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12УДК 57.086.3:57.012.4:582.282.123.4:611.24:599.323.41
ультраструктурные аспекты взаимодействия между альвеолярными макрофагами мышей и клетками гиф Aspergillus fumigatus
''Степанова А.А. (зав. лаб.)*, Васильева Н.В. (директор института, зав. кафедрой), 2Ямагучи М. (адъюнкт-профессор), 2Чибана Ш. (профессор), 'Босак И.А. (с.н.с.)
1 НИИ медицинской микологии им. П.Н. Кашкина, СевероЗападный государственный медицинский университет им. И.И. Мечникова, Санкт-Петербург, Россия; 2 Центр исследований по медицинской микологии, Университет г. Чиба, Япония
©Коллектив авторов, 2016
Представлены ультраструктурные данные о моделях взаимодействия между клетками гиф Aspergillus fumigatus и макрофагами легких мышей при изучении экспериментального аспергиллеза. Показано присутствие трех типов макрофагов в легких мышей: 1) с «поглощенными» клетками гиф и часто на ранних стадиях этого процесса (98%); 2) неактивные (1%) и 3) на различных стадиях деструкции (1%). Выявлено три модели взаимодействия между макрофагами легких мышей и клетками гиф A. fumigatus в ходе которых: 1) клетки гиф убивают макрофаги (80%); 2) макрофаги убивают клетки гиф (19%); 3) «ничья» между грибами и макрофагами (1%). На основании анализа моделей взаимодействий между альвеолярными макрофагами мышей и гифами A. fumigatus показано, что клетки иммунной системы находятся в подавленном состоянии, а грибная инфекция доминирует.
Ключевые слова: Aspergillus fumigatus, взаимодействие, гифы, легкие мышей, макрофаги, трансмиссионная электронная микроскопия, in vivo, ультраструктура
ultrastructural aspects of the interactions between the murine lungs macrophages and the Aspergillus fumigatus hyphal cells
1Stepanova A.A. (head of the laboratory), 1Vasilyeva N.V. (director of the institute, head of the chair), 2Yamaguchi M. (grand-fellow), 2 Chibana H. (associated professor), iBosak i.A. (senior scientific collaborator)
1Kashkin Research Institute of Medical Mycology of Northwest State Medical University named after I.I. Mechnikov, St. Petersburg, Russia; 2Medical Mycology Research Center, Chiba University, Chiba, Japan
©Collective of authors, 2016
The ultrastructural data about the patterns of interactions between the Aspergillus fumigatus hyphal cells and murine lungs macrophages in the experimental aspergillosis were presented. Three types of macrophages demonstrated in murine lungs: 1) with «ingested» hyphal cells and often in
Контактное лицо: Степанова Амалия Аркадьевна, тел.: (812) 303-51-40
the early stage of this process (98%); 2) inactive (1%) and 3) in different stage of destruction (1%). The three patterns of interactions revealed between the murine lungs macrophages and the hyphal cells of A. fumigatus in which: 1) the hyphal cells kill macrophages (80%); 2) macrophages kill hyphal cells (19%); 3) «draw» between the fungi and macrophages (1%). On the basis of analysis of the patterns of interactions between the murine alveolar macrophages and A. fumigatus hyphae we demonstrated that the cells of immune system were in suppressed condition and fungal infection was dominated.
Key words: Aspergillus fumigatus, hyphae, transmission electron microscopy, interactions, in vivo, macrophages, murine lungs, ultrastructure
INTRODUCTION
Previously we investigated the ultrastructure of this pathogenic fungi species in the tissue [1] and cytological peculiarities of early stage of development of Aspergillus fumigatus infection - penetration of hyphae through the cells of murine tracheobronchial epithelium cells [2]. The aim of the present work was continue this investigation and study the pattern of interactions between the murine lungs macrophages and the hyphal cells of Aspergillus fumigatus.
MATERIALS AND METHODS
The model of experimental aspergillosis was reproduced by the technique presented in the work of Balloy V. with coauthors [3] on mice - 5 males of the Balb strain weighing 18-20 g. According to Latge J.-P. [Latge J.-P. // Clin. Microbiol. Rev. - 1999. - Vol. 12, №2] A. fumigatus becomes pathogenic only at the immunocompromised host. Therefore for immunity suppression, the hydrocortisone suspension was given intraperitoneal in the amount of 0,01 ml per 1 g of animal weight.
Mice were infected intranasal with conidia (1x107 in 0,05 ml of the sterile distilled water) of clinical A. fumigatus isolate (RKPGF-1172, Russian collection of pathogenic fungi of P. N. Kashkin Research Institute of Medical Mycology). The fungal cultures were grown for 7 days at 37 °C on Saburo's agar (pH 5,7).
Earlier [4] we shown that after 6 hours of mature conidia incubation at 37 °C on liquid Capek's medium of the frequency of the germinated conidia of this aspergillus strain were equal 90,6%. Autopsy of the mice was carried out after 5 days of the experiments, so that we note the death of 20% them from total number. The pieces of the murine lungs were fixed with glutaraldehyde-osmium by the technique described previously [5]. Ultrathin sections were cut on the LKB V ultramicrotome, contrasted with uranyl acetate and lead citrate and then examined in a TEM Jem 100SX II (Japan, Tokyo).
RESULTS AND DISCUSSION
In investigated murine lungs the cells of immune system were presented in a large amount. For comparison in human lungs with aspergillosis in immunocompromised patients situation may be opposite. As example, in the human lungs with hypha in catabolic condition the cells of immune system present in minor quantity and were inactive [6] and in the case with active living A. fumigatus they absent [7]. Thus we «lucky» so that used of in our experiments doses of hydrocortisone was optimal for development of fungi and activation of the cells of immune system.
We have investigated more than 150 macrophages in murine lungs. This fact showed that we deal with «far come» infection process. Besides the studying of A. fumiga-tus in the murine lungs tissues of this experiment show its
active and living condition with formation of pictures of radial growth [1]. In investigated case we revealed 3 types of macrophages: 1) with «ingested» hyphal cells and often on in the early stage of this process (98 %); 2) inactive (1 %); 3) in different stage of destruction (perhaps «waste» one, 1 %).
In inactive macrophages one large (4,0 x 1,5 ^m) irregular nucleus occupied its central part and main cell volume (Fig. 1 a). Condensed chromatin localized near nuclear envelope. Cytosol medial electron density, small number of small mitochondria, vesicular elements and without storage substances. Pseudopodia were numerous and long sized. The fungal elements were absent. In the destroying macrophages the volume of nucleus and cytosol was identical (Fig. 1 b). Nucleus (2,0 ^m) with regular form, with rare small (0,2 - 0,3 ^m) lipid inclusions, small vacuoles and destroyed mitochondria. The pseudopodia, hyphae and plasma membrane (in some sites) were absent.
Among the 1-st types of macrophages we observed three types of interactions between the fungal hyphal cells and macrophages when: 1) fungi kill macrophages (80 %); 2) macrophages kill fungi (19 %); 3) «draw» between the fungi and macrophage (1 %).
In all described types of scenaries of A. fumigatus interactions with murine lungs macrophages participated only hyphal cells. The mature and swollen conidia in murine lungs were absent.
The 1-st type of interaction. The rare pictures of initial stage of ingesting (Fig. 1 c, d) demonstrated that macrophages have contact with intact hyphal elements. Figure 1 c (arrow) demonstrated the early stage of hyphal ingestion by macrophage which posses with two nucleus and numerous mitochondria what was the attributes of its activity. Another criterion of its activity was the possibility to dissolve the extracellular matrix (double arrows) which contain melanin. It was obvious that macrophages at the first time «come» in contact with more young part of in vivo growing «mycelium» - one or more (less than three) hyphal terminal cells (Fig. 1 e) so that it was more active (in terms of speed of growth and distribution of infection and synthesis and secretion of hydrolytic ferments). Directly in the hyphal apex (Fig. 1 e, arrow) extracellular matrix around the cell wall absent. The hyphal cells contain several single spherical (1,0 ^m) or ellipsoidal nuclei (0,9 x 1,2 ^m), higher electron density cytosol, median number of small (0,2 - 0,3 ^m) polymorphic mitochondria with dark matrix and light cristae. As a rule the localized in some distance from apical dome. Several small single or in small group vacuoles randomly distributed in hyphal cells. They with light content or with one dark protein globule. The component of endomembrane system practically absent. Lipid globules present as exclusion. Hyphal cell wall was thin (0,15-0,20 ^m) as in the same which infected murine lungs tissue [1]. The extracellular matrix was very thin (0,20,7 ^m) and sometimes irregular in thickness or absent. In the content of macrophages the number of hyphasomes [2] varied from 1 to 4. We very often visualized the presence of hyphal elements (Fig. 1 g) or terminal hyphal segments (Fig. 1 e-f) near macrophagal nucleus which were in different stages of destruction. It was created impression that presents some growing tropism of hyphal cells in direction of this cell component.
We interpreted this situation as «fast purposeful shot in heart» so that fungal metabolites destroyed of main cell com-
ponent break the normal coordination inside macrophages. In macrophages with two nucleus the morphological characters of destroying process may be different (Fig. 1 h) what support this suggestion. It was obvious that the presence of two nucleus in some macrophages give them some «cart blanche» in opposition of fungal infection.
The macrophagal nuclei may significantly reduce in size (Fig. 1 f, g). Nucleolar envelop was formed light irregular spaces (Fig. 1 a, arrow). Condensed chromatin formed large irregular dark aggregations or concentrated near nucleolar envelope (Fig. 1 i). Later nucleoplasm and cytosol were clarifyed. The number of mitochondria, storage substances and another cell component inside macrophages significantly reduce. Mitochondrial matrix and cristae loose (Fig. 1 j). Numerous vesicular elements with variable size appear in cytosol. The rupturing the vacuolar tonoplast initiate the global autolysis of content of alveolar macrophages. Rare between hyphasome and macrophagal plasma membrane (Fig. 1 h, arrow) possible observed the wide light halo.
The 2-nd type of interaction. It was important note that in a whole the ultrastructural «portrait» of A. fumigatus hyphal cells (Fig. 1 l) in this variant of interactions was identical with the described for 1-st type of interaction and young part fungal colonies in the murine lungs [1]. Sometimes possible find hyphae which was in close contact (Fig. 1 m, arrow) and formed co-called «biofilm» in content of macrophage. According the different pictures of the zones of contact between the hyphal cells and plasma membrane of macrophages (Fig. 2 a-d) of this variant of interaction the «opposition» between the pathogen and cell of immune system was more intense. Figures 2 a-d demonstrated that around the hyphasomes formed asymmetrically (Fig. 2 b) or symmetrically (Fig. 2 a, c, d) halo with different morphological peculiarity (thin-fibrillar base and dark variable in size and morphology inclusions). It was obvious that visible in this sites small dark granules (Fig. 2 a, arrows) or lamel-las (Fig. 2 c, d) present the debris of disintegrated extracellular matrix. Around the halo present median size vesicles (Fig. 2 a) or microvacuoles (Fig. 2 g) with thin-fibrillar content which identical with the halo content around hyphasomes. Gradually the thickness of extracellular matrix was reduced (Fig. 2 e, f) and finally loose (Fig. 2 g). After this the cell wall destruction started (Fig. 2 g). Figure 2 h very representative so that demonstrated sequential stages of hyphal cell elimination (arrows route). After the «stripping» from cell wall hyphal cell protoplast was dark and surround with macrophagal plasma membrane. Then plasmolysis of fungal cell protoplast and its subsequent lysis occur. Several times possible see membranous fragments and vesicular elements inside the macrophagal plasma membrane lumen which long time preserve the hyphal spherical form. Thus, the alveolar macrophages together with the cells of the murine tracheobronchial epithelium [2] able fully disintegrated hyphal extracellular matrix and cell wall. For comparison light-micro-scopic investigations of Cryptococcus neoformans yeast cells interaction with murine macrophages in vitro condition [8; Tucker S.C., Casadeval A. // Proc. Natl. Acad. Sci. - 2002. - Vol. 99, №5] and transmission electron microscopic data on example of human brain macrophages [9] demonstrated that fungal cell walls not «utilize» with the cells of immune system and extruded from them.
In some rare cases the space between the hyphal cells
Fig. 1. Transmission electron microscopy of inactive (a), destroying (b) and with 1-st type of interaction macrophages (c - m) in murine lungs. Explanation for this and figure 2: CC - condensed chromatin; CW - cell wall, EM - extracellular matrix; P -pseudopodia; H - halo; HC - hyphal cell(s); LI - lipid inclusion(s), M - mitochondrium(ia), Mc - macrophage; Mv - microvacuole; N - nucleus, V - vacuoles, Vs - vesicles. Scale: a - 1,5 |m; b, l - 1 |m; c-f, h-k - 2 |m; g - 0,5 |m
Fig. 2. Fragments (a-h) and (a-g) and general view of macrophages with 2-nd (a-h) and 3-rd type (i, j) interactions with hyphal cells A. fumigatus. Scale: a - 0,5 |m; b, e, g, i, j - 1 |m; c, d, f - 0,5 |m; h - 1,5 |m
and macrophagal plasma membrane and secretory vesicles and microvacuoles absent (Fig. 1 h, i).
The 3-rd type of interaction which not possible named as «type» possible interpreted as «draw» (Fig. 2 i, j) with some elements of «friendship». It was evident the reduced macrophagal size, its large nuclear-cytoplasmic relation and presence minor number young hyphal cell without extracellular matrix. Macrophagal nucleus localized asymmetrical near plasma membrane (Fig. 2 i, j). Halo, secretory vesicles and microvacuoles around hyphal segment absent what were the indicators of absence the destructive process, macrophagal plasma membrane in tight contact with fungal cell wall. It was obvious that the absence of extracellular matrix and «stripping» condition of fungal hyphae not activated macrophages with this scenario and presence of extracellular matrix around hyphal cells play signaling role for activation of macrophages. In literature present data about the latent staying of the C. neoformans yeast cells in the macrophages, reported in numerous papers, carry out on light microscopic level in vitro on example of culture of murine alveolar macrophages [10-12; Harrison T.S., Lev-itz S.M. Cryptococcus neoformans and macrophages, 2002; Lee S.C., et al. // Lab. Invest. - 1995. - Vol.73]. As believed Harrison T.S. and Levitz S.M. [Harrison T.S., Levitz S.M. Cryptococcus neoformans and macrophages, 2002], during this type of relations between the macrophages and fungal cells they are in condition of equilibrium, that allow the last to stay in intact state indeterminate time. Before we show that latent condition of murine lungs macrophages which ingest C. neoformans cells determinate with the presence of desiccated yeast cells [13].
Thus differences of described 3-rd type with latent condition described for macrophages which ingest yeast cells C. neoformans [13] consist in fact that in our case hyphal cells were in intact condition. It is important to note that for content of macrophages with latent condition in present case and with C. neoformans [13] typical presence of lipid inclusions. According our opinion the number of storage substances demonstrated the time of staying of macrophages in latent condition. Perhaps presence of storage lipids in this types macrophages need for «opposition» during possible in future time hyphal activation and growth.
RESUME
Thus, provided analysis of the different «scenaries» of interactions between the murine alveolar macrophages
and A. fumigatus hyphae, demonstrated that in a whole the cells of immune system were in suppressed condition what correlated with presence of numerous intact hyphae in lungs and in circulatory system [1]. The pictures of early stages of ingestion demonstrated absence the macrophagal pseudopodia in this process. Possible suggest that macrophagal plasma membrane with part of cytosol «obduce» the part of the hypha and formed hyphasome [2]. The same pattern was revealed also for A. fumigatus hyphal cell interaction with the cells of murine tracheobronchial epithelium cells [2].
The pictures of early and another stages of interactions between A. fumigatus hyphae and alveolar murine macrophages demonstrated that last have contact with intact and interact with young hyphal elements. So that possible conclude about the selective ability of macrophages ingest at the first time terminal young and apically actively growing hyphal cells. If true for cells of immune system «contest» with growing part of hyphae more difficult in comparison with yeast cells, including budding one so that its task was double: killing of moving and one of the actively secreted hydrolytic ferments part of infection agent.
According our data the ultrastructure of intact hyphal cells inside murine alveolar macrophages was identical and independent of the type of its interactions and it's final. We not revealed the cytological characters of fungal activity even for 1-st type during which they kill macrophages. The cells of murine tracheobronchial epithelium cells and alveolar macrophages in analyzed experiments may destroy hyphal extracellular matrix, cell wall and internal part of its cell, but the cytological appearance of this process was different.
During the process interaction between the murine alveolar macrophages and A. fumigatus hyphae the last not may destroy the hyphasomal plasma membrane what significantly differ this pathogen from C. neoformans yeast cells [13] which may destroy the phagosomal one. Unfortunately, but we have not data about the level of virulence of investigated A. fumigatus strain. Perhaps higher virulent strain be able, but need additional investigations. Other differences between these two patterns of interactions consist in possibility of macrophages in case of A. fumigatus and immunosuppressed with hydrocortisone mouse fully disintegrated chitin of hyphal cells walls contrary with example with not suppressed mouse and C. neoformans yeast cells [13]. Arise impression, that macrophages in immuno-suppressed mouse more aggressive than in not suppressed.
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Поступила в редакцию журнала 20.12.2015
Рецензент: Сергеева Л.Ю.
