IL-33 facilitates rapid expulsion of the parasitic nematode Strongyloides ratti from the intestine via ILC2- and IL-9-driven mast cell activation.

Parasitic helminths are sensed by the immune system via tissue-derived alarmins that promote the initiation of the appropriate type 2 immune responses. Here we establish the nuclear alarmin cytokine IL-33 as a non-redundant trigger of specifically IL-9-driven and mast cell-mediated immunity to the intestinal parasite Strongyloides ratti. Blockade of endogenous IL-33 using a helminth-derived IL-33 inhibitor elevated intestinal parasite burdens in the context of reduced mast cell activation while stabilization of endogenous IL-33 or application of recombinant IL-33 reciprocally reduced intestinal parasite burdens and increased mast cell activation. Using gene-deficient mice, we show that application of IL-33 triggered rapid mast cell-mediated expulsion of parasites directly in the intestine, independent of the adaptive immune system, basophils, eosinophils or Gr-1+ cells but dependent on functional IL-9 receptor and innate lymphoid cells (ILC). Thereby we connect the described axis of IL-33-mediated ILC2 expansion to the rapid initiation of IL-9-mediated and mast cell-driven intestinal anti-helminth immunity.

Cutting Edge: the BTLA-HVEM regulatory pathway interferes with protective immunity to intestinal Helminth infection.

Helminths exploit intrinsic regulatory pathways of the mammalian immune system to dampen the immune response directed against them. In this article, we show that infection with the parasitic nematode Strongyloides ratti induced upregulation of the coinhibitory receptor B and T lymphocyte attenuator (BTLA) predominantly on CD4(+) T cells but also on a small fraction of innate leukocytes. Deficiency of either BTLA or its ligand herpes virus entry mediator (HVEM) resulted in reduced numbers of parasitic adults in the small intestine and reduced larval output throughout infection. Reduced parasite burden in BTLA- and HVEM-deficient mice was accompanied by accelerated degranulation of mucosal mast cells and increased Ag-specific production of the mast cell-activating cytokine IL-9. Our combined results support a model whereby BTLA on CD4(+) T cells and additional innate leukocytes is triggered by HVEM and delivers negative signals into BTLA(+) cells, thereby interfering with the protective immune response to this intestinal parasite.

Litomosoides sigmodontis induces TGF-ß receptor responsive, IL-10-producing T cells that suppress bystander T-cell proliferation in mice.

Helminth parasites suppress immune responses to prolong their survival within the mammalian host. Thereby not only helminth-specific but also nonhelminth-specific bystander immune responses are suppressed. Here, we use the murine model of Litomosoides sigmodontis infection to elucidate the underlying mechanisms leading to this bystander T-cell suppression. When OT-II T cells specific for the third-party antigen ovalbumin are transferred into helminth-infected mice, these cells respond to antigen-specific stimulation with reduced proliferation compared to activation within non-infected mice. Thus, the presence of parasitic worms in the thoracic cavity translates to suppression of T cells with a different specificity at a different site. By eliminating regulatory receptors, cytokines, and cell populations from this system, we provide evidence for a two-staged process. Parasite products first engage the TGF-ß receptor on host-derived T cells that are central to suppression. In a second step, host-derived T cells produce IL-10 and subsequently suppress the adoptively transferred OT-II T cells. Terminal suppression was IL-10-dependant but independent of intrinsic TGF-ß receptor- or PD-1-mediated signaling in the suppressed OT-II T cells. Blockade of the same key suppression mediators, i.e. TGF-ß- and IL-10 receptor, also ameliorated the suppression of IgG response to bystander antigen vaccination in L. sigmodontis-infected mice.

Foxp3⁺ regulatory T cells delay expulsion of intestinal nematodes by suppression of IL-9-driven mast cell activation in BALB/c but not in C57BL/6 mice.

Accumulating evidence suggests that IL-9-mediated immunity plays a fundamental role in control of intestinal nematode infection. Here we report a different impact of Foxp3⁺ regulatory T cells (Treg) in nematode-induced evasion of IL-9-mediated immunity in BALB/c and C57BL/6 mice. Infection with Strongyloides ratti induced Treg expansion with similar kinetics and phenotype in both strains. Strikingly, Treg depletion reduced parasite burden selectively in BALB/c but not in C57BL/6 mice. Treg function was apparent in both strains as Treg depletion increased nematode-specific humoral and cellular Th2 response in BALB/c and C57BL/6 mice to the same extent. Improved resistance in Treg-depleted BALB/c mice was accompanied by increased production of IL-9 and accelerated degranulation of mast cells. In contrast, IL-9 production was not significantly elevated and kinetics of mast cell degranulation were unaffected by Treg depletion in C57BL/6 mice. By in vivo neutralization, we demonstrate that increased IL-9 production during the first days of infection caused accelerated mast cell degranulation and rapid expulsion of S. ratti adults from the small intestine of Treg-depleted BALB/c mice. In genetically mast cell-deficient (Cpa3-Cre) BALB/c mice, Treg depletion still resulted in increased IL-9 production but resistance to S. ratti infection was lost, suggesting that IL-9-driven mast cell activation mediated accelerated expulsion of S. ratti in Treg-depleted BALB/c mice. This IL-9-driven mast cell degranulation is a central mechanism of S. ratti expulsion in both, BALB/c and C57BL/6 mice, because IL-9 injection reduced and IL-9 neutralization increased parasite burden in the presence of Treg in both strains. Therefore our results suggest that Foxp3⁺ Treg suppress sufficient IL-9 production for subsequent mast cell degranulation during S. ratti infection in a non-redundant manner in BALB/c mice, whereas additional regulatory pathways are functional in Treg-depleted C57BL/6 mice.

T-cell-derived, but not B-cell-derived, IL-10 suppresses antigen-specific T-cell responses in Litomosoides sigmodontis-infected mice.

IL-10, a cytokine with pleiotropic functions is produced by many different cells. Although IL-10 may be crucial for initiating protective Th2 responses to helminth infection, it may also function as a suppressive cytokine preventing immune pathology or even contributing to helminth-induced immune evasion. Here, we show that B cells and T cells produce IL-10 during murine Litomosoides sigmodontis infection. IL-10-deficient mice produced increased amounts of L. sigmodontis-specific IFN-γ and IL-13 suggesting a suppressive role for IL-10 in the initiation of the T-cell response to infection. Using cell type-specific IL-10-deficient mice, we dissected different functions of T-cell- and B-cell-derived IL-10. Litomosoides sigmodontis-specific IFN-γ, IL-5, and IL-13 production increased in the absence of T-cell-derived IL-10 at early and late time points of infection. In contrast, B-cell-specific IL-10 deficiency did not lead to significant changes in L. sigmodontis-specific cytokine production compared to WT mice. Our results suggest that the initiation of Ag-specific cellular responses during L. sigmodontis infection is suppressed by T-cell-derived IL-10 and not by B-cell-derived IL-10.

Nematode-induced interference with the anti-Plasmodium CD8+ T-cell response can be overcome by optimizing antigen administration.

Malaria is still responsible for up to 1 million deaths per year worldwide, highlighting the need for protective malaria vaccines. Helminth infections that are prevalent in malaria endemic areas can modulate immune responses of the host. Here we show that Strongy-Ioides ratti, a gut-dwelling nematode that causes transient infections, did not change the efficacy of vaccination against Plasmodium berghei. An ongoing infection with Litomosoides sigmodontis, a tissue-dwelling filaria that induces chronic infections in BALB/c mice, significantly interfered with vaccination efficacy. The induction of P. berghei circumspor-ozoite protein (CSP)-specific CD8(+) T cells, achieved by a single immunization with a CSP fusion protein, was diminished in L. sigmodontis-infected mice. This modulation was reflected by reduced frequencies of CSP-specific CD8(+) T cells, reduced CSP-specific IFN-y and TNF-a production, reduced CSP-specific cytotoxicity, and reduced protection against P. berghei challenge infection. Implementation of a more potent vaccine regime, by first priming with CSP-expressing recombinant live Salmonella prior to CSP fusion protein immunization, restored induction of CSP-specific CD8(+) T cells and conferred almost sterile immunity to P. berghei challenge infection also in L. sigmodontis-infected mice. In summary, we show that appropriate vaccination regimes can overcome helminth-induced interference with vaccination efficacy.

Pathogenic nematodes suppress humoral responses to third-party antigens in vivo by IL-10-mediated interference with Th cell function.

One third of the human population is infected with helminth parasites. To promote their longevity and to limit pathology, helminths have developed several strategies to suppress the immune response of their host. As this immune suppression also acts on unrelated third-party Ags, a preexisting helminth infection may interfere with vaccination efficacy. In this study, we show that natural infection with Litomosoides sigmodontis suppressed the humoral response to thymus-dependent but not to thymus-independent model Ags in C57BL/6 mice. Thereby, we provide evidence that reduced humoral responses were mediated by interference with Th cell function rather than by direct suppression of B cells in L. sigmodontis-infected mice. We directly demonstrate suppression of Ag-specific proliferation in OVA-specific Th cells after adoptive transfer into L. sigmodontis-infected mice that led to equally reduced production of OVA-specific IgG. Transferred Th cells displayed increased frequencies of Foxp3(+) after in vivo stimulation within infected but not within naive mice. Helminth-mediated suppression was induced by established L. sigmodontis infections but was completely independent of the individual worm burden. Using DEREG mice, we rule out a central role for host-derived regulatory T cells in the suppression of transferred Th cell proliferation. In contrast, we show that L. sigmodontis-induced, host-derived IL-10 mediated Foxp3 induction in transferred Th cells and significantly contributed to the observed Th cell hypoproliferation within infected mice.

A novel and divergent role of granzyme A and B in resistance to helminth infection.

Granzyme (gzm) A and B, proteases of NK cells and T killer cells, mediate cell death, but also cleave extracellular matrices, inactivate intracellular pathogens, and induce cytokines. Moreover, macrophages, Th2 cells, regulatory T cells, mast cells, and B cells can express gzms. We recently reported gzm induction in human filarial infection. In this study, we show that in rodent filarial infection with Litomosoides sigmodontis, worm loads were significantly reduced in gzmA × B and gzmB knockout mice during the whole course of infection, but enhanced only early in gzmA knockout compared with wild-type mice. GzmA/B deficiency was associated with a defense-promoting Th2 cytokine and Ab shift, enhanced early inflammatory gene expression, and a trend of reduced alternatively activated macrophage induction, whereas gzmA deficiency was linked with reduced inflammation and a trend toward increased alternatively activated macrophages. This suggests a novel and divergent role for gzms in helminth infection, with gzmA contributing to resistance and gzmB promoting susceptibility.

Filarial infections compromise influenza vaccination efficacy: Lessons from the mouse.

Helminth parasites infect more than a quarter of the human population and inflict significant changes to the immunological status of their hosts. Several human studies report impaired responses to vaccinations in helminth-infected individuals. Analysing the impact of helminth infections on the efficacy of influenza vaccinations in the mouse system helps to elucidate the underlying immunological processes. Concurrent infection with the parasitic nematode Litomosoides sigmodontis reduced the quantity and quality of antibody responses to vaccination against seasonal influenza in BALB/c and C57BL/6 mice. This led to impaired vaccination-induced protection against challenge infections with the human pathogenic 2009 pandemic H1N1 influenza A virus in helminth-infected mice. Impaired responses were also observed if vaccinations were performed after immune-driven or drug-induced clearance of a previous helminth infection. Mechanistically, the suppression was associated with a systemic and sustained expansion of IL-10-producing CD4+CD49b+LAG-3+ type 1 regulatory T cells and partially abrogated by in vivo blockade of the IL-10 receptor. In summary, these findings raise the concern that individuals in helminth-endemic areas may not always benefit from vaccinations, even in the absence of an acute and diagnosable helminth infection.

Retinoic acid drives intestine-specific adaptation of effector ILC2s originating from distant sites.

Adaptation of immune cells to tissue-specific microenvironments is a crucial process in homeostasis and inflammation. Here, we show that murine effector type 2 innate lymphoid cells (ILC2s) from various organs are equally effective in repopulating ILC2 niches in other anatomical locations where they adapt tissue-specific phenotypes of target organs. Single-cell transcriptomics of ILC2 populations revealed upregulation of retinoic acid (RA) signaling in ILC2s during adaptation to the small intestinal microenvironment, and RA signaling mediated reprogramming of kidney effector ILC2s toward the small intestinal phenotype in vitro and in vivo. Inhibition of intestinal ILC2 adaptation by blocking RA signaling impaired worm expulsion during Strongyloides ratti infection, indicating functional importance of ILC2 tissue imprinting. In conclusion, this study highlights that effector ILC2s retain the ability to adapt to changing tissue-specific microenvironments, enabling them to exert tissue-specific functions, such as promoting control of intestinal helminth infections.

Strongyloides ratti infection modulates B and T cell responses to third party antigens.

It is estimated that over one third of the world population is infected with helminths, Strongyloides ssp. accounting for approximately 30-100 million cases. As helminth infections often result in a modulation of the host's immune system, infected people may display impaired responses to concurrent infections and to third party antigens. Here, we employ the experimental system of murine Strongyloides ratti infection to investigate the impact of helminth infections on experimental vaccinations. We demonstrate that concurrent infection with S. ratti strongly affected the humoral response to a thymus dependent model antigen, whereby predominantly Th1 associated IgG2b production was suppressed. We provide evidence that this suppression was due to modulation of T helper cell and not B cell function as the responses to a thymus independent model antigen remained unchanged in S. ratti infected mice. Moreover, using an adoptive transfer system, we show that infection with S. ratti directly interfered with antigen-specific proliferation of T cell receptor transgenic CD4(+) T helper cells in vivo. Finally, using IL-10 deficient mice and mice that selectively lack T helper cell derived IL-10 we rule out a role for host-derived IL-10 in mediating the suppression of thymus dependent model antigen response in S. ratti infected mice.

Pre-existing helminth infection impairs the efficacy of adjuvanted influenza vaccination in mice.

The world health organization estimates that more than a quarter of the human population is infected with parasitic worms that are called helminths. Many helminths suppress the immune system of their hosts to prolong their survival. This helminth-induced immunosuppression "spills over" to unrelated antigens and can suppress the immune response to vaccination against other pathogens. Indeed, several human studies have reported a negative correlation between helminth infections and responses to vaccinations. Using mice that are infected with the parasitic nematode Litomosoides sigmodontis as a model for chronic human filarial infections, we reported previously that concurrent helminth infection impaired the vaccination-induced protection against the human pathogenic 2009 pandemic H1N1 influenza A virus (2009 pH1N1). Vaccinated, helminth-infected mice produced less neutralizing, influenza-specific antibodies than vaccinated naïve control mice. Consequently helminth-infected and vaccinated mice were not protected against a challenge infection with influenza virus but displayed high virus burden in the lung and a transient weight loss. In the current study we tried to improve the vaccination efficacy using vaccines that are licensed for humans. We either introduced a prime-boost vaccination regimen using the non-adjuvanted anti-influenza vaccine Begripal or employed the adjuvanted influenza vaccine Fluad. Although both strategies elevated the production of influenza-specific antibodies and protected mice from the transient weight loss that is caused by an influenza challenge infection, sterile immunity was not achieved. Helminth-infected vaccinated mice still had high virus burden in the lung while non-helminth-infected vaccinated mice rapidly cleared the virus. In summary we demonstrate that basic improvements of influenza vaccination regimen are not sufficient to confer sterile immunity on the background of helminth-induced immunosuppression, despite amelioration of pathology i.e. weight loss. Our findings highlight the risk of failed vaccinations in helminth-endemic areas, especially in light of the ongoing vaccination campaign to control the COVID-19 pandemic.

Corrigendum: Eosinophils and Neutrophils Eliminate Migrating Strongyloides ratti Larvae at the Site of Infection in the Context of Extracellular DNA Trap Formation.

[This corrects the article DOI: 10.3389/fimmu.2021.715766.].

Elucidating different pattern of immunoregulation in BALB/c and C57BL/6 mice and their F1 progeny.

Helminths are large multicellular parasites that infect one quarter of the human population. To prolong their survival, helminths suppress the immune responses of their hosts. Strongyloides ratti delays its expulsion from the gut by induction of regulatory circuits in a mouse strain-specific manner: depletion of Foxp3+ regulatory T cells (Treg) improves the anti-S. ratti immunity in BALB/c but not in C57BL/6 mice. In the current study we compare the hierarchy of immunoregulatory pathways in BALB/c, C57BL/6 mice and their F1 progeny (BALB/c × C57BL/6). Using multicolor flow cytometry, we show that S. ratti induces a distinct pattern of inhibitory checkpoint receptors by Foxp3+ Treg and Foxp3- T cells. Intensity of expression was highest in C57BL/6 and lowest in BALB/c mice, while the F1 cross had an intermediate phenotype or resembled BALB/c mice. Treg subsets expanded during infection in all three mouse strains. Similar to BALB/c mice, depletion of Treg reduced intestinal parasite burden and increased mucosal mast cell activation in S. ratti-infected F1 mice. Our data indicate that Treg dominate the regulation of immune responses in BALB/c and F1 mice, while multiple regulatory layers exist in C57BL/6 mice that may compensate for the absence of Treg.

A Combination of Deworming and Prime-Boost Vaccination Regimen Restores Efficacy of Vaccination Against Influenza in Helminth-Infected Mice.

Helminths still infect a quarter of the human population. They manage to establish chronic infections by downmodulating the immune system of their hosts. Consequently, the immune response of helminth-infected individuals to vaccinations may be impaired as well. Here we study the impact of helminth-induced immunomodulation on vaccination efficacy in the mouse system. We have previously shown that an underlying Litomosoides sigmodontis infection reduced the antibody (Ab) response to anti-influenza vaccination in the context of a systemic expansion of type 1 regulatory T cells (Tr1). Most important, vaccine-induced protection from a challenge infection with the 2009 pandemic H1N1 influenza A virus (2009 pH1N1) was impaired in vaccinated, L. sigmodontis-infected mice. Here, we aim at the restoration of vaccination efficacy by drug-induced deworming. Treatment of mice with Flubendazole (FBZ) resulted in elimination of viable L. sigmodontis parasites in the thoracic cavity after two weeks. Simultaneous FBZ-treatment and vaccination did not restore Ab responses or protection in L. sigmodontis-infected mice. Likewise, FBZ-treatment two weeks prior to vaccination did not significantly elevate the influenza-specific Ig response and did not protect mice from a challenge infection with 2009 pH1N1. Analysis of the regulatory T cell compartment revealed that L. sigmodontis-infected and FBZ-treated mice still displayed expanded Tr1 cell populations that may contribute to the sustained suppression of vaccination responses in successfully dewormed mice. To outcompete this sustained immunomodulation in formerly helminth-infected mice, we finally combined the drug-induced deworming with an improved vaccination regimen. Two injections with the non-adjuvanted anti-influenza vaccine Begripal conferred 60% protection while MF59-adjuvanted Fluad conferred 100% protection from a 2009 pH1N1 infection in FBZ-treated, formerly L. sigmodontis-infected mice. Of note, applying this improved prime-boost regimen did not restore protection in untreated L. sigmodontis-infected mice. In summary our findings highlight the risk of failed vaccinations due to helminth infection.

Eosinophils and Neutrophils Eliminate Migrating Strongyloides ratti Larvae at the Site of Infection in the Context of Extracellular DNA Trap Formation.

Parasitic nematodes such as hookworms actively penetrate the skin of their hosts, encountering skin-resident innate immune cells that represent the host´s first line of defense. Here we use Strongyloides ratti as a model for an intestinal helminth parasite with tissue migrating stages. We show that interception and killing of migrating larvae in mice during a 1st infection occurred predominantly in skin and muscle tissue before larvae migrated via lung and head tissue to the intestine. Inhibition of larval migration was even more efficient in immune mice during a 2nd infection where larvae barely left the site of entry i.e. the foot. Using cell-deficient mice we show that interception in the tissue was predominantly mediated by neutrophils and eosinophils while basophils and mast cells were dispensable in vivo. Likewise, neutrophils and eosinophils inhibited S. ratti L3 motility in vitro in the context of ETosis. Thereby eosinophils were strictly dependent on the presence of anti-S. ratti antibodies while neutrophils inhibited L3 motility as such. Also, MPO and MMP-9 were released by neutrophils in response to L3 alone, but immune plasma further stimulated MPO release in an antibody-dependent manner. In summary, our findings highlight the central role of the skin as first line of defense against helminth parasites in both, innate and adaptive immunity.

Limited role of mast cells during infection with the parasitic nematode Litomosoides sigmodontis.

Mast cells are innate effector cells that due to their localization in the tissue form the first line of defense against parasites. We have previously shown that specifically mucosal mast cells were essential for the termination of the intestinal Strongyloides ratti infection. Here, we analyze the impact of mast cells on the immune response and defense against the tissue-dwelling filarial nematode Litomosoides sigmodontis using mast cell-deficient Cpa3cre mice. Despite an increase and an activation of mast cells at the site of infection in wildtype BALB/c mice the outcome of L. sigmodontis infection was not changed in mast cell-deficient BALB/c Cpa3cre mice. In Cpa3cre mice neither vascular permeability induced by blood-sucking mites nor the migration of L3 was altered compared to Cpa3 wildtype littermates. Worm burden in the thoracic cavity was alike in the presence and absence of mast cells during the entire course of infection. Although microfilaremiae in the peripheral blood increased in mast cell-deficient mice at some time points, the infection was cleared with comparable kinetics in the presence and absence of mast cells. Moreover, mast cell deficiency had no impact on the cytokine and antibody response to L. sigmodontis. In summary, our findings suggest that mast cells are not mandatory for the initiation of an appropriate immune response and host defense during L. sigmodontis infection in mice.

Helminth Infections Suppress the Efficacy of Vaccination against Seasonal Influenza.

Helminth parasites infect more than a quarter of the human population and inflict significant changes to the immunological status of their hosts. Here, we analyze the impact of helminth infections on the efficacy of vaccinations using Litomosoides sigmodontis-infected mice. Concurrent helminth infection reduces the quantity and quality of antibody responses to vaccination against seasonal influenza. Vaccination-induced protection against challenge infections with the human pathogenic 2009 pandemic H1N1 influenza A virus is drastically impaired in helminth-infected mice. Impaired responses are also observed if vaccinations are performed after clearance of a previous helminth infection, suggesting that individuals in helminth-endemic areas may not always benefit from vaccinations, even in the absence of an acute and diagnosable helminth infection. Mechanistically, the suppression is associated with a systemic and sustained expansion of interleukin (IL)-10-producing CD4+CD49+LAG-3+ type 1 regulatory T cells and partially abrogated by in vivo blockade of the IL-10 receptor.

Rbpj expression in regulatory T cells is critical for restraining TH2 responses.

The transcriptional regulator Rbpj is involved in T-helper (TH) subset polarization, but its function in Treg cells remains unclear. Here we show that Treg-specific Rbpj deletion leads to splenomegaly and lymphadenopathy despite increased numbers of Treg cells with a polyclonal TCR repertoire. A specific defect of Rbpj-deficient Treg cells in controlling TH2 polarization and B cell responses is observed, leading to the spontaneous formation of germinal centers and a TH2-associated immunoglobulin class switch. The observed phenotype is environment-dependent and can be induced by infection with parasitic nematodes. Rbpj-deficient Treg cells adopt open chromatin landscapes and gene expression profiles reminiscent of tissue-derived TH2-polarized Treg cells, with a prevailing signature of the transcription factor Gata-3. Taken together, our study suggests that Treg cells require Rbpj to specifically restrain TH2 responses, including their own excessive TH2-like differentiation potential.

Interleukin-9 promotes early mast cell-mediated expulsion of Strongyloides ratti but is dispensable for generation of protective memory.

IL-9 is a cytokine with pleiotropic function that mediates allergic inflammation and immunity to intestinal helminth parasites. Accumulating evidence suggests that IL-9 acts via both, initiation and regulation of adaptive immune responses and direct activation of intestinal effector pathways. Here we use IL-9 receptor deficient mice on BALB/c and C57BL/6 genetic background to dissect effector and regulatory functions of IL-9 during infection with the parasitic nematode Strongyloides ratti. IL-9 receptor-deficient mice displayed increased intestinal parasite burden and prolonged infection irrespective of the genetic background of the mice. Increased parasite burden was correlated to a reciprocally reduced early degranulation of mucosal mast cells, reduced intestinal IL-13 expression and caused by IL-9 receptor deficiency on hematopoietic cells. We observed additional significant changes in the adaptive immune response to S. ratti infection in the absence of the IL-9 receptor that depended on the mouse strain. However, the generation of protective memory to a second infection was intact in IL-9 receptor-deficient mice, irrespective of the genetic background. In summary, our results support a central role for IL-9 as an early mast cell activating effector cytokine during intestinal helminth infection while non-redundant functions in the initiation and amplification of adaptive immune responses were not apparent.