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 1␣ and interleukin-1␤ (IL-1␤) 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, CD8␣, 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.The processes leading to the induction of polarized T-helper lymphocyte populations following exposure of the host to infectious pathogens are believed to involve interactions between the innate and acquired immune systems at the initial site of infection (24). In this context, the skin is a major barrier across which many infectious agents, including the parasitic helminth Schistosoma mansoni, gain entry to the host. This site is populated by an array of immune-competent accessory cells acting as sentinels, which are an important source of cytokines and chemokines responsible for amplifying the inflammatory response by activating resident cells and recruiting additional cells (9, 49). Cytokines also have an important influence on Th cell development, with interleukin-12 (IL-12), IL-18, tumor necrosis factor alpha, and IL-1␤ being key factors in the differentiation of Th1 cells (20,29).In our study, we examined innate immune responses elicited in the skin of mice exposed to radiation-attenuated (RA) larvae of S. mansoni. In this vaccine model, RA larvae induce Th1-type acquired immunity, which is characterized by the persistence of gamma interferon (IFN-␥)-secreting Th cells in the skin-draining lymph nodes (sdLN) (26) and confers 60 to 70% protection against a challenge infection (11). There is clear evidence that IL-12 is a critical component of this Th1-mediated protection (2, 47). In contrast,...

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 ongoing progress in fluorescence labeling and in microscope instrumentation allows the generation and the imaging of complex biological samples that contain increasing numbers of fluorophores. For the correct quantitative analysis of datasets with multiple fluorescence channels, it is essential that the signals of the different fluorophores are reliably separated. Due to the width of fluorescence spectra, this cannot always be achieved using the fluorescence filters in the microscope. In such cases spectral imaging of the fluorescence data and subsequent linear unmixing allows the separation even of highly overlapping fluorophores into pure signals. In this chapter, the problems of fluorescence cross talk are defined, the concept of spectral imaging and separation by linear unmixing is described, and an overview of the microscope types suitable for spectral imaging are given.

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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.