Adrian P. Mountford

Sm16, a major component of Schistosoma mansoni cercarial excretory/secretory products, prevents macrophage classical activation and delays antigen processing

BackgroundSchistosoma mansoni cercariae penetrate the skin by releasing excretory/secretory (E/S) products known as 0-3hRP, which are associated with immune modulation through Toll like receptor (TLR) signalling. Furthermore, these secretions contain Sm16, which when given to cells as a recombinant protein inhibits human monocyte derived cytokine responses to TLR4 and TLR3 ligands. Nonetheless, the extent and mechanism(s) of these inhibitory effects remain largely uncharacterized.MethodsMurine bone marrow derived macrophages were exposed to different fractions of 0-3hRP, obtained via ultracentrifugation, or recombinant Sm16. These cells were exposed to the parasite molecules in combination with different TLR ligands, or Interferon gamma, and tested for the production of the cytokines IL-10 and IL-12p40, and their ability to process antigen.ResultsThe immunomodulatory function of 0-3hRP is enriched predominantly in the pellet fraction, which contains a greater proportion of Sm16, also corroborating the ability of recombinant Sm16 to inhibit macrophage activation in response to TLR ligands. We further demonstrate that Sm16 blocks classical activation of macrophages to LPS or IFN-γ stimulation in vitro, and that inhibition of macrophage classical activation is independent of TLR2 recognition. Finally we show that Sm16 shares the altered intracellular processing observed for 0-3hRP, and is able to delay antigen processing by macrophages.ConclusionsCollectively, our findings show that Sm16 is a major component of S. mansoni cercarial E/S products, and is partly responsible for its immune-regulatory properties. Moreover, we propose that the mechanism employed by Sm16 to exert its inhibitory function is likely to be linked with alteration of endosomal trafficking and is not dependent on particular TLR receptors. Finally, we suggest that accumulation of Sm16 in the skin after percutaneous infection with S. mansoni cercariae could contribute to limiting dermal inflammation.

Immunity induced by the radiation-attenuated schistosome vaccine

As a paradigm for the development of a vaccine against human schistosomiasis, the radiation‐attenuated (RA) vaccine has enabled the dissection of different immune responses as putative effector mechanisms. This review considers advances made in the past, and updates our knowledge with reference to recent studies that have provided new information relevant particularly to the early innate events after vaccination, and to the nature of the protective effector mechanism. Priming of a protective response by RA larvae is a highly co‐ordinated series of events starting in the skin, draining lymph nodes and lungs, leading to the development of various effector responses, ranging from Th1‐associated cell‐mediated activity, to anti‐parasitic antibodies, all of which contribute to the elimination of challenge larvae to varying extents. In this respect, the RA vaccine elicits a multifaceted immune response, from which we can derive valuable insights relevant to the future design of novel delivery systems and adjuvants for recombinant and subunit vaccines.

Modulation of the host's immune response by schistosome larvae.

Schistosomes appear to have evolved several strategies to down‐regulate the hosts immune response in order to promote their own survival. For the host, down‐regulation is also beneficial as it can limit the extent of pathology. It is widely accepted that schistosomes modulate the immune response during the chronic phase of infection after egg deposition has started. However, there is increasing evidence that modulation of the immune response can occur much earlier at the time infective cercariae penetrate the host skin. In this review, we explore the various lines of evidence that excretory/secretory (ES) molecules from cercariae down‐regulate the hosts immune response. We highlight the immunological factors that are produced and may be involved in regulating the immune system (e.g. IL‐10, and eicosanoids), as well as speculating on possible mechanisms of immune modulation (e.g. mast‐cell activation, T‐cell apoptosis, and/or the skewed activation of antigen‐presenting cells [APCs]). Finally, we draw attention to several molecules of schistosome origin that have the potential to stimulate the regulatory response (e.g. glycans) and link these to potential host receptors (e.g. TLRs and C‐type lectins).

Interleukin-12 p40 Secretion by Cutaneous CD11c+ and F4/80+ Cells Is a Major Feature of the Innate Immune Response in Mice That Develop Th1-Mediated Protective Immunity to Schistosoma mansoni

ABSTRACT Radiation-attenuated (RA) schistosome larvae are potent stimulators of innate immune responses at the skin site of exposure (pinna) that are likely to be important factors in the development of Th1-mediated protective immunity. In addition to causing an influx of neutrophils, macrophages, and dendritic cells (DCs) into the dermis, RA larvae induced a cascade of chemokine and cytokine secretion following in vitro culture of pinna biopsy samples. While macrophage inflammatory protein 1α and interleukin-1β (IL-1β) were produced transiently within the first few days, the Th1-promoting cytokines IL-12 and IL-18 were secreted at high levels until at least day 14. Assay of C3H/HeJ mice confirmed that IL-12 secretion was not due to lipopolysaccharide contaminants binding Toll-like receptor 4. Significantly, IL-12 p40 secretion was sustained in pinnae from vaccinated mice but not in those from nonprotected infected mice. In contrast, IL-10 was produced from both vaccinated and infected mice. This cytokine regulates IL-12-associated dermal inflammation, since in vaccinated IL-10−/− mice, pinna thickness was greatly increased concurrent with elevated levels of IL-12 p40. A significant number of IL-12 p40+ cells were detected as emigrants from in vitro-cultured pinnae, and most were within a population of rare large granular cells that were Ia+, consistent with their being antigen-presenting cells. Labeling of IL-12+ cells for CD11c, CD205, CD8α, CD11b, and F4/80 indicated that the majority were myeloid DCs, although a proportion were CD11c− F4/80+, suggesting that macrophages were an additional source of IL-12 in the skin.

Cercarial Dermatitis Caused by Bird Schistosomes Comprises Both Immediate and Late Phase Cutaneous Hypersensitivity Reactions

Avian schistosomes are the primary causative agent of cercarial dermatitis in humans, but despite its worldwide occurrence, little is known of the immune mechanism of this disease. Using a murine model, hosts were exposed to primary (1×) and multiple (4×) infections of Trichobilharzia regenti via the pinna. Penetration of larvae into the skin evoked immediate edema, thickening of the exposure site, and an influx of leukocytes, including neutrophils, macrophages, CD4+ lymphocytes, and mast cells. A large proportion of the latter were in the process of degranulating. After 1× infection, inflammation was accompanied by the release of IL-1β, IL-6, and IL-12p40. In contrast, in 4× reinfected animals the production of histamine, IL-4, and IL-10 was dramatically elevated within 1 h of infection. Analysis of Ag-stimulated lymphocytes from the skin-draining lymph nodes revealed that cells from 1× infected mice produced a mixed Th1/Th2 cytokine response, including abundant IFN-γ, whereas cells from 4× reinfected mice were Th2 polarized, dominated by IL-4 and IL-5. Serum Abs confirmed this polarization, with elevated levels of IgG1 and IgE after multiple infections. Infection with radiolabeled cercariae revealed that almost 90% of larvae remained in the skin, and the majority died within 8 days after infection, although parasites were cleared more rapidly in 4× reinfected mice. Our results are the first demonstration that cercarial dermatitis, caused by bird schistosomes, is characterized by an early type I hypersensitivity reaction and a late phase of cutaneous inflammation, both associated with a polarized Th2-type acquired immune response.

The profile of IgG1 and IgG2a antibody responses in mice exposed to Schistosoma mansoni

The segregation of IgG2a and IgG1 immunoglobulin isotypes as markers for Th1 and Th2 lymphocytes respectively, was investigated in mice exposed to normal or optimally‐irradiated S. mansoni cercariae. Using a panel of ELISAs, soluble antigens from lung‐stage schistosomula, adult worms, or eggs, were probed with serum samples collected at biweekly intervals. Infected mice developed increased IgG1 responsiveness to all three antigens, especially between weeks five and seven, whereas IgG2a responses were lower, particularly to egg antigens. This confirms that Th2 responses are dominant after the onset of patency in infected mice. In comparison, vaccinated mice developed lower levels of IgG1, and higher levels of IgG2a to larval and worm antigens. Thus, they had balanced expression of IgG1 and IgG2a, despite having a dominant Th1 lymphocyte population. An elevated IgG1 response to egg antigens in vaccinated mice challenged with normal parasites, occurred two weeks later than in normal mice. Mice exposed to male‐only cercariae developed IgG1 and IgG2a antibodies to larval and worm antigens. However, they also had elevated IgG1 to egg antigens from week five, despite a total absence of eggs. Therefore, adult worm antigens may cross react with the egg and stimulate the switch to Th2 dominated responsiveness.

In the absence of IL-12, the induction of Th1-mediated protective immunity by the attenuated schistosome vaccine is impaired, revealing an alternative pathway with Th2-type characteristics.

Vaccination of mice with irradiated Schistosoma mansoni larvae confers high levels of immunity which is mediated by Th1‐type lymphocytes. To investigate a possible role for IL‐12 in the induction of protection, we have compared the immune response of IL‐12 p40‐deficient (KO) mice and their C57BL/6 (WT) counterparts following vaccination. Cultured lymph node cells from KO mice had markedly altered cytokine profiles with significantly decreased production of IFN‐γ increased IL‐4. Correspondingly, KO mice had enhanced levels of IgE. After challenge, cells recovered from the lungs of KO mice secreted abundant IL‐4 and IL‐5 but little IFN‐γ, while flow cytometric and histological analysis of lung cell populations recorded a very high proportion of eosinophils. The levels of protection in KO mice were substantially lower than in their WT counterparts, demonstrating the importance of IL‐12 and Th1‐mediated immune responses. This conclusion is reinforced by the administration of rIL‐12 to KO mice immediately after vaccination which led to increased IFN‐γ and the restoration of protective immunity. Nevertheless, the data also indicated that the limited levels of protection induced in KO mice occur via an IL‐12‐independent pathway, possibly mediated by Th2 cells.

Antigen localization and the induction of resistance in mice vaccinated with irradiated cercariae of Schistosoma mansoni

The fate of 75Se-labelled parasites and their released pre-synthesized macromolecules has been followed in three murine infection models. Parasite numbers in specific tissues were determined by autoradiography, and released material was estimated by gamma-counting of tissues, with adjustment for the presence of parasite-associated radiolabel. Marked differences were found between the three models. The pattern of migration of normal schistosomula was similar to that previously reported. In addition we have described the transit of parasites through the lymph nodes draining the infection site. Significant quantities of released material were detected in the skin, draining lymph nodes, bloodstream and liver. The circulating material was of parasite origin, macromolecular, and hence potentially antigenic. In comparison to the normal infection, radiation-attenuated parasites (inducing a high level of resistance to challenge) persisted in the skin, draining lymph nodes and lungs, releasing a proportionally greater amount of material in the nodes. In mice exposed to attenuated parasites and treated with the compound RO11-3128 at 24 h (inducing a low level of resistance) there was an early death and rapid clearance of the parasites whilst still in the skin. This situation resulted in the highest levels of released material in the skin, bloodstream and liver, but negligible levels in the draining lymph nodes. We suggest that the persistence of radiation-attenuated parasites in the skin and draining lymph nodes, together with the prolonged release of antigen in the latter site, compared to the normal situation, are major factors in the induction of resistance.

CD4+CD25+ regulatory cells contribute to the regulation of colonic Th2 granulomatous pathology caused by schistosome infection

Eggs of the helminth Schistosoma mansoni accumulate in the colon following infection and generate Th2-biassed inflammatory granulomas which become down- modulated in size as the infection proceeds to chronicity. However, although CD4+CD25+FoxP3+regulatory T cells (Tregs) are known to suppress Th1-mediated colitis, it is not clear whether they control Th2 –associated pathologies of the large intestine which characterise several helminth infections. Here we used a novel 3D-multiphoton confocal microscopy approach to visualise and quantify changes in the size and composition of colonic granulomas at the acute and chronic phases of S. mansoni infection. We observed decreased granuloma size, as well as reductions in the abundance of DsRed+ T cells and collagen deposition at 14 weeks (chronic) compared to 8 weeks (acute) post-infection. Th2 cytokine production (i.e. IL-4, IL-5) in the colonic tissue and draining mesenteric lymph node (mLN) decreased during the chronic phase of infection, whilst levels of TGF-β1 increased, co-incident with reduced mLN proliferative responses, granuloma size and fibrosis. The proportion of CD4+CD25+FoxP3+Tregs: CD4+ cells in the mLN increased during chronic disease, while within colonic granulomas there was an approximate 4-fold increase. The proportion of CD4+CD25+FoxP3+Tregs in the mLN that were CD103+ and CCR5+ also increased indicating an enhanced potential to home to intestinal sites. CD4+CD25+ cells suppressed antigen-specific Th2 mLN cell proliferation in vitro, while their removal during chronic disease resulted in significantly larger granulomas, partial reversal of Th2 hypo-responsiveness and an increase in the number of eosinophils in colonic granulomas. Finally, transfer of schistosome infection-expanded CD4+CD25+Tregs down-modulated the development of colonic granulomas, including collagen deposition. Therefore, CD4+CD25+FoxP3+Tregs appear to control Th2 colonic granulomas during chronic infection, and are likely to play a role in containing pathology during intestinal schistosomiasis.

The Mannose Receptor (CD206) is an important pattern recognition receptor (PRR) in the detection of the infective stage of the helminth Schistosoma mansoni and modulates IFNγ production.

In this study, infective larvae of the parasitic helminth Schistosoma mansoni were shown to contain a large number of glycosylated components specific for the Mannose Receptor (MR; CD206), which is an important pattern recognition receptor (PRR) of the innate immune system. MR ligands were particularly rich in excretory/secretory (E/S) material released during transformation of cercariae into schistosomula, a process critical for infection of the host. E/S material from carboxyfluorescein diacetate succinimidyl ester (CFDA-SE)-labelled cercariae showed enhanced binding by cells lines that over-express the MR. Conversely, uptake was significantly lower by bone marrow-derived macrophages (MΦ) from MR(-/-) mice, although they were more active as judged by enhanced pro-inflammatory cytokine production and CD40 expression. After natural percutaneous infection of MR(-/-) mice with CFDA-SE-labelled parasites, there were fewer cells in the skin and draining lymph nodes that were CFDA-SE(+) compared with wild-type mice, implying reduced uptake and presentation of larval parasite antigen. However, antigen-specific proliferation of skin draining lymph node cells was significantly enhanced and they secreted markedly elevated levels of IFNγ but decreased levels of IL-4. In conclusion, we show that the MR on mononuclear phagocytic cells, which are plentiful in the skin, plays a significant role in internalising E/S material released by the invasive stages of the parasite which in turn modulates their production of pro-inflammatory cytokines. In the absence of the MR, antigen-specific CD4(+) cells are Th1 biased, suggesting that ligation of the MR by glycosylated E/S material released by schistosome larvae modulates the production of CD4(+) cell specific IFNγ.