Immunoendocrine abnormalities in the female reproductive system, and lung steroidogenesis during experimental pulmonary tuberculosis

Abstract

Introduction

Tuberculosis (TB) caused by Mycobacterium tuberculosis mainly affects the lungs, but can spread to other organs. TB chronically activates the immune and endocrine systems producing remarkable functional changes.So far, it is unknown whether pulmonary non-disseminated TB cause changes in the female reproductive system and lung endocrinology.

Objective

To investigate whether pulmonary TB produces immunoendocrine alterations of the female mice reproductive organs, and lung estradiol synthesis.

Methods

BALB/c mice were infected intratracheally with Mycobacterium tuberculosis (Mtb) strain H37Rv. Groups of six non-infected and infected animals were euthanized on different days. Bacillary loads were determined in the lungs, ovaries and uterus. Immunohistochemistry and morphometry studies were performed in histological sections. Serum estradiol wasassayed, and supernatantfrom cultured lung cells was analyzed by Thin Layer Chromatography (TLC).

Results

Mtb only grew in lung tissue. Histopathology revealed abnormal folliculogenesis and decreased corpora lutea. Altered ovarian expression of IL-6, IL-1β was found. The infection increased serum estradiol. Estradiol synthesis by infected lung cells triplicate after 30 pi days.Aromatase immunostaining was found in the alveolar and bronchial epithelium, being stronger in the infected lungs, mainly in macrophages.

Conclusion

Pulmonary TB affects the histophysiology of the female reproductive system in absence of its local infection, and disturbslung endocrinology.

Introduction

Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis (MTB) that represents one of the main problems for public health [1]. TB is the leading cause of death in the world by a single bacterial agent. In 2019, 10 million people became ill with TB and 1.4 million died from this disease [1].

Especially in developing countries, TB is twice more common in men than in women (male/female ratio of 1.9for the world case notification) [1]. This difference has been attributed to biological, genetic and hormonal mechanisms involved in sex-specific immune responses, epidemiological, cultural and socioeconomic factors [[2], [3], [4], [5]]that also influence TB biomarkers [6]. Fromthe biological perspective, it is important to consider the regulatory activities that the steroid hormones have on the immune cellsand the role of cytokines in the neuro-immune-endocrine interactions [7].These hormones participate in macrophages and lymphocytes development and function, as well as in the outcome of infectious diseases Females have higher antibodyconcentrations in sera and produce stronger immune responses after immunization than malesBothamley, Bouman 2004, Taneja 2018, Bini[6,[8], [9], [10]]. Moreover, females usually show lower rates of infection and mortality than men, which has been associated withimportant differences in the inflammatory response, particularly the activity of estrogens to drive pro-inflammatory Th-1 immune responses and that of testosterone to inhibit it, which at least in murine models partially explain the better control of MTB growth by females [11].Although TB is more frequent in men, this infectious disease is the third leading cause of death in women between 15 and 44 years old, therefore approximately 500,000 women per year die, most of them ofreproductive age [1].

Pulmonary TB is an infectious disease in which the growth control of the bacteria required not only the activity of the immune system, but also the endocrine system[Kleynhans L 2017 [12]] Although bi-directional interactions between the immune and neuroendocrine systemsin TB are well known, the particular immunoendocrine abnormalities that occur in the course of the disease have been only described in men [13]and in male mice [14,15].

In the case of women, TB produces alterations related to the functionality of the hypothalamus-pituitary axis that results in alterations inthe menstrual cycle, intermenstrual bleeding, polycystic ovary, as well as problems in pregnancy, with the consequent increase in stress [16].

Women with pulmonary TB presented a medical history of alterations in the menstrual cycle, amenorrhea and hypomenorrhea [17,18]. So far, no studies have been found that investigate the causes of these alterations or attempt to explain the mechanism by which they occur.

Inthis regard, the success or failure of the immune response that is mounted against the infectious challenge is in part related to sex hormones. There are other infectious diseases where the involvement of the endocrine system together with the immune system isessential to control them and that generates reproductive and/or hypothalamic-pituitary-adrenal axis (EHHS) alterations. This is the case in women and men with neurocisticercosis [19,20] or malaria [21]] and in toxoplasmosis and trypanosomiasis where alterations in reproduction-related hormones have been found [22].

It is well established that in females, 17β-estradiol modulates the structure and function of reproductive tissues, such as the uterus and oviduct [20]. Ciliated cells of the oviduct are extensions of the cylindrical epithelial cells that transport both gametes and embryos. Fluctuations in circulating estradiol are responsible for the characteristic female cycling pattern through their activity on the hypothalamus-pituitary-ovaryaxis. Numerous reports have revealed that severe reproductive defects occur when their synthesis is blocked. Estrogens are synthesized by the enzyme P450-aromatase encoded by the Cyp19a1 gene, from androgen precursors such as Androstenedione (A₄) and testosterone. This enzyme is expressed in the ovary by granulosa cells and found in a number of extragonadal sites, including bone, breast, adipose tissue and brain [23,24]. The androgenic substrate for aromatase is produced by theca cells of the ovarian follicles, and is also directly involved in the regulation of P450-aromatase, the ovarian estrogens are fundamental for the female reproductive cycle [25,26].

The ovarian follicle is the functional unit of the ovary; basically, it consists of oocytes surrounded by two types of somatic cells, the granulose and theca cells. The process of follicular development involves maturation steps called folliculogenesis, which goes through a primordial follicle, transformed into a primary follicle, an antral follicle, and finally into a mature dominant follicle that will release an oocyte at ovulation. This process is regulated in part by the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) that stimulate the synthesis of estradiol and progesterone Some follicles do not reach maturation to antral follicles and go to atresia. Ovarian follicular atresia is a hormonally controlled apoptotic process and occurs at all stages of follicle development. The estrogen/androgen ratio seems to play a role in atresia [25], besides sex steroids, several intraovarian factors can also induce atresia [[27], [28], [29]].

It is well known that inflammation is involved in beneficial and harmful effects. On the one hand, it is part of many physiological processes in reproductive physiology, including ovulation, menstruation, and implantation. On the other hand, uncontrolled inflammation can negatively affect fertility as a result of an alteration of hormone production and ovulation [30,31].

Studies have shown that cytokines such as IL-1 and IL-6 can alter the production of steroid hormones by the ovary. Several reports showed that these cytokines increase the synthesis of estrogens acting for instance on aromatase, the enzyme that catalyzes the transformation of androgens to estrogens [32,33].

In infections such as TB, crosstalk between sex hormones and the activity of the immune system occur that can affect the immune response and gonadal functions, but as far as we know there are no studies about the functional and anatomical changes of the non-infected mice female reproductive system during the course of pulmonary TB.

In the present study, we demonstrate that in the absence of local infection or growth of bacteria in the mice female reproductive system, it undergoes significant abnormalities in folliculogenesis, as well as alterations in IL-1β and IL-6 expression in the ovaries. Interestingly, we also show that the infection modifies the lung endocrine capacity, in terms of female sexual hormones production.