Identification of Piecemeal Degranulation and Vesicular Transport of MBP-1 in Liver-Infiltrating Mouse Eosinophils During Acute Experimental Schistosoma mansoni Infection

Eosinophils have been long associated with helminthic infections, although their functions in these diseases remain unclear. During schistosomiasis caused by the trematode Schistosoma mansoni, eosinophils are specifically recruited and migrate to sites of granulomatous responses where they degranulate. However, little is known about the mechanisms of eosinophil secretion during this disease. Here, we investigated the degranulation patterns, including the cellular mechanisms of major basic protein-1 (MBP-1) release, from inflammatory eosinophils in a mouse model of S. mansoni infection (acute phase). Fragments of the liver, a major target organ of this disease, were processed for histologic analyses (whole slide imaging), conventional transmission electron microscopy (TEM), and immunonanogold EM using a pre-embedding approach for precise localization of major basic protein 1 (MBP-1), a typical cationic protein stored pre-synthesized in eosinophil secretory (specific) granules. A well-characterized granulomatous inflammatory response with a high number of infiltrating eosinophils surrounding S. mansoni eggs was observed in the livers of infected mice. Moreover, significant elevations in the levels of plasma Th2 cytokines (IL-4, IL-13, and IL-10) and serum enzymes (alanine aminotransferase and aspartate aminotransferase) reflecting altered liver function were detected in response to the infection. TEM quantitative analyses revealed that while 19.1% of eosinophils were intact, most of them showed distinct degranulation processes: cytolysis (13.0%), classical and/or compound exocytosis identified by granule fusions (1.5%), and mainly piecemeal degranulation (PMD) (66.4%), which is mediated by vesicular trafficking. Immunonanogold EM showed a consistent labeling for MBP-1 associated with secretory granules. Most MBP-1-positive granules had PMD features (79.0 ± 4.8%). MBP-1 was also present extracellularly and on vesicles distributed in the cytoplasm and attached to/surrounding the surface of emptying granules. Our data demonstrated that liver-infiltrating mouse eosinophils are able to degranulate through different secretory processes during acute experimental S. mansoni infections with PMD being the predominant mechanism of eosinophil secretion. This means that a selective secretion of MBP-1 is occurring. Moreover, our study demonstrates, for the first time, a vesicular trafficking of MBP-1 within mouse eosinophils elicited by a helminth infection. Vesicle-mediated secretion of MBP-1 may be relevant for the rapid release of small concentrations of MBP-1 under cell activation

Schistosomiasis Mansoni-Recruited Eosinophils: An Overview in the Granuloma Context

Eosinophils are remarkably recruited during schistosomiasis mansoni, one of the most common parasitic diseases worldwide. These cells actively migrate and accumulate at sites of granulomatous inflammation termed granulomas, the main pathological feature of this disease. Eosinophils colonize granulomas as a robust cell population and establish complex interactions with other immune cells and with the granuloma microenvironment. Eosinophils are the most abundant cells in granulomas induced by Schistosoma mansoni infection, but their functions during this disease remain unclear and even controversial. Here, we explore the current information on eosinophils as components of Schistosoma mansoni granulomas in both humans and natural and experimental models and their potential significance as central cells triggered by this infection

Histological assessment of granulomas in natural and experimental <i>Schistosoma mansoni</i> infections using whole slide imaging

<div><p>The pathology of schistosomiasis mansoni, a neglected tropical disease of great clinical and socioeconomic importance, results from the parasite eggs that become trapped in host tissues, particularly in the liver and intestines. Continuous antigenic stimulation from these eggs leads to recruitment of inflammatory cells to the sites of infection with formation of periovular granulomas. These complex structures have variable size and composition and are the most striking histopathological feature of schistosomiasis mansoni. However, evaluation of granulomas by conventional microscopy methods is time-consuming and limited, especially in large-scale studies. Here, we used high resolution Whole Slide Imaging (WSI), which allows fast scanning of entire histological slides, and multiple morphometric evaluations, to assess the granulomatous response elicited in target organs (liver, small and large intestines) of two models of schistosomiasis mansoni. One of the advantages of WSI, also termed virtual microscopy, is that it generates images that simultaneously offer high resolution and a wide field of observation. By using a model of natural (<i>Nectomys squamipes</i>, a wild reservoir captured from endemic areas in Brazil) and experimental (Swiss mouse) infection with <i>Schistosoma mansoni</i>, we provided the first detailed WSI characterization of granulomas and other pathological aspects. WSI and quantitative analyses enabled a fast and reliable assessment of the number, evolutional types, frequency and areas of granulomas and inflammatory infiltrates and revealed that target organs are differentially impacted by inflammatory responses in the natural and experimental infections. Remarkably, high-resolution analysis of individual eosinophils, key cells elicited by this helminthic infection, showed a great difference in eosinophil numbers between the two infections. Moreover, features such as the intestinal egg path and confluent granulomas were uncovered. Thus, WSI may be a suitable tool for detailed and precise histological analysis of granulomas and other pathological aspects for clinical and research studies of schistosomiasis.</p></div

Intensity of the granulomatous response in target organs of rodents naturally or experimentally infected with <i>S</i>. <i>mansoni</i>.

<p>(A) Mean percentage of granulomatous response in livers and intestines (mean ± SEM). (B-J) Representative virtual slides of tissues with different levels of granuloma formation. In the liver (B, E, H), the lowest response is shown by the natural infection in <i>N</i>. <i>squamipes</i> (B) compared to the experimental infection in mice (E, H), while in the small intestines (C, F, I), the highest granuloma formation was observed in this wild reservoir (C). Different letters indicate significant differences between the means (<i>P</i> < 0.0001 for all comparisons between different letters in the respective groups). Bar = 1000 μm (B, D, F); 950 μm (H); 750 μm (C); 650 μm (E, G, I, J).</p

Liver and intestine areas taken by inflammatory infiltrates in the natural and experimental infections with <i>S</i>. <i>mansoni</i>.

<p>(A) Representative image of hepatic tissue from a mouse experimentally infected. After acquisition of whole slide images, areas with inflammatory infiltrates (marked in red) outside typical granulomas (marked in green) were measured. In (B), morphometric analyses reveal a very low incidence of infiltrates in the liver of infected wild rodent (natural infection) while mice experimentally infected show a very high proportion of infiltrates. In the small intestine, infiltrates are moderately higher in the natural compared to the acute experimental infection. Different letters indicate significant differences between the means (<i>P</i> < 0.0001 for all comparisons between different letters in the respective groups).</p

Sequence of steps to acquire and analyze whole slide images.

<p>(A) After loading the slides in the scanner, image acquisition starts with a “prescan” step in which the equipment takes a low resolution grayscale image followed by an automated setup process termed “profile” in which the tissue is detected and focused. (B) The equipment scans all regions of the slide defined by the profile and generates a virtual slide, which is saved on disk. These first steps take just few minutes and many slides can be sequentially scanned. (C, Ci) The operator can then review the virtual slide(s) and work on each one by selecting the area(s) of interest with the use of morphometric software. In the present work, the number and area of granulomas and area of inflammatory infiltrates were quantitated by using <i>Pannoramic Viewer 1</i>.<i>15</i>.<i>2 SP2 RTM</i> software. A representative digital slide shows a section from the small intestine of an <i>S</i>. <i>mansoni</i>-infected mouse in which the above parameters were manually delineated for subsequent automatic quantification.</p

Eosinophil numbers in the natural and experimental infections with <i>S</i>. <i>mansoni</i>.

<p>(A) Representative image of a hepatic granuloma from a naturally infected wild reservoir (<i>N</i>. <i>squamipes</i>) showing accumulation of eosinophils (indicated in high magnification in Ai by arrowheads). (B) Quantitative analyses revealed a lower number of eosinophils per granuloma area (μm²) in all target organs in the natural infection compared to the experimental infections. Different letters indicate significant differences between the means (<i>P</i> < 0.0001 for all comparisons between different letters in the respective groups). (C) Proportion of eosinophils within hepatic granulomas in each model of the infection. (****) <i>P</i> < 0.0001. Eosinophil numbers were quantitated per granuloma area, considering all types of granuloma (B) or per most frequent type of hepatic granuloma (C). Data represent mean ± SEM. Morphometric evaluation was done with the use of <i>Histoquant</i> software. Bar = 400 μm (A), 100 μm (Ai).</p

Granuloma area analyzed in digital slides from target organs of <i>S</i>. <i>mansoni</i>-infected animals.

<p>Granuloma area analyzed in digital slides from target organs of <i>S</i>. <i>mansoni</i>-infected animals.</p

Representative types of granulomas and their frequencies in target organs of rodents naturally or experimentally infected with <i>S</i>. <i>mansoni</i>.

<p>Four types of granulomas were identified in virtual slides: Pre-granulomatous exudative (<b>PE</b>); necrotic-exudative (<b>NE</b>); exudative-productive (<b>EP</b>) and productive (<b>P</b>) as described in material and methods. Bar = 250 μm.</p

Confluent granulomas in livers and small and large intestines of <i>Nectomys squamipes</i> and Swiss mice infected with <i>Schistosoma mansoni</i>.

<p>(A-H) General morphology of confluent granulomas in different organs. Graph I shows the proportions of confluent granulomas in livers and in intestines taken from naturally infected <i>N</i>. <i>squamipes</i> (at day one of capture after confirmation of the infection) and from experimentally infected Swiss mice at days 55 (acute phase) and 120 (chronic phase) of infection. Confluent granulomas were quantified using Pannoramic Viewer software. (***) indicates significant differences between the means (<i>P</i> < 0.001). Bar = 100 μm (A, D), 220 μm (B, C); 150 μm (E, F, G, H).</p