecting millions of people, but represents a huge obstacle during mass drug administration programmes targeting other filarial diseases. Even less is known about mansonellosis, potentially the most widespread of the human filariases, but underestimated due to the lack of clinical symptoms. Large scale intervention as well as mass drug administration programmes are undertaken with the long term goal of eliminating the filarial diseases lymphatic filariasis and onchocerciasis. However, there is still neither a vaccination nor short term macrofilaricidal treatments available. The following chapter will encompass the different filarial diseases, the biology of the parasite and their vector, the epidemiology as well as pathology of the filariases, highlighting the impact of these diseases is still immense and further research in understanding and combating these diseases is needed.

The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in the C57BL/6 strain. Here, we provide a comprehensive analysis of immune cells in the pleural cavity using both C57BL/6 and BALB/c mice. Unlike C57BL/6 mice, naïve tissue-resident Large Cavity Macrophages (LCM) of BALB/c mice failed to fully implement the tissue residency program. Following infection with a pleural-dwelling nematode these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6 but not BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte to macrophage conversion required both T cells and IL-4Rα signalling. Host genetics are therefore a key influence on tissue resident macrophage biology, and during nematode infection Th2 cells control the differentiation pathway of tissue resident macrophages.Graphical Abstract

Nippostrongylus brasiliensis is a well-defined model of type-2 immunity but the early lung-migrating phase is dominated by innate IL-17A production and neutrophilia. Using N. brasiliensis infection we confirm previous observations that Il17a-KO mice exhibit an impaired type-2 immune response. Neutrophil depletion and reconstitution studies demonstrated that neutrophils contribute to the subsequent eosinophilia but are not responsible for the ability of IL-17A to promote type-2 cytokine responses. Transcriptional profiling of the lung on day 2 of N. brasiliensis infection revealed an increased Ifnγ signature in the Il17a-KO mice confirmed by enhanced IFNγ protein production. Depletion of early IFNγ rescued type-2 immune responses in the Il17a-KO mice demonstrating that IL-17A-mediated suppression of IFNγ promotes type-2 immunity. Notably, when IL-17A was blocked later in infection, the type-2 response increased. IL-17A regulation of type-2 immunity was lung-specific and infection with Trichuris muris, revealed that IL-17A promotes a type-2 immune response in the lung even when a parasite lifecycle is restricted to the intestine. Together our data reveal IL-17A as a major regulator of pulmonary type-2 immunity which supports the development of a protective type-2 immune response but subsequently limits the magnitude of that response.

Nippostrongylus brasiliensis is a well-defined model of type-2 immunity but the early lung-migrating phase is dominated by innate IL-17A production. In this study, we confirm previous observations that Il17a-KO mice infected with N. brasiliensis exhibit an impaired type-2 immune response. Transcriptional profiling of the lung on day 2 of N. brasiliensis infection revealed an increased Ifng signature in Il17a-KO mice confirmed by enhanced IFNγ protein production in lung lymphocyte populations. Depletion of early IFNγ rescued type-2 immune responses in the Il17a-KO mice demonstrating that IL-17A-mediated suppression of IFNγ promotes type-2 immunity. Notably, later in infection, once the type-2 response was established, IL-17A limited the magnitude of the type-2 response. IL-17A regulation of type-2 immunity was lung-specific and infection with Trichuris muris revealed that IL-17A promotes a type-2 immune response in the lung even when infection is restricted to the intestine. Together our data reveal IL-17A as a major regulator of pulmonary type-2 immunity such that IL-17A supports early development of a protective type-2 response by suppression of IFNγ but subsequently limits excessive type-2 responses. A failure of this feedback loop may contribute to conditions such as severe asthma, characterised by combined elevation of IL-17 and type-2 cytokines.

IL-6 has a wide range of biological activities that includes anti- and pro-inflammatory aspects. In this study, we investigated the role of IL-6 in immune responses to the rodent filarial nematode Litomosoides sigmodontis, a model for human filarial infections. IL-6-/- mice had a significantly increased worm burden after natural infection compared with wild type controls at early time points p.i. Given that the worm burden in IL-6-/- mice was already increased at the time point the infective larvae reached the pleural cavity, immune responses that may facilitate the migration from the site of infection (skin) via the lymphatics to the pleural cavity were analysed. Increased vascular permeability may facilitate larval migration, but blocking of histamine receptors had no effect on worm burden and vascular permeability was similar between IL-6-/- mice and wild type controls. In contrast, blocking mast cell degranulation reduced the worm burden in IL-6-/- mice partially, suggesting that release of mast cell-derived mediators improves larval migration to some degree. Protective immune responses within the skin were involved, as bypassing the skin barrier by inoculating infective L3s subcutaneously resulted in a comparable worm recovery in both mouse strains. Analysis of the cellular composition by flow cytometry and PCR array in the skin after exposure to filarial extract or L3s, respectively, indicate that the absence of IL-6 results in a delayed recruitment of neutrophils and macrophages to the site of initial infection. These results demonstrate that IL-6 is essentially involved in protective immune responses within the skin that impair migration of infective L3s.

IL-13 is tissue protective during acute lung injury caused by nematode migration and specifically regulates type 2 epithelial cell effector functions., IL-13 is implicated in effective repair after acute lung injury and the pathogenesis of chronic diseases such as allergic asthma. Both these processes involve matrix remodelling, but understanding the specific contribution of IL-13 has been challenging because IL-13 shares receptors and signalling pathways with IL-4. Here, we used Nippostrongylus brasiliensis infection as a model of acute lung damage comparing responses between WT and IL-13-deficient mice, in which IL-4 signalling is intact. We found that IL-13 played a critical role in limiting tissue injury and haemorrhaging in the lung, and through proteomic and transcriptomic profiling, identified IL-13-dependent changes in matrix and associated regulators. We further showed a requirement for IL-13 in the induction of epithelial-derived type 2 effector molecules such as RELM-α and surfactant protein D. Pathway analyses predicted that IL-13 induced cellular stress responses and regulated lung epithelial cell differentiation by suppression of Foxa2 pathways. Thus, in the context of acute lung damage, IL-13 has tissue-protective functions and regulates epithelial cell responses during type 2 immunity.

Key Points Nlrp3−/− mice have enhanced early antihelminth immunity in the lung. Type 2 immunity and repair responses are dysregulated in Nlrp3−/− mice. NLRP3 plays an inflammasome-independent role during Nippostrongylus infection., Alternatively activated macrophages are essential effector cells during type 2 immunity and tissue repair following helminth infections. We previously showed that Ym1, an alternative activation marker, can drive innate IL-1R–dependent neutrophil recruitment during infection with the lung-migrating nematode, Nippostrongylus brasiliensis, suggesting a potential role for the inflammasome in the IL-1–mediated innate response to infection. Although inflammasome proteins such as NLRP3 have important proinflammatory functions in macrophages, their role during type 2 responses and repair are less defined. We therefore infected Nlrp3−/− mice with N. brasiliensis. Unexpectedly, compared with wild-type (WT) mice, infected Nlrp3−/− mice had increased neutrophilia and eosinophilia, correlating with enhanced worm killing but at the expense of increased tissue damage and delayed lung repair. Transcriptional profiling showed that infected Nlrp3−/− mice exhibited elevated type 2 gene expression compared with WT mice. Notably, inflammasome activation was not evident early postinfection with N. brasiliensis, and in contrast to Nlrp3−/− mice, antihelminth responses were unaffected in caspase-1/11–deficient or WT mice treated with the NLRP3-specific inhibitor MCC950. Together these data suggest that NLRP3 has a role in constraining lung neutrophilia, helminth killing, and type 2 immune responses in an inflammasome-independent manner.

Animal studies demonstrated that administration of helminth products can protect from autoimmune diseases. However, the success of such administrations is limited in the case of type 1 diabetes, as protection is only provided if the administration is started before the development of insulitis. In this study we investigated whether inclusion of helminth antigen administrations to an antigen-specific treatment with proinsulin improves the protective effect by triggering non-specific regulatory immune responses. Using a combination therapy of intraperitoneal Litomosoides sigmodontis antigen and intranasal pro-insulin, onset of diabetes was prevented in NOD mice after insulitis started, while either administration alone failed to protect. This protection was associated with an increased frequency of regulatory T cells within the pancreatic lymph nodes and a reduced inflammation of the pancreatic islets. This suggests that inclusion of helminth antigens improve the protective effect provided by antigen-specific therapies and represent a new potential therapeutic approach against autoimmune diseases.

It is estimated that by the year 2035 almost 600 million people will suffer from diabetes. In the case of type 2 diabetes, the strongest increase of diabetes incidence occurs in developing and newly industrialized countries. This increase correlates not only with a progressing sedentary lifestyle and nutritional changes, but also environmental changes. Similarly, the increase of type 1 diabetes incidence in industrialized countries over the past decades cannot be explained by genetic factors alone, suggesting that environmental changes are also involved. One such environmental change is a reduced exposure to pathogens because of improved hygiene. Parasitic helminths modulate the immune system of their hosts and induce type 2 as well as regulatory immune responses. As pro-inflammatory immune responses are crucial for the onset of both type 1 and type 2 diabetes, helminth-induced immunomodulation may prevent diabetes onset and ameliorate insulin sensitivity. Several epidemiological studies in human and experimental animal models support such a protective effect of helminths for autoimmune diabetes. Recent studies further suggest that helminths may also provide such a beneficial effect for type 2 diabetes. In this review we summarize studies that investigated parasitic helminths and helminth-derived products and their impact on both type 1 and type 2 diabetes highlighting potential protective mechanisms.

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) recognizes muramyl dipeptide (MDP) of bacterial cell walls, triggering NFκB-induced pro-inflammation. As most human pathogenic filariae contain Wolbachia endobacteria that synthesize the MDP-containing cell wall precursor lipid II, NOD2’s role during infection with the rodent filaria Litomosoides sigmodontis was investigated. In NFκB reporter-cells, worm-extract containing Wolbachia induced NOD2 and NOD1. NOD2-deficient mice infected with L. sigmodontis had significantly more worms than wildtype controls early in infection. Increased worm burden was not observed after subcutaneous infection, suggesting that protective NOD2-dependent immune responses occur within the skin. Flow cytometry demonstrated that neutrophil recruitment to the skin was impaired in NOD2−/− mice after intradermal injection of third stage larvae (L3), and blood neutrophil numbers were reduced after L. sigmodontis infection. PCR array supported the requirement of NOD2 for recruitment of neutrophils to the skin, as genes associated with neutrophil recruitment and activation were downregulated in NOD2−/− mice after intradermal L3 injection. Neutrophil depletion before L. sigmodontis infection increased worm recovery in wildtype mice, confirming that neutrophils are essential against invading L3 larvae. This study indicates that NOD-like receptors are implemented in first-line protective immune responses against filarial nematodes.

Helminths induce type 2 immune responses and establish an anti-inflammatory milieu in their hosts. This immunomodulation was previously shown to improve diet-induced insulin resistance which is linked to chronic inflammation. In the current study, we demonstrate that infection with the filarial nematode Litomosoides sigmodontis increased the eosinophil number and alternatively activated macrophage abundance within epididymal adipose tissue (EAT) and improved glucose tolerance in diet-induced obese mice in an eosinophil-dependent manner. L. sigmodontis antigen (LsAg) administration neither altered the body weight of animals nor adipose tissue mass or adipocyte size, but it triggered type 2 immune responses, eosinophils, alternatively activated macrophages, and type 2 innate lymphoid cells in EAT. Improvement in glucose tolerance by LsAg treatment remained even in the absence of Foxp3+ regulatory T cells. Furthermore, PCR array results revealed that LsAg treatment reduced inflammatory immune responses and increased the expression of genes related to insulin signaling (Glut4, Pde3b, Pik3r1, and Hk2) and fatty acid uptake (Fabp4 and Lpl). Our investigation demonstrates that L. sigmodontis infection and LsAg administration reduce diet-induced EAT inflammation and improve glucose tolerance. Helminth-derived products may, therefore, offer new options to improve insulin sensitivity, while loss of helminth infections in developing and developed countries may contribute to the recent increase in the prevalence of type 2 diabetes.

It is estimated that by the year 2035 almost 600 million people will suffer from diabetes. In the case of type 2 diabetes, the strongest increase of diabetes incidence occurs in developing and newly industrialized countries. This increase correlates not only with a progressing sedentary lifestyle and nutritional changes, but also environmental changes. Similarly, the increase of type 1 diabetes incidence in industrialized countries over the past decades cannot be explained by genetic factors alone, suggesting that environmental changes are also involved. One such environmental change is a reduced exposure to pathogens because of improved hygiene. Parasitic helminths modulate the immune system of their hosts and induce type 2 as well as regulatory immune responses. As pro-inflammatory immune responses are crucial for the onset of both type 1 and type 2 diabetes, helminth-induced immunomodulation may prevent diabetes onset and ameliorate insulin sensitivity. Several epidemiological studies in human and experimental animal models support such a protective effect of helminths for autoimmune diabetes. Recent studies further suggest that helminths may also provide such a beneficial effect for type 2 diabetes. In this review we summarize studies that investigated parasitic helminths and helminth-derived products and their impact on both type 1 and type 2 diabetes highlighting potential protective mechanisms.

Background\ud \ud Interactions of the Th2 cytokine IL-33 with its receptor ST2 lead to amplified Type 2 immune responses. As Type 2 immune responses are known to mediate protection against helminth infections we hypothesized that the lack of ST2 would lead to an increased susceptibility to filarial infections.\ud \ud Methodology/Principal Finding\ud \ud ST2 deficient and immunocompetent BALB/c mice were infected with the filarial nematode Litomosoides sigmodontis. At different time points after infection mice were analyzed for worm burden and their immune responses were examined within the thoracic cavity, the site of infection, and systemically using spleen cells and plasma. Absence of ST2 led to significantly increased levels of peripheral blood microfilariae, the filarial progeny, whereas L. sigmodontis adult worm burden was not affected. Development of local and systemic Type 2 immune responses were not impaired in ST2 deficient mice after the onset of microfilaremia, but L. sigmodontis infected ST2-ko mice had significantly reduced total numbers of cells within the thoracic cavity and spleen compared to infected immunocompetent controls. Pronounced microfilaremia in ST2-ko mice did not result from an increased microfilariae release by adult female worms, but an impaired splenic clearance of microfilariae.\ud \ud Conclusions/Significance\ud \ud Our findings suggest that the absence of ST2 does not impair the establishment of adult L. sigmodontis worms, but is important for the splenic clearance of microfilariae from peripheral blood. Thus, ST2 interactions may be important for therapies that intend to block the transmission of filarial disease.

Nippostrongylus brasiliensis is a well-defined model of type-2 immunity but the early lung migrating phase is dominated by innate IL-17A production. In this study, we confirm previous observations that Il17a-KO mice infected with N. brasiliensis exhibit an impaired type-2 immune response. Transcriptional profiling of the lung on day 2 of N. brasiliensis infection revealed an increased Ifn γ signature in Il17a-KO mice confirmed by enhanced IFN γ protein production in lung lymphocyte populations. Depletion of early IFNγ rescued type-2 immune responses in the Il17a-KO mice demonstrating that IL-17A-mediated suppression of IFNγ promotes type-2 immunity. Notably, later in infection, once the type 2 response was established, IL-17A limited the magnitude of the type 2 response. IL-17A regulation of type-2 immunity was lung-specific and infection with Trichuris muris, revealed that IL-17A promotes a type-2 immune response in the lung even when infection is restricted to the intestine. Together our data reveal IL-17A as a major regulator of pulmonary type-2 immunity such that IL-17A supports early development of a protective type-2 response by suppression of IFN γ but subsequently limits excessive type 2 responses. A failure of this feedback loop may contribute to conditions such as severe asthma, characterised by combined elevation of IL-17 and type-2 cytokines.

Additional file 2: Table S1. Mean adult worm burden and microfilariae (MF) counts per 50 Âľl of blood and standard deviation (SD) as well as minimum and maximum adult worm/microfilariae counts are shown as well as the frequency of animals developing microfilaremia. Statistical significance was determined by Mann-Whitney U-test.