Immunomodulatory Effect of Lactoferrin on Mucosal Immunity of Uterus in Pregnant Rat

Immunity stabilization in early pregnancy is one of the most reasons for the establishment of gestation. Lactoferrin is an iron-binding glycoprotein which proved to has a positive effect on immunity including broad-spectrum antimicrobial activity, regulation of cell growth and differentiation, and intonation of inflammatory as well as humoral and cellular immune responses. So, this study aimed to examine some immune parameters, cells and cytokines during early Pregnancy. Twenty-four mature female and six male rats for mating were subjected to this investigation. They were grouped into two groups, the control and lactoferrin group treated daily with Lactoferrin with a dose of 50µgm/kg body weight orally starting from one week before and persisted for one week after mating. Blood samples were collected from each female rat at 5hrs, 1day,3days, and 7 days after mating for hematological and biochemical assay Extracted RNA was subjected to real-time PCR to determine mRNA levels for immune- related genes interleukin1A(IL1A) and interleukin 10(IL10). Histopathological examination was done on the uterus. Results showed a significant increase in white blood cell count, mainly neutrophil, eosinophil, and basophil, mainly at 5hrs and 7-days of the lactoferrin group. Similarly, C- reactive protein and progesterone in the lactoferrin group at 3 and 7 days were significantly increased. Also, significant increases in the levels of interleukin 1 A at all experimental periods were noticed, while interleukin 10 was increased at 1day and 3 days after mating in the Lactoferrin treated group. TNF increased significantly at 5hrs, 1days, and 7 days. In contrast, estrogen decreased significantly at 5hrs and 1 day while Total Antioxidant Capacity (TAC) decreased at 5hrs and 3 days and 7 days of Lactoferrin treated group. Moreover, histopathological examination revealed high leukocytic infiltration, especially neutrophil and eosinophil, at 5hrs hours after mating in the Lactoferrin treated group. This research demonstrated that using Lactoferrin during Pregnancy has a good impact on some immune cytokines and parameters that may improve immune status during early pregnancy.


Introduction
Disturbances in early Pregnancy and endometrial receptivity are the major causes of subfertility and also impact placental improvement and fetal growth [1], which has implications for the phenotype of the offspring and capacity to withstand health challenges in later life [2]. Key regulators of the conception and implantation events are maternal tract cytokines and immune cells within the female reproductive tract, while the receptivity of the endometrium is dependent on immune cells present at the implantation site [3]. In mice and rats, implantation was between days 4 and 5 of Pregnancy, considering the first day of Pregnancy as the day on which a vaginal plug or spermatozoa are present in the vagina [4]. Lactoferrin (LF) was a very important part of the human body's natural defense system [5]. Lactoferrin is a cellsecreted mediator that links innate and adaptive immune function in mammals. It is a pleiotropic molecule that directly supports the influence of presenting cells for the development of T-helper cell polarization [6].
In vitro, Lactoferrin stimulates the growth of lymphocytes [7], natural killer activity [8], and the release of interleukin-8 (IL-8) from neutrophils. Further, Lactoferrin stimulates the release of IL 1, IL 2, and tumor necrosis factor (TNF) from leukocytes or complement activation [9]. LF and its derivatives have pleiotropic functions, including broad-spectrum antimicrobial activity, regulation of cell growth and differentiation, and intonation of inflammatory as well as humoral and cellular immune responses [10]. During Pregnancy, the peripheral-specific immune response is shifted away from a type 1 cellular immune response towards a type 2 humoral immune response [11]. During rat pregnancy, both monocyte and granulocytes increased in number in circulation [12].
There is an absolute monocities during Pregnancy, especially in the first trimester, but decreases as gestation advances. Monocytes help in preventing fetal allograft rejection by infiltrating the decidual tissue from seven to twenty weeks of gestation, possibly through prostaglandin E2 mediated immunosuppression [13].
In fact, the uteroplacental tissue produces an array of antiinflammatory cytokines as interleukin 10 that are expressed in the uterus, oviducts, and ovaries of cycling mice and during Pregnancy in the uterine myometrium and cervix [14][15][16][17][18][19][20]. Thus, the balance between pro-inflammatory cytokines as tumor necrosis factor (TNF) and interleukin 1a and anti-inflammatory cytokines is essential to a successful pregnancy outcome [21]. IL1A is one of the major cytokines that participate in the local regulation of many reproductive affairs. IL1A also plays a role in regulating ovulation [22]. TNF-α has been shown to influence hormone synthesis, placental architecture, embryonic and follicle development, steroidogenesis, uterine cyclicity, placental differentiation, and parturition [23,24]. [25][26][27][28][29][30] hypothesized that Pregnancy is a stressful condition as it leads to increased levels of oxidants and therefore reduces Total Antioxidant Capacity (TAC). C-Reactive

Protein (CRP) is a sensitive marker of systemic inflammation and
is primarily synthesized in hepatocytes in response to infection and tissue injury. Production of CRP is stimulated by the release of proinflammatory cytokines, including interleukin-1, interleukin-6, and tumor necrosis factor-alpha. Although sometimes referred to as an acute-phase reactant, CRP accompanies both acute and chronic inflammatory disorders [31]. [32] showed that progesterone reduced macrophage migration into the murine uterus, while [33][34][35][36] showed that estrogen, progesterone, and human chronic gonadotrophin did not affect migration of macrophage cells in guinea pigs. [37][38][39][40][41][42][43][44][45] showed that progesterone(p4) withdrawal is associated with the initiation of labor. The effect of P4 on immunity has been revealed, mainly at pregnancy concentrations. These effects are primarily mediated via the intracellular P4 receptors (PR), PR-A, and PR-B, which act as transcription factors [46]. Upon Pregnancy, estrogen is shown to promote uterine blood flow, myometrial growth, stimulate breast growth, and at term, promote cervical softening and expression of myometrial receptors. Also, estrogen was suggested to affect different immune cell populations in their number and function and thereby contribute to fetal tolerance [47]. Leukocytic infiltration during early Pregnancy indicated that decidual natural killer cells makeup around 30% of the uterine stromal compartment, mainly at the implantation site where they are found scattered throughout the stroma and thickly clustered around glands and spiral arteries in the uterus [48]. So, the present study aimed to evaluate the immunological effect of Lactoferrin on some cytokines, immune cells, and mucosal immunity of uterine tissue in the rat during pregnancy.

Materials and Methods
All procedures of laboratory work carried out under guidelines for biosecurity and infection control according to Guidelines for

Experimental Animals
The study was conducted on twenty-four mature female Sprague Dawley rats and six mature male rats weighing 250 g. B.
wt, divided into two groups, twelve female rats in each group. All animals were purchased from a laboratory animal house in, Helwan -Egypt. They were housed in separate cages in the physiology department, Faculty of veterinary medicine, Mansoura University.
They were kept in a controlled environment for two weeks before the experiment for accommodation; maintained under normal condition, and rats were fed a basal control diet; water and diet were given ad-libitum throughout the experimental period. All females were examined for the detection of the stage of the estrus cycle by taking vaginal smear and examine it under the microscope to determine which female in the estrous phase to determine the best time of insemination according to the methods of [49].

Diet and Additives
The basal diet was prepared by the Faculty of Agriculture, Mansoura University, according to standard levels of NRC. The diet was produced in the form of pellets and was admitted to all groups of the study.

Lactoferrin
Bovine Lactoferrin (BLF), an immune-modulatory substance, was purchased from Sigma company; the commercial name is L9507 in the form of a powder that was dissolved by distilled water and was prepared to be given to twelve mature female rats (lactoferrin group) in a dose of 50microgram/kg orally by stomach tube every day before mating by one week and persisted after mating till one week according to (Yamauchi et al., 2000). Oligonucleotide primers used in SYBR Green real-time PCR: They are shown in Table 1.  according to [52].

2-progesterone:
Progesterone was diagnostic by using IMMULITE and IMMULITE 1000 Analyzer for the quantitative measurement of progesterone in serum, as an aid in the differential diagnosis of amenorrhea, and monitoring of ovulation induction with and without stimulating in Assisted Reproductive Technology (ART) according to [53].

3-TAC (Total Antioxidant Capacity):
Total antioxidant capacity is measured by the colorimetric determination method by ready-made diagnostic kits provided by Bio-diagnostic, according to [54].

5-CRP (C-Reactive Protein):
The rapid latex agglutination test is for the qualitative screening and quantitative determination of C-Reactive Protein (CRP) in serum presented by ATLAS MEDICAL LATEX Kit, according to [56].

Tissue Sampling
Both uterine horns were excised from all pregnant rats and one sample from each horn was taken and fixed immediately in 10% neutral buffered formalin solution. All specimens were routinely processed in ascending grades of ethyl alcohol (80%, 90%, 100%),

Histopathological Examination and Statistical Analysis
The data of histopathological examination of morphometric measurements of epithelial height and diameter of uterine glands were subjected to student T-test to differentiate between control and lactoferrin treated group to compare the difference between means. Data were expressed as means ± standard errors. The difference between means was considered significant when (P <

Effect of Lactoferrin on Total Leukocytic Count (WBCS×10 3 /µl)
As shown in Table 2 results indicated an increase in Wbcs at 5hrs, 1day, and 3 days after mating in the lactoferrin group compared with the control group at the same time.

Effect of Lactoferrin on Neutrophil%
As shown in Table 3, results indicated an increase in neutrophil percent in the lactoferrin group at 5hrs, 3 days, and 7 days after mating compared with the control group.

Effect of Lactoferrin on Lymphocyte%
As shown in Table 4, results demonstrated a decrease in lymphocyte percent in the lactoferrin group at 5hrs, 1day, 3 days, and7 days after mating.

Effect of Lactoferrin on Eosinophil%
As shown in Table 5, results indicated that the eosinophil percent increase in Lactoferrin at 5hrs, 1day, and 7 days.

Effect of Lactoferrin on Basophil%
As shown in Table 6, results indicated that basophil decrease in the lactoferrin group at 5hrs, 1 day, and 7 days.

Effect of Lactoferrin on Monocyte%
As shown in Table 7, results indicated that the monocyte percent decrease in the lactoferrin group at 5hrs, 1day, 3days, and 7 days.

Effect of Lactoferrin on Estrogen(ng/ml)
As shown in Table 8, results indicated that estrogen decrease in the lactoferrin group at 5hrs and 1 day when compared to the control group.

Effect of Lactoferrin on Progesterone (ng/ml)
As shown in Table 9, results indicated an increase in progesterone hormone in the lactoferrin group at 5hrs, 1 day, 3 days, and 7 days after mating when compared to the control group. Effect of Lactoferrin on Total Antioxidant Capacity (TAC) (µ mol/L): As shown in Table 10, results indicated a decrease in the lactoferrin group at 5hrs, 1day, 3days, and 7 days when compared with the control group.

Effect of Lactoferrin on Tumor Necrosis Factor (TNF) (pg/ml)
As shown in Table 11, results indicated an increase in TNF at 5hrs, 1 day, 3 days, and 7 days in the lactoferrin group when compared to the control group.

Effect of Lactoferrin on C-Reactive Protein (mg/L)
As shown in Table 12, results indicated an increase in C reactive protein in the lactoferrin group at 5hrs, 1day, 3days, and 7 days when compared to the control group.

Effect of Lactoferrin on IL1a and IL10 by Real-Time PCR:
As illustrated from Figure 1, results indicated an increase in interleukin 1A in the lactoferrin group at 5hrs, 1 day, and 3 days after mating when compared to the control group. At the same time, results indicated an increase in interleukin 10 in the lactoferrin group at 5hrs and 1 day after mating but decreased at 3 days and 7 days when compared with the control group.

Rat Uterus After 5 Hours of Gestation
As illustrated from Figure

Rat Uterus at 3 Days of Gestation
As illustrated from Figure

Rat Uterus at 7 Days of Gestation
As illustrated from Figure (5hrs, 1day, 3days, and 7

days) of Pregnancy
The effects of lactoferrin administration to pregnant rats on eosinophil count in uterine tissue at a different time (5hrs, 1day, 3days, and 7 days) of Pregnancy is shown and summarized in  as well as identifying cell targets and receptors and this was important in the maintenance of immune system homeostasis [59].
Neutrophils increased at 5hrs, 3days, and 7 days after insemination, while basophil and eosinophil increased at 5hrs, 1 day, and 7 days after insemination in the lactoferrin group.
While, Lymphocyte and monocyte showed a significant decrease in the lactoferrin group at 5hrs, 1 day, 3 days, and 7 days after insemination. This result agreed with [60], who showed that Lactoferrin had a role in regulating innate and adaptive immune response, regulating the secretion of cytokines and regulated growth, differentiation, and activation of different leukocyte cells and so increased the number of circulated leukocytes. This agrees with [61], who showed that both monocytes and granulocytes increased and immune systems activated during Pregnancy in the rat. [62] reported that Lactoferrin regulated the immune system by stimulating endometrial and stromal cell proliferation and stimulating immune cells against antigen as well as [63] reported that Lactoferrin increased recruitment of neutrophil in mice circulation. Besides, [64] recorded that Lactoferrin increased the number of natural killer cells and modulated myelopoiesis with increased phagocytosis against a specific antigen. In the same respect, [65] showed that WBCs increased during rat pregnancy, and this was inconsistent with the significant increase in the number of white blood cells as presented in (Table 2).
Estrogen increased in the lactoferrin group, particularly at 7 days after insemination, and showed decreased at 5hrs and 1day after insemination. Estrogen was considered an immune modulator substance that could improve the immune response of the female reproductive tract in rodents, as shown by [66]. This agreed with (Hamid et al. 2012) showed that rats implantation period was initiated on day 5 and completed by day 7 of the pregnancy and during this implantation period, the dominant hormone modulator to morohological and functional changes were estrogen and progesterone and this may explain why estrogen increased at our study at 7 days taking into consideration the difference between species as difference between rat and human in time of endometrium changes and hormones secretion as shown [67] showed that estrogen played a pivotal role in early Pregnancy in mice as, during the first two days after insemination, the preovulatory estrogenstimulated proliferation of the luminal and glandular epithelial cells in addition to potentiated with progesterone hormone that secreted from corpus luteum and stimulated stromal cell proliferation on 4 days which was the day of the implantation process. Furthermore, [68] showed that there was a close relationship between estrogen and Lactoferrin as an estrogen-regulated expression of Lactoferrin in the endometrial epithelium of rat, particularly along all stages of the estrus cycle as well as lactoferrin levels changed with the change in estrogen level and so this could explain the increase in estrogen hormone in lactoferrin group after insemination.
Progesterone increased at 5hrs, 3 days, and 7 days in the lactoferrin group compared to the control group, as showed in Table   10. [69] reported that progesterone hormone inhibited estrogen hormone and decreased lactoferrin gene expression in uterine epithelial and stromal cells by controlling progesterone receptor and affecting estrogen receptors in vitro using uterine rat tissue.
Also, [70] reported that progesterone was secreted in hamsters at the preovulatory stage from an extra luteal tissue source for allowed characteristics appearance of estrus behavior, and this could explain the increase in progesterone hormone in the lactoferrin group.
Total Antioxidant Capacity(TAC) increased in the lactoferrin group at 1 day after insemination when compared to the control group as showed in Table 10 and this agrees with [71] who showed that Lactoferrin (LF) was believed to be safer than the drugs currently used for hypertension treatment due to its antioxidant effect. It has been reported that two weeks of lactoferrin supplementation has been able to increase the hydrophilic antioxidant capacity in healthy humans [72]. Besides, [73] suggested that Lactoferrin contributed to oxidoreductive reactions at the cell membrane, and LF had an antioxidant effect on red blood cells through inhibition of lipid peroxidation and hemolysis. In the same respect, LF was an important specialized iron scavenger, and its antioxidant activity was most likely related to its ability to bind ferrous and ferric ions.
Thus, LF may inhibit the iron-catalyzed formation of hydroxyl radicals.
Tumor necrosis factor (TNF) decreased at 3 days and then increased at 5hrs, 1 day, and 7 days in the lactoferrin group as presented in Table 11. This increase may be due to secretion of TNF at early gestation as that different cytokine profiles might be beneficial or harmful at different stages of Pregnancy as TNF-a which was proinflammatory cytokines seem to be crucial during the implantation process, whereas high levels of these cytokines may be harmful later in Pregnancy. Also, showed that tumor necrosis factor was multi potent cytokines and had an essential role during  Table 12

Conclusion
Lactoferrin increases the number of leukocytes, especially lymphocytes, eosinophil, and basophil, and increases TNF and C-reactive protein in serum. In addition to that, increase the concentration of interleukin 1 A and interleukin 10, especially with the advancement of pregnancy at 3 and 7 days of gestation.
So Lactoferrin has a positive effect on stimulating the general and mucosal immune system during pregnancy.