Modeling the inflammatory process in the reproductive organs of female mice of the C57BL/6 and I/ST inbred strains with aerosolized Mycobacterium tuberculosis

Currently, both pulmonary and extrapulmonary tuberculosis in humans has been widely studied and described in detail [1–3]. Mice are one of the most commonly used experimental animal models of pulmonary tuberculosis [4–11]. Until recently, animal models of genital tuberculosis were developed using the intravaginal, intrauterine, alimentary, and intraperitoneal routes of M. tuberculosis administration [12, 13]. These techniques are not identical to the naturally occurring involvement of the genitals in the tuberculosis process in humans. Current literature is lacking experimental studies investigating changes in the reproductive organs during pulmonary tuberculosis, including the use of female mice [14].

The most notable feature of laboratory mice for studying reproductive problems is a complete identity of female reproductive systems of humans and mice. Female mice have genitals, including ovaries, fallopian tubes, uterus, and vagina. The histological structure of the mouse genital organs is also similar to that of humans [15]. The effect of tuberculosis infection on the reproductive system represents significant scientific interest, and its investigation will contribute to elucidating the pathogenesis of reproductive disorders in active pulmonary tuberculosis.

This study aimed to develop a model of the inflammatory process induced by aerosolized M. tuberculosis in the reproductive organs of female mice with different genetically determined susceptibilities to tuberculosis infection.

Material and methods

Animal experiments were conducted on female mice of I/St and C57BL/6 inbred strains that were kept in the laboratory animal facility at the Central Research Institute for Tuberculosis. Mice were bred under the guidelines of the Ministry of Health of the Russian Federation No. 755, INH Office of Laboratory Animal Welfare (OLAW). All experimental procedures were approved by the Ethics Committee of the Central Research Institute for Tuberculosis (IACUC protocols No. 4, 9, 14). Female mice weighing 20-22 g that did not have any contact with males were infected in a Glas-Col aerosol exposure chamber (USA) at a dose of 100 CFU/lung. The virulent strain H37Rv of Mycobacterium tuberculosis was kindly provided by Gilles Marchal (Pasteur Institute, Paris) and kept in the immunology department of the Central Research Institute of Cytology and Genetics. The total number of mice (n = 27) was divided into group 1 comprised of intact mice (n = 3); group 2 including mice of the C57BL/6 strain (n = 12), which were sequentially removed from the experiment after 3 (n = 3), 7 (n = 3), 15 (n = 3), and 35 (n = 3) days after the moment of infection; group 3 including mice of I/St strain (n = 12), which were sequentially removed from the experiment through 3 (n = 3), 7 (n = 3), 15 (n = 3), and 35 (n = 3) days after the moment of infection.

The vaginal flora was analyzed using a real-time polymerase chain reaction-based Femoflor test [14, 16]. For this, before infection (day 0), as well as on day 3, 7, 15, and 35 after infection, vaginal smears were taken from each mouse and placed in guanidine thiocyanate-phenol-chloroform (Trizol). For histological examination, one uterine horn was placed in 10% formalin. Sections were prepared from fixed samples and stained with hematoxylin-eosin. Morphological examinations of the reproductive organs were synchronized with the phase of the estrous cycle. The most objective data were obtained in metestrus and diestrus mice. During histological examination, the degree of endometritis severity was determined according to the method by Ellinidi V.N. et al. [17] taking into account the following morphological characters: the presence of inflammatory infiltrates consisting of lymphocytes, macrophages, and eosinophils located around the glands and blood vessels; the presence of plasma cells in the infiltrates; the presence of focal fibrosis of the endometrial stroma; the presence of sclerotic changes in the spiral artery walls of the of the endometrium; the formation of capillary and sinusoid types of blood vessels.

Each morphological trait scored 1 point. The endometrial injury was evaluated in 10 fields of view. The grade of endometritis was categorized according total severity score as mild (1-4), moderate (5-7), and severe (8-10). For microbiological and molecular genetic studies for the presence of Mt band Mtb DNA, another uterine horn was placed in phosphate buffer and homogenized; 100 ml of homogenate was added to 1 ml of trizole.

Statistical analysis was performed using the Microsoft Excel and the IBM SPSS Statistics v.21 (2012) statistical package. The median (Me) and quartiles (Q3; Q1) were reported as measures of central tendency and variability. The results are presented as Me (Q3; Q1). The statistical significance of between-group differences for unpaired continuous variables was tested with the Mann–Whitney test. Differences between the groups were considered statistically significant at p<0.05.

Results and discussion

Vaginal flora of animals in group 1 (control group), was characterized by a total bacterial mass (TBM) of 103-104 genome-equivalent/ml, the presence of scanty lactobacilli, and aerobic and anaerobic biotopes. Microbiological and molecular genetic studies of vaginal smears, lungs, uterine horns revealed the absence of Mtb and Mtb DNA. A morphological analysis of sections of the lungs and uterine horns revealed a normal histological structure of these organs.

Vaginal flora of animals in group 2 (C57BL/6) showed a significant increase in TBM already on day 3 after Mtb infection from 2.8 (2.5; 3.2) to 5.2 (4.6; 6,0) (p = 0.043), slightly decreasing by day 35 after infection to TMB of 3.8 (4.6; 3.5). Among mice of group 3 (I/St), TMB remained unchanged during the entire observation period from baseline 3.8 (3.0; 4.9) to 3.2 (2 , 8; 3.6) by day 35 after Mtb infection. Vaginal microbiocenosis in female mice group 2 (C57BL/6) on day 3 after Mtb infection was characterized by a significant increase in Enterobacteriaceae spp. from 2.2 (1.0; 2.5) to 4.2 (3.3; 4.9) (p = 0.017), followed by a decrease to 2.3 (2.2; 3.2) by day 35 after Mtb infection. On day 35 after Mtb infection, there was a growth of Eubacterium spp from 1.2 (0.0; 2.6) to 2.3 (0.0; 2.6); Megasphaera spp. from 0.0 (0.0; 0.0) to 1.2 (0; 1.4); Gardnerella vaginalis from 1.4 (0.0; 2.8) to 3.2 (0; 3.5), without a statistically significant difference. On the contrary, in group 3 (I/St), a decrease in Enterobacteriaceae spp was observed by day35 after infection compared with baseline from 3.4 (2.9; 3.8) to 1.6 (0.0; 2.4) and Candida spp. from 3.0 (3.0; 3.1) to 2.6 (0.0; 3.0); slight increase in mass of Lachnobacterium spp. / Clostridium spp. from 1.2 (0.3; 2.3) to 1.7 (0.0; 2.6), without a statistically significant difference. In mice of both strains, regardless of the timing of infection, we did not detect Lactobacillus spp., Peptostreptococcus spp., Sneathia spp. / Leptotrihia spp. / Fusobacterium spp., Atopobium vaginae spp., and Ureaplasma urealyticum.

Therefore, vaginal microbiota of both strains of female mice underwent different changes showing a statistically significant imbalance with a tendency to moderate dysbiosis in the early stages of Mtb infection in C57BL/6 mice and the absence of statistically significant changes in I/St mice, regardless of the timing of Mtb infection (Fig. 1-2).

A morphological study of the reproductive organs of mice from groups 2 and 3 (C57BL/6, I/St) was performed on day three after infection with aerosolized Mtb. In both groups, endometrium had normal structure with the mucosa covered by a high-prismatic epithelium and the presence of proliferative-type glands. Epithelial cells were located at the base of the cell, with intense staining and few mitoses. The cytoplasm was basophilic. The apical margin of epithelial cells was smooth and regular. The stroma consisted of spindle-shaped cells, interstitial edema, congestion of the blood vessels and moderately expressed perivascular and periglandular lymphocytic-plasmacytic inflammatory infiltration.

On day7 after infection with aerosolized Mtb, mice group 3 (I/St strain) had a pattern of chronic nonspecific endometritis with pronounced periglandular lymphocytic and eosinophilic infiltrates with an admixture of plasmocytes (Fig. 3, a) consistent with chronic endometritis of mild severity (1 + 2). There was a focal inflammatory process; in most specimens, inflammatory changes were minimal. No similar histological changes were observed in the reproductive organs of mice in group 2 (C57BL/6 strain) (Fig. 3b).

On day 15 after infection with aerosolized Mtb, mice in groups 2 and 3 (C57BL/6 and I/St) had periglandular, perivascular and focal exudate, and the interstitial edema showed an increase in eosinophils located mainly around the vessels in places contacting with pericytes. Interlinear changes were of mild intensity.

On day 35 after infection with aerosolized Mtb, in mice from group 3 (I/St strain), endometrial inflammatory reaction was more pronounced and widely spread. The endometrial mucosa was covered with high-prismatic epithelium. Subnuclear vacuoles were found in the epithelium of some glands. The stroma was thick, loosened, and consisted of spindle-shaped cells; cell nuclei were large, round; there were focuses of fibrosis and perivascular, periglandular lymphoplasmocytic infiltration. The vessels were somewhat convoluted, located on the surface of the endometrium, periglandularly and perivascularly, with focal fibrosis. Focal accumulation of lymphocytes in the stroma was noted in some of the places, according to the type of formation of lymphoid follicles (Fig. 3, c). In this group, morphological changes in the endometrium were consistent with severe chronic endometritis (4 + 3) with a widely spread inflammatory process in the endometrium. In mice of group 2 (C57BL/6 strain), the histological signs of inflammation in the reproductive organs were less pronounced (Fig. 3, d); there were no sclerotic changes in the walls of the spiral endometrial arteries (3 + 3), which is consistent with chronic endometritis of moderate severity. In the lungs of mice from groups 2 and 3, histological signs of tuberculosis were detected on day 35 after infection with aerosolized Mtb (Fig. 3, e, f).

No Mtb and Mtb DNA were identified by microbiological and molecular genetic analysis in the reproductive organs of mice in groups 2 and 3 (C57BL/6 and I/St). Microbiological examination of the lung tissue of mice in groups 2 and 3 showed a high bacterial load on day 35 after infection with aerosolized Mtb - 5.12 (4.98; 5.32) for C57BL/6 and 6.39 (6.21; 6.47) for I/St.

Therefore, we identified histological signs of the inflammatory process in the reproductive organs of female mice in group 3 (I/St strain) against the background of conditional normocenosis on day 35 after infection with aerosolized Mtb. In female mice from group 2 (C57BL/6 strain), the inflammatory process in the reproductive organs was less pronounced during the same period against the background of an imbalance of the vaginal flora with a tendency to the formation of moderate dysbiosis. Inflammatory changes in the reproductive organs of mice in groups 2 and 3 increased along with the progression of tuberculosis infection in the lungs.

Conclusion

Tuberculosis is a systemic infection characterized by the development of para-specific reactions in various body systems, including reproductive organs. Infection of laboratory animals with aerosolized Mtb brings the experimental model of pulmonary tuberculosis development closer to the real pathogenetic mechanism in humans. Of great importance is individual susceptibility to tuberculosis infection, which has a genetic predisposition. The use of two inbred strains of mice exhibiting opposite susceptibility to tuberculosis (resistant and sensitive) provides insights into individual differences in the speed and intensity of the inflammatory process in the reproductive organs. At the same time, an important factor is the sequence of processes in the relationship of macro-and microorganisms: a progressive increase in the bacterial load and the formation of tuberculous lesions in the lungs; changes in the vaginal microbiota from conditional normocenosis to moderate dysbiosis; sequential development of the inflammatory process in the reproductive organs (uterus and uterine horns). It should be noted that, according to the results of the study of the vaginal microbiota, it is still difficult to conclude, but the C57BL/6 and I/St mice showed inter-strain differences in this trait in the early stages of Mtb infection. Multiple microbiological, molecular-genetic and morphological studies of the reproductive organs of female mice in our earlier experimental models of tuberculosis convincingly demonstrate the consistent development of the inflammatory process, regardless of the method of infection with Mtb and the choice of a mouse strain.