Short Communication Alterations induced in the ileum of mice upon inoculation with different species of Leishmania : a preliminary study

Introduction: Leishmania species cause skin, mucosal, and disseminated lesions. We studied the effects of three Leishmania species on ileal morphology in mice. Methods: BALB/c mice were intraperitoneally inoculated with Leishmania (Leishmania) amazonensis , Leishmania (Viannia) braziliensis , and Leishmania (Leishmania) major (4 animals/group) . After 72h, the ilea were collected and histologically processed. Results: Following inoculation, the goblet cell and intraepithelial lymphocyte populations increased, while Paneth cell number and crypt width decreased. In addition, enterocyte size, villi height, and mucosa, submucosa, and muscular tunic thickness increased. Conclusions: Leishmania modified the quantity of cells in and morphology of mice ilea.

Leishmaniasis is a parasitic disease that manifests in cutaneous, mucosal, and visceral forms and causes several clinical sequelae, including disseminated lesions that depend on the parasitic species and parasite-host interactions 1 . Several species of Leishmania, including Leishmania (Leishmania) amazonensisis (LLA), are able to induce cutaneous and visceral leishmaniasis 2 . Leishmania (Viannia) braziliensis (LVB) and Leishmania (Leishmania) major (LLM) are associated with the mucocutaneous form of the disease 3 . The different forms of leishmaniasis occur from migration of the parasite from the skin to other sites far from the inoculated area via lymphatic or hematogeneous dissemination 4 . The parasite is also able to invade and remain in organs associated with the phagocytemononuclear cell system 5 .
In addition to cutaneous lesions, the parasite is found in lymph nodes, liver, and spleen 6 . However, little is known about the effects of the Leishmania infection on the gastrointestinal tract. Thus, the present study investigated the effects of inoculation with three Leishmania species on the morphology and morphometry of the ileum in mice.

Ethical considerations
The present study was approved by the Comitê de Conduta Ética no Uso de Animais of the State University of Maringá [Universidade Estadual de Maringá (UEM); 039/2013] and performed in accordance with the Guidelines of the Brazilian Control and Experimentation. Sixteen female Balb/c mice (25-40g) from the central animal facility of UEM were used. The animals were kept under a controlled temperature (22ºC) and 12h/12h light/dark cycle with food and water available ad libitum. The mice were randomly divided into four groups with four animals per group: the control group and three groups that were inoculated with 0.1ml (2 × 10 8  Promastigotes were obtained from the parasite bank of the Leishmaniasis Laboratory of UEM. The parasites were cultured in 199 medium (Gibco Laboratories, Grand Island, NY, USA) supplemented with 1% L-glutamine, 1% human urine, and 10% fetal bovine serum. The parasites were then harvested in the stationary phase of culture and inoculated into the peritoneal cavity to ensure the intestine was not contaminated.
The mice were euthanized under deep anesthesia with halothane vapor (Tanohalo, Cristália, Itapira, Brazil). After laparotomy, 1cm of the distal ileum was collected and processed for histology. Semi-serial 4-µm transverse sections were cut and stained with hematoxylin and eosin for morphometric analysis of the intestinal wall and enterocytes and quantification of Paneth cells and intraepithelial lymphocytes (IELs). Periodic acid Schiff (PAS) and Alcian blue (AB) pH 1.0 and 2.5 were used to stain and evaluate goblet cells.
For cellular quantification, each section was divided into four quadrants and Paneth cells that were present in eight crypts in each quadrant per animal per group were quantified. Goblet cells and IELs were quantified by counting a total of 2,500 epithelial cells from each animal and the ratios of goblet cells and IELs to 100 epithelial cells were calculated.
Morphometric analysis was performed using images captured with a digital camera (Pro series 3CCD camera) coupled to an optical microscope (Olympus BX50). Measurements were made using Image-Pro Plus software (Media Cybernetics). The width and height of villi, width and depth of crypts, thickness of the mucosa, submucosa, and muscular tunic, and total thickness of the ileum wall were measured from the images. We performed 80 measurements for each parameter throughout the circumference of the ileum for each animal. We evaluated the width and height of enterocytes and also captured images of 10 Paneth cells per quadrant per animal per group and counted the number of granules inside the Paneth cells. Using these same images, we measured the areas of the Paneth cells and their granules.
The data were analyzed using BioEstat 5.3 software. The data distributions were evaluated using D' Agostinho-Pearson normality tests and the Bartlett test was used to check the homogeneity of the variances. Data with normal distributions were statistically analyzed using analysis of variance (ANOVA) followed by Tukey's multiple-comparison tests. Data with non-normal distributions were analyzed using Kruskal-Wallis followed by Dunn's test. A p < 0.05 was considered to indicate statistically significant differences. Data are presented as mean ± standard error.
The LVB group displayed an increase in enterocyte width and height. The LLM group presented a decrease in enterocyte height. The number of Paneth cells decreased in the LLA and LVB groups. Further, there was an increase in the area of the Paneth cells in the LVB group and in the number of granules inside them in the LLM and LVB groups. There was also an increase in the number of sialomucin-producing goblet cells, based on AB staining (pH 2.5), in the LLA, LLM, and LVB groups. The LLM group also presented with an increase in the number of sulfomucin-secreting cells, as shown by AB staining (pH 1.0). The LLA and LVB groups had more neutral mucin cells based on PAS staining. The number of IELs increased in all three inoculated groups (Table 1 and Figure 1). (Figure 1 and Figure 2) indicated that all infected groups exhibited an increase in the height of the villi and thickness of the mucosa, submucosa, and muscular tunic, reflecting an increase in the total thickness of the ileal wall. The LVB group also had increased villi width. A decrease in the width of the crypts was observed in all of the infected groups. The depth of the crypts decreased in the LLM group.  In the peritoneal cavity, immune cells, some of which are phagocytic, can recognize pathogens and initiate an early response against parasites 8 . In response to infection or chemical stimuli, these phagocytic cells can migrate from the peritoneal cavity to the lamina propria 9 , perhaps transporting the parasite.

Morphometric analysis of the ileal wall
During the innate immune response, the release of several mediators can cause tissue injury depending on the intensity of the inflammatory response. The intestinal response depends on the infection and inflammation in remote locations and may endure after treatment 10 . Although we did not detect the parasite in the ileum, inflammation triggered by Leishmania inoculation may be responsible for the alterations observed in this study. In a previous study, histopathological changes caused by some isolates of L. (V.) braziliensis were observed even when no amastigotes were found in the tissue of interest 7 .
Enterocytes play an important role in the mucosal immune response and may control local inflammation 11 .We observed variable changes in enterocytes in the different inoculated groups. These differences may be related to the susceptibility of Balb/c mice to different species of Leishmania. Paneth cells play an important role in the intestinal innate defense system by releasing substances stored within their granules 12 . A decrease in the number of Paneth cells may be related to the inflammatory state triggered after parasite inoculation. By contrast, an increase in the area and number of granules may reflect a compensatory response to the reduction in the number of Paneth cells.
Goblet cells are responsible for mucin production and release, which provides lubrication and protection against pathogens 13 . We observed an increase in the number of goblet cells, possibly reflecting a defense mechanism to protect the intestinal epithelium.
Intraperitoneal inoculation of Leishmania species increased the number of IELs, indicating that more immune cells migrated to the epithelium. These cells can produce cytokines, which are important for controlling infections 14 . Furthermore, the proliferation and recruitment of immune cells to the intestine may have contributed to the increase in the thickness of the submucosa and villi 15 . Additionally, according to Mawe 10 , local inflammation alters the metabolism of intestinal cells, especially smooth muscle cells and epithelial cells, which may have contributed to the observed hypertrophy.
In summary, the present study found that inoculation with different species of Leishmania induced alterations in the small intestine in mice. L. (V.) braziliensis provoked the most pronounced alterations. These findings may spur further studies on host-parasite interactions that are not limited to the skin, but also likely involve distal organs such as the intestine.