Analysis of Spleen Cells in Susceptible and Resistant Mice with Experimental Lagochilascariosis

Lagochilascariosis is an emerging parasitic disease caused by the helminth Lagochilascaris minor. The experimental mouse model has been used to study the immune response against L. minor infection. In the present work, immunohistochemistry analysis of spleen cells populations was evaluated in susceptible (C57BL/6) and resistant (BALB/c) mice experimentally infected with L. minor. The BALB/c mice exhibited increased spleen cell indexes as follows: F4/80+ at 100 days after infection (DPI), CD4+ at 100 and 250 DPI, CD8+ at 35 and 100 DPI, and CD19+ at 100, 150, and 250 DPI. In the spleens of the infected C57BL/6 mice, increased indexes of the following spleen cells were observed: F4/80+ cells at 250 DPI, CD4+ cells at 150 DPI, CD8+ cells at 35, 150, and 250 DPI, and CD19+ cells at 150 to 250 DPI. The index of spleen cells confirmed the differences between the control and infected groups at several time points following the infection. These data demonstrate an association between a preferential increase in the number of CD4+ and CD19+ spleen cells and resistance to experimental lagochilascariosis in BALB/c mice and between a preferential increase in the number of CD8+ spleen cells and susceptibility in C57BL/6 mice.


Introduction
Lagochilascariosis is caused by Lagochilascaris minor, which can be the aetiological agent of the human infection; eventually, it is also found in domestic animals, such as dogs and cats [1]. Although it does not constitute a public health problem, lagochilascariosis is an emerging disease in Brazil; 90% of the described cases worldwide occur in this country [2].
Lagochilascariosis is a chronic disease characterised by the presence of granulomatous lesions in the oronasopharyngeal region that cause exudative abscesses with the presence of eggs, larvae, and adult parasites and indicate the occurrence of an autoinfection. is infection can be fatal if the parasite invades the lungs and the central nervous system [3,4].
e natural life cycle and mechanism of infection of L. minor are still unclear because little is known about the biology of this parasite. Wild animals, such as dogs, felines, and rodents, are considered to be likely natural reservoirs of L. minor [2,5]. e experimental heteroxenic life cycle of the parasite was described using mice as intermediate hosts and domestic cats as de�nitive hosts [6,7]. e extraordinary capacity of L. minor to migrate to different human tissues has also been observed in experimental models [8]. In mice inoculated with infective parasite eggs containing third-stage larvae (L3), hatched larvae were observed during migration to the intestinal tract. A dissemination of these larvae to other organs, such as the lungs, skeletal muscles, and subcutaneous tissue, was observed. When cats were fed the carcasses of infected mice, the L3 larvae hatched from the stomach cysts and ascended to the oropharynx, where fourth-stage larvae (L4) were found. L. minor eggs were found in the faeces of infected cats [9].
e host-parasite relationship during experimental infection with L. minor has been studied in strains of inbred mice with different genetic backgrounds. In previous studies, aer observing the cumulative mortality rate within one year of infection, it was possible to classify the mouse strains as resistant (A/J, BALB.xid, and BALB/c) or susceptible (C57BL/6 and B10.A). e susceptible strains displayed reduced survival, more intense granulomatous lesions, and higher numbers of L3 larvae and adult parasites [8,10].
It is evident that the difference in the susceptibility of mice to experimental lagochilascariosis can be in�uenced by the individual immune response, which plays an essential role in the progression of the infection by reducing or blocking the pathological processes [8,11].
We have previously demonstrated that the serum levels of interleukin-10 (IL-10) were similar in L. minor-infected C57BL/6 and BALB/c mice at 90 DPI; however, the sera from the BALB/c mice contained higher levels of interferon-(IFN-) than those from C57BL/6 mice. During this period, less severe pulmonary lesions and fewer subcutaneous nodules containing L3 larvae were observed in BALB/c mice than in C57BL/6 mice [12]. Moreover, we have recently demonstrated that both strains produce different classes of antibodies in response to crude extract and soluble antigens from the parasite [11,13].
Based on these previous results, we considered it important to evaluate the populations of spleen cells during experimental infection with L. minor. Because the pro�les of spleen cells during experimental lagochilascariosis have never been studied, we accomplished the immunophenotyping of F4/80+, CD4+, CD8+, and CD19+ cells in the spleens of control mice and BALB/c and C57BL/6 mice that were experimentally infected with L. minor; the goal of this study was to identify alterations in the cellular populations during experimental lagochilascariosis infection.

Parasite Eggs.
Parasite eggs were isolated from the faeces of Felis domesticus individually fed the carcasses of mice infected with 30 to 100 nodules of infective third-stage larvae (L3) from a human L. minor isolate. e faeces of infected cats were subjected to Hoffman's method of sedimentation and stored in a formalin solution (1%) at room temperature for 40 days [14]. Aer the infective eggs containing L3 developed, faecal samples were subjected to Faust's method to optimally recover eggs that were free of faecal debris [15]. e egg suspensions were washed �ve times (20 minutes/4000 rpm) with phosphate-buffered saline (PBS, pH 7.4) and transferred to a graduated centrifuge tube. e eggs were counted on microscope slides, and the �nal concentration was adjusted to 10 4 eggs/mL and used to inoculate the mice [16].

Mice and Experimental Infection.
Mice were obtained from the Butantan Institute animal facilities. e animals were given food and water ad libitum and handled according to the local regulations. e research protocols were approved by the Ethics Committees of the Federal University of Goiás and the Butantan Institute.
A total of 20 BALB/c and 20 C57BL/6 mice were orally inoculated with a suspension of 10 3 ± 200 L. minor eggs per animal. Groups of �ve animals were sacri�ced at each time point (35, 100, 150, and 250 DPI) and submitted to necropsy. e spleens were harvested for immunohistochemistry and analysis of the cell populations. A total of 20 BALB/c and 20 C57BL/6 mice received saline orally and were used as uninfected controls at the same time points.

2.3.
Immunohistochemistry. e mouse spleens were collected, and fragments were put into a mould and covered with OCT, an inclusion tissue compound. e fragments were then immersed in cooled isopentane (Vetec, Brazil), snap-frozen in liquid nitrogen, and stored at −80 ∘ C until use. e fragments were sectioned with a cryostat, and the tissue sections were �xed in cold acetone for 10 minutes; they were then stored at −80 ∘ C. e sections were subjected to peroxidase blocking with 30 volumes of H 2 O 2 diluted 1/1000 in methanol (15 minutes) and then incubated with normal goat serum (15 minutes) to block nonspeci�c binding. Aer the excess serum was removed, the sections were incubated with primary rat anti-mouse monoclonal antibodies (mAb) diluted in PBS containing 2% foetal bovine serum in a humid chamber for 18 h at 4 ∘ C. e following rat anti-mouse mAb were used: anti-F4/80 (macrophage), clone A3-1 (Serotec); anti-CD19 (B lymphocytes), clone 1D3 (BD Biosciences); anti-CD4 (T-helper lymphocytes), clone H129.19 (BD Biosciences); and anti-CD8 (cytolytic T lymphocytes), clone 53-6.7 (BD Biosciences). e slides were washed in PBS and incubated with a secondary biotinylated anti-rat IgG (BD Biosciences) for 45 min. Aer another wash in PBS was performed, avidin-peroxidase was added for 30 minutes (room temperature), and the slides were washed again in PBS. Subsequently, the slides were stained with a diaminobenzidine substrate solution for 3 minutes and counterstained in Mayer's hematoxylin solution (Merck, Germany).

2.�. �uanti�cation of �pleen �ell �u�populations.
e �elds for quanti�cation of splenic cells were imaged using a camera (Cyber shot DSC-S85) coupled to a microscope and a computer for digitisation. e cell populations were analysed using the Image J soware program (NIH-EUA). e positively stained cells were quanti�ed, in the 30 crossings of the grating, in each of 30 analysed �elds.
e accumulated median was calculated [17], and the results were presented as the median and medium deviation. e index of the stained cells (CIs) was calculated as the ratio of the mean number of positively stained cells in the infected animals versus the mean number of positively stained cells in the uninfected control animals in the different groups.

Statistical
Analysis. e statistical analysis was performed with the GraphPad Prism 4.0 soware program.
All of the variables were tested for normal distribution and homogeneous variance. e means and standard deviations were used for the analysis of one variable followed by the Mann-Whitney test, and the ANOVAs were followed by Tukey's test for multiple comparisons. e differences were considered signi�cant when .

Results
e spleen immunohistochemical analysis revealed a predominance of F4/80+ cells in the red pulp and a predominance of CD4+, CD8+, and CD19+ cells in the white pulp ( Figure 1). e BALB/c animals did not present a signi�cant variation in the number of F4/80+ cells during the course of infection (Figure 2(a)). On the other hand, the infected C57BL/6 animals displayed reduced numbers of F4/80+ spleen cells at 100 DPI ( ) and increased numbers of these cells at 250 DPI ( ) compared to the controls (Figure 2(b)). However, we detected an increased CI of F4/80+ spleen cells in the BALB/c mice at 100 DPI (CI = 1.2) and in the C57BL/6 animals at 250 DPI (CI = 1.4) (Figures  2(a) and 2(b)). e CI of the stained cells was consistent with the results that were obtained from the median but also revealed some important differences among some statistically insigni�cant differences among samples.
e number of CD4+ spleen cells increased in the infected BALB/c mice compared to the control mice at 100 DPI ( ) (Figure 3(a)); in the C57BL/6 mice, increased numbers of CD4+ cells were observed at 150 DPI ( ) (Figure 3(b)). We detected an increased CI of the CD4+ spleen cells in the BALB/c mice at 100 (CI = 1.2) and 250 (CI = 1.3) DPI and in the C57BL/6 animals at 150 DPI (CI = 1.4) (Figures 3(a) and 3(b)). Again, the results obtained from the CI of the stained cells were consistent with those obtained by the median but also showed some important differences among samples that were not statistically signi�cant.
e number of CD8+ cells in the spleens of infected BALB/c mice was signi�cantly increased only at 100 DPI when compared with the controls ( ) (Figure 4(a)). In the infected C57BL/6 mice, the number of CD8+ cells increased in comparison with the controls at 150 DPI ( ; Figure 4(b)). e CI of the CD8+ cells in the spleen increased at 35 (CI = 1.1) and 100 DPI (CI = 1.3) in the BALB/c mice and at 35 (CI = 1.2), 150 (CI = 1.5), and 250 (CI = 1.1) DPI in the C57BL/6 mice (Figures 4(a) and 4(b)). Again, the CI of the stained cells are consistent with the results that were obtained by the median but also reveals some important differences among samples that are not statistically signi�cant.
e number of CD19+ cells in the spleens was increased at 100, 150, and 250 DPI ( ) in the infected BALB/c mice in comparison with the controls (Figure 5(a)); in the C57BL/6 mice, the number of CD19+ cells increased signi�cantly ( ) at 250 DPI ( Figure 5(b)). e spleen CI of the CD19+ cells increased at 100 (CI = 1.2), 150 (CI = 1.3), and 250 DPI (CI = 1.6) in the BALB/c mice and at 150 (CI = 1.1) and 250 DPI (CI = 1.1) in the C57BL/6 mice (Figures 5(a) and 5(b)). e CI of the stained cells is consistent with the results obtained by the median and shows some important differences among samples that are not statistically signi�cant.

Discussion
e number of F4/80+ cells in the spleens of infected mice increased signi�cantly at 250 DPI in the C57BL/6 mice. e index of the F4/80+ cells in the spleens of the infected mice increased at 100 DPI in the BALB/c mice and at 250 DPI in the C57BL/6 mice. e macrophages of the red pulp were F4/80+, and their role is to remove antigens and red blood cells. Certain subsets of mature macrophages residing in the T-cells areas of the spleen express low levels of F4/80. We should consider that resident macrophages with different phenotypic and functional characteristics, and their precursors in the blood, the monocytes that in�ltrate the tissues, can be answering to the helminth in the infection site and the expression of F4/80 can vary during the activation and macrophage maturation [18,19]. During experimental infection with L. minor, F4/80+ cells can play an important role in the orchestration of the immune response by producing proin�ammatory cytokines with neutrophilactivating potential, regulatory cytokines that modulate the in�ammation, and cytokines that aid in the differentiation of CD4+ lymphocyte subsets.
In mice spleens, a macrophage population characterised by an F4/80(high) Mac-1(low) phenotype produces suppressor cytokines, such as TGF-and IL-10 [20]. Some studies have demonstrated that alternatively activated macrophages (AAMs) are associated with resistance to parasitic infections and the production of cytokines and angiogenic growth factors during helminth infections [21]. Classically activated macrophages (CAMs) that produce nitric oxide (NO), which is cytotoxic to metazoan larvae [22], also seem to be important in helminthosis resistance [23].
e BALB/c mice exhibited an increased CI of the CD4+ spleen cells at 100 and 250 DPI, and such an increase was observed in the C57BL/6 mice only at 150 DPI. It has been suggested that these CD4+ cells are resident in the spleen or are lymphocytes that have migrated from the blood to the marginal zone (MZ) of the spleen [24]. e modulation of different CD4+ lymphocyte subsets has been observed in many experimental models of helminthosis [25]. e fact that higher levels of IFN-are found in the serum of BALB/c mice than in that of C57BL/6 mice suggests the participation of cells with a 1 pro�le in resistance to infection [12]. Several studies have described the participation of 17 cells in the aggravation of the pathology caused by schistosomiasis [26][27][28]; in fact, the presence of neutrophils in the granulomatous lesions that was observed in the C57BL/6 mice suggests the participation of these 17 cells in this strain [12]. Our results demonstrated an increase in the CI of the CD8+ spleen cells in the C57BL/6 mice at 35, 150, and 250 DPI. e susceptibility of mice chronically infected with Trichuris muris is associated with high numbers of CD8+ T cells [29]. e CD8 molecule can be expressed in the CD8 con�guration in T lymphocytes [30], and these cells have been described in other cases of helminthosis [31]. It is necessary to further evaluate the participation of CD8+ noncytolytic lymphocytes that produce in�ammatory and regulatory cytokines during the course of L. minor infection. e preferential increase of these cells in C57BL/6 mice is apparently associated with the susceptibility to experimental infection.
e BALB/c mice displayed increased indexes of CD19+ spleen cells at 100, 150, and 250 DPI. e CD19+ molecule is present at the surface of practically all B lymphocytes; it operates as a coreceptor to amplify the BCR signal cascade [32]. B lymphocytes possess a variety of immune functions that contribute to protective immunity during helminth infections, including the production of antibodies and cytokines, and the presentation of antigens [33]. During experimental infection with Heligmosomoides polygyrus, antibodies block the intestinal invasion in the earliest days of the infection [34]. BALB/c mice have been demonstrated to produce IgM, IgG, IgA, and IgE against the parasite's crude extract (CE) and secreted-excreted (SE) antigens, with a higher production of IgA, IgM, and IgG in response to the CE antigens than to the SE products of L. minor [11]. ese results suggest that the large number of CD19+ cells in the spleens of BALB/c mice is associated with the increased production of antibodies that participate in the resistance to L. minor infection.
At 100 DPI, an increased CI of the F4/80+, CD4+, CD8+, and CD19+ spleen cells was demonstrated in the BALB/c mice. At this time point, we observed less intense lung lesions and fewer subcutaneous encysted L3 larvae in the BALB/c strain compared to the infected C57BL/6 strain.   e information gained from the immunohistochemistry regarding the mean, standard deviation, and the CI of the stained cells demonstrated an association between the preferential increase in the number of CD4+ and CD19+ spleen cells with resistance to experimental lagochilascariosis in the BALB/c mice and between the preferential increase of CD8+ spleen cells and susceptibility in the C57BL/6 mice.