Immunosuppressant Cyclosporin ABased Regulation of Lipopolysaccharide-Triggered Pro-and Anti-Inflammatory Cytokines in the Genital Tract of Female Rabbits

In this study, we evaluated the effects of Cyclosporine A (CsA) on Lipopolysaccharide (LPS)-induced cytokine production in the genital tract of female rabbits. Twelve sexually mature and healthy female rabbits were randomly divided into four groups (n = 3 each). The rabbits in the LPS group were given an intrauterine infusion of Escherichia coli LPS (4 mg/kg body weight (BW)). Rabbits in the CsA group were given CsA (20 mg/kg BW). Rabbits in the LPS + CsA group were given LPS (4 mg/kg BW) and CsA (20 mg/kg BW). The control group received only LPS and CsA carrier. The gene expression and protein levels of proand anti-inflammatory cytokines were observed using qRT-PCR and immuno-histochemical (IHC) assay, respectively. Our study showed that IL-1β, IL-6, IL-8, TNF-α, IFN-γ, IL-4, IL-10, IL-13, and TGF-β were expressed in female genital organs. The LPS challenge increased the mRNA expression of IL-6 and TNF-α in the uterine body and IL-1β in the uterotubal junction compared to the control group. CsA increased the basal mRNA expression of anti-inflammatory cytokines (i.e., IL-4 in the uterine body, uterotubal junction, and oviductal ampulla; IL-10 in the cervix, oviductal isthmus, and ampulla; and TGF-β in the uterotubal junction and oviductal ampulla) and pro-inflammatory cytokines (i.e., IL-6 and IL-8 in the cervix; IL-1β in the oviductal isthmus; TNF-α in the oviductal ampulla; and IFN-γ in the uterine body compared to the control group). In addition, CsA inhibited the mRNA expression of pro-inflammatory cytokines, such as IL-6 in the uterine body, uterotubal junction, and oviductal isthmus; TNF-α in the uterine body; and IFN-γ in the uterotubal junction and oviductal isthmus induced by the LPS challenge. The IHC assay showed the LPS-induced increase in protein production of IL-6 in the uterine body and oviductal isthmus. CsA increased the protein production Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 November 2018 doi:10.20944/preprints201811.0289.v1 © 2018 by the author(s). Distributed under a Creative Commons CC BY license.


Introduction
Female infertility, such as tubal factor infertility, is an increasing concern around the world.Most of the cases of tubal factor infertility are associated to undiagnosed and untreated sexually transmitted diseases.These diseases ascend through the female reproductive tract and are implicated in causing damage and inflammation in these organs.Past evidence has demonstrated that several pathogenic bacteria species are involved in causing reproductive tract disorders in females, such as tubal factor infertility and pelvic inflammatory disease [1].Moreover, asymptomatic, undiagnosed, and untreated genital infections may have serious complications related to reproductive health in both humans and domestic animals [1][2][3][4].Clinical and sub-clinical infections induced by Gram-negative bacteria such as Pasteurella, Escherichia coli, and Salmonella are common in rabbits and other female domestic animals [5] and reportedly cause severe economic losses to the farmers as a consequence of the resulting sub-fertility, infertility, increased culling rates due to repeated failure to conceive, reduced production, and increased rearing expenditures [2,[6][7][8].
Lipopolysaccharide (LPS) is an essential building element of the outer membrane of the cell wall of Gram-negative bacteria which is released upon bacterial division and lysing and are recognized by mammalian cells via Toll-like receptors (TLR) 2 and 4 [9,10].LPS is widely used to trigger the in vivo immune inflammatory response in several cells and organs [9,[11][12][13][14] and could be used to elucidate the underlying mechanisms by which infections and/or inflammation impair occurs in the reproductive system in females [15].Toll-like receptors (TLRs) are the members of pathogen-related molecular pattern receptors that are involved in recognizing microbial agents [9,[16][17][18].From these, TLR4, along with some adapter proteins (CD14 and MD2), recognizes the LPS on the surface of host immune cells.The interaction between TLR4 and LPS triggers a cascade of intracellular signaling pathways, resulting in production of immune cytokines such as interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) [15,[19][20][21][22]. This, in turn, stimulates the production of chemokines and other mediators of inflammation, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) [23][24][25].The expression of TLR4 on the surface of epithelial and stromal cells in female reproductive organs, i.e., vagina, cervix, uterus, and oviduct of women and other animal species, has been observed [15].
Cyclosporine A (CsA) is a widely used, powerful immunosuppressant.It has been reported that CsA greatly ameliorates immunosuppressive therapy in organ transplantation and results in increased organ survival rate [52].Moreover, it has been used to treat several autoimmune diseases such as rheumatoid arthritis [53], nephrotic syndrome [54,55], and systemic lupus erythematosus [56].In addition, some previous studies have focused on the implications of immune therapy in reproduction related disorders, such as spontaneous miscarriage and induced pre-eclampsia [57][58][59].
Furthermore, previous studies in humans and mice have shown that CsA could inhibit the pro-inflammatory cytokines IL-1β, IL-6, IL-8, TNF-α, and IFN-γ and increase the production of anti-inflammatory cytokines IL-4 and IL-10 in response to LPS stimulation [19,[59][60][61][62][63][64].Recently Hu and colleagues, using a pregnant rat model, have demonstrated that CsA significantly ameliorates the clinical signs of LPS-induced pre-eclampsia and inhibits the inflammatory response [59].However, little is known about the potential effects of CsA on LPS-induced immune cytokine production in the female genital tract in mammalian species, and further studies are awaited to continually explore the potential implication of this powerful immunosuppressant in regulating immune cytokine production in female reproductive organs.
In this study, using rabbit a model, we explored and elucidated the expression profile and localization of pro-and anti-inflammatory cytokines in female reproductive organs-i.e., cervix, uterine body, uterotubal junction, oviductal isthmus, and ampulla-and assessed whether LPS-induced gene expression and protein levels of pro-and anti-inflammatory cytokines could be affected by CsA in female rabbits.

Tract
The results of CsA's effect on the mRNA expression profile of immune cytokines in various female reproductive tissues following the LPS challenge are shown in Figure 4. Briefly, CsA significantly decreased the expression of LPS-induced pro-inflammatory cytokines IL-6 in uterine body, uterotubal junction, and oviductal isthmus (p < 0.05; Figure 4b), TNF-α in the uterine body (p < 0.05; Figure 4d), and IFN-γ in the uterotubal junction and oviductal isthmus (p < 0.05; Figure 4e).However, the expressions of pro-inflammatory cytokines IL-1β (p > 0.05; Figure 4a) and IL-8 (p > 0.05; Figure 4c) and anti-inflammatory cytokines (p > 0.05; Figures 4f-4i) were not affected by CsA after the LPS challenge.

Discussion
The organs of the female genital tract are more likely to encounter invasion by microorganisms compared to other internal organs.The infection caused by Gram-negative bacteria is one of the major causes of infertility and sub-fertility in humans and domestic animals [1,2,4,65,66].These infections and inflammatory conditions have relevance to several female reproduction-related complications, such as intrauterine growth retardation [67], premature birth, intrauterine fetal death and abortion [68], and fetal resorption [69,70].The progression of these complications is reportedly mediated through endotoxins, including LPS, which triggers the production of immune cytokines and other mediators of inflammation by immune and non-immune cells [13,71].However, at present, the exact underlying molecular mechanisms through which LPS-induced inflammatory responses are mediated in female reproductive organs are not yet sufficiently understood and require further elucidation [15].
The immune system of an organism is charged with defending the host against pathogenic microbes, however, this response must be appropriate and measured [26].The success of the host immune response is by and large the outcome of both pro-and anti-inflammatory components that are cautiously tuned with the aim of scavenging pathogenic microbes and minimizing host damage [26].Experimental studies are important to revealing the identities of pro-and anti-inflammatory players in immune responses and identifying the aftermaths of interrupting their balance [26].The ultimate aim is to harness knowledge and isolate factors regulating this dynamic balance to furnish strategies for better patient results.For instance, analysis of models can identify immunomodulatory factors that, combined with antibiotics, enable patients to achieve pathogen clearance.While we now better understand how a balance of pro-and anti-inflammatory mediators might be needed to control infection, we have not yet fully applied that concept for therapeutic development [26].In the present study, using rabbits as a model, we have demonstrated the CsA mediated regulation of immune and inflammatory responses following a local inoculation of LPS.
Our results showed that LPS significantly increased the mRNA expression of pro-inflammatory cytokines IL-6 and TNF-α in the uterine body and IL-1β in the uterotubal junction.However, in the cervix and oviduct, the expression of pro-inflammatory cytokines was unaffected.These results are in agreement with our previous studies demonstrating the LPS-induced up-regulated expressions of IL-1β, IL-6, and TNF-α in the uterine body and horn but not in the cervix following LPS challenge [19,72].The tissue-specific differential expressions of pro-inflammatory cytokines may be attributed to the higher natural elicitation of an immune response and, perhaps, the relative anatomical location and histo-architecture of the cervix [72].The findings are further supported by our present study which demonstrated that the expression of pro-inflammatory cytokine IL-6 in the cervix and oviduct of control group was higher compared to uterine body and uterotubal junction.Conversely, the expression of anti-inflammatory cytokine IL-10 contrasted that of IL-6 (Figure 17).
Even though there is certainly a commonality in the mucosal immune responses, there is also manifestations of a degree of location-specific immune mechanisms, dependent upon the physiological role and anatomical position of an organ [73].There is also evidence that the upper "sterile" and the lower "non-sterile" female reproductive tract compartments may sustain a different immunological surveillance milieu and might also respond differentially to a pathogen challenge [73].Albeit, the TLR patterns on endocervical epithelium are similar to the lower tract, the constitutive and induced cytokine profile in immortalized endocervical epithelium is considerably higher compared to matched immortalized ectocervical and vaginal epithelial cells [73].Therefore, it reasonable to suggest that future research should also focus on clarifying the underlying mechanisms dictating the site-specific elicitation or up-regulation of immunological responses in specific reproductive tract sites.Moreover, a deeper apprehension of organ-specific, epithelial-derived early immune responses in the upper and lower reproductive tracts has significant implications for developing vaccines or immune-based remedies, since the elicitation and consequence of an acquired immune response are determined by the specific early immunological events which occur at a given site [73].
As for the LPS-induced expression of pro-inflammatory cytokines in uterine tissues, the findings of our study are also supported by results of previous in vitro studies reporting the increased expression of pro-inflammatory cytokine genes in cultured bovine endometrial epithelial and stromal cells, as well as in human endometrial epithelial cells following treatment with LPS [21,60,74,75].Furthermore, few other reports have demonstrated that the intra-uterine infusion of LPS may induce an up-regulated expression of pro-inflammatory cytokine genes in mice and bovine animals [4,14].
Furthermore, it is well established that LPS is recognized by TLR4, which then triggers the expression of pro-inflammatory cytokines.Previous studies have demonstrated that LPS administration induced an increase in mRNA expression of TLR4 in the uterus, while no up-regulation in expression was found in the oviducts [15,72].The differential expressions of TLR4 may be associated with different types of uterine and oviductal cells and/or the influx of inflammatory cells into the uterus and oviducts [15].Additionally, it can be influenced by the concentrations of the reproductive hormones [76,77].Recently, Menchetti and colleagues have reported a stronger signal for TLR4 protein in uterine stromal cells of LPS-challenged rabbits compared to the control group, indicating that these cells, in addition to epithelial cells, may have implications in triggering the inflammatory response to LPS challenge or invading microorganisms [15].Additionally, it has also been reported that, following LPS challenge, mRNA expression of IL-1β is increased both in the uterus and oviduct, whereas the up-regulated expression of TNF-α was only observed in the rabbit uterus [15].Therefore, it is reasonable to speculate that differential expression of immune cytokines in the uterus and oviduct following LPS challenge is tissue-and cell-specific and might be linked to the differential expression of TLR4 in these tissues.Nevertheless, studies are awaited to explicate the putative mechanisms by which LPS induces the differential expression of Inflammation elicited by the action of pro-inflammatory cytokines, such as IL-1, TNF, IFN-γ, and IL-6, is adjudicated by anti-inflammatory cytokines (i.e., IL-4, IL-10, IL-13, and TGF-β) [78].Our results demonstrated that the expression of anti-inflammatory cytokines did not change in all tissues analyzed after LPS-challenge.These observations are consistent with the findings of Laura and colleagues who reported that LPS-treated endometrial cells demonstrated a significant increase in the mRNA expression of IL-1β, IL-6, and IL-8 but not IL-10 [79].There is a dearth of information regarding the potential effects of LPS on the elicitation of anti-inflammatory cytokine expression.To our knowledge, our study is the first to report the effects of intrauterine administration of LPS on the protein and mRNA expression of anti-inflammatory cytokines in the uteruses and oviducts of female rabbits.
To understand the mode of action of CsA in regulating the immune and inflammatory responses, a thorough comprehension is needed of the underlying mechanisms by which it targets biochemical processes that have potential involvement in the stimulation of specified subsets of immune cells.Also needed is an understanding of how the interaction of these different subsets is mediated by a pathogen's antigens to result in a physiologically appropriate response [80].Moreover, it has been demonstrated that CsA has implications in the inhibition of the essential antigen-dependent signal needed to stimulate T cells, as well as the production of interleukins by T cells and T cell clones.In addition to this, CsA is also implicated in inhibiting the antigen presentation by macrophages.The manifestation of such distinct activities warns one against an overly simplistic view of how CsA acts and of what immunoregulatory capacity it might possess [80].
CsA has been widely used as an anti-inflammatory agent in inflammatory conditions.The importance of CsA as an immunosuppressant is associated with its efficacy in suppressing the elicitation of immune responses at doses that do not impair the functioning of the hematopoietic system [80].
In the present study, we observed that CsA up-regulated the basal production of anti-inflammatory cytokines and down-regulated the LPS-triggered production of pro-inflammatory cytokines in reproductive organs of female rabbits.These enticing findings indicate that CsA might act as a regulator of the negative feedback between pro-inflammatory and anti-inflammatory cytokines to maintain tissue homeostasis [65,81].To the best of our knowledge, no in vivo study investigated the effects of CsA on the production of inflammatory cytokines in the female genital tracts of mammals prior to this study.However, the results of our study conform with previous studies reporting the effect of CsA on the expression of inflammatory cytokines in other cell lines.For instance, consistent with our findings, it has been reported that CsA might inhibit the production of IL-6, IFN-γ, and TNF-α and up-regulate IL-10 and IL-4 expression in response to LPS stimulation in dendritic cells [60,61], macrophages [63], and PBMCs [59].Interestingly, we further observed that CsA also up-regulated the basal production of pro-inflammatory cytokines.These findings may support the conjecture that CsA has potential implications in maintaining a delicate balance between pro-and anti-inflammatory cytokines, both in the resolution of inflammation and normal homeostasis.However, we are unaware of other precedents that report the implication of CsA in regulating pro-inflammatory and anti-inflammatory cytokines following LPS challenge in mammalian female genitals.Nevertheless, further research will be necessary to validate these findings.
In addition, previous research has indicated that CsA may act as immunomodulatory agent, however, this activity largely depends on the concentration of the antigen and dose of CsA used [80].
There have been reports that low and moderate concentrations of CsA increased, while therapeutic and high concentrations decreased, the basal production of pro-inflammatory cytokines in immune cells [82,83] and non-immune cells [84].Furthermore, it has been demonstrated that high concentrations of CsA could decrease the viability of cells [85], thus decreasing pro-inflammatory cytokine production [84].García and colleagues have shown that low and intermediate concentrations of CsA decreased, while high concentrations increased, the basal production of pro-inflammatory cytokines in human alveolar macrophages [62].The discrepancy in these observations might be ascribed to diverse activities and differential effects of CsA on distinct cell lines [65,86,87].Therefore, future studies focusing on this caveat are warranted to better comprehend the underlying complex feedback mechanisms, and they will add to the known effects of this potent immunosuppressant.

Conclusions
Interactions between the immune cascades and reproductive system have important consequences for successful reproduction and fertility.Our results show that CsA increased both mRNA expression of anti-and pro-inflammatory cytokines and inhibited the mRNA and protein expression of pro-inflammatory cytokines following LPS challenge.Our findings imply the potential role of CsA in downregulating the excessive production of pro-inflammatory cytokines as a part of the innate host immune response, enabling the inflammatory cascades to eliminate the invading agents through complex feedback mechanisms without compromising the immune system.Taken together, the results presented here will serve as a foundation for future research focused on the potential implications of CsA in regulating the delicate balance between pro-inflammatory and anti-inflammatory cytokines, particularly in the context of Gram-negative bacteria-induced localized and sub-clinical infections invading the female genital tract that consequently affect overall female reproduction and fertility.Moreover, properly powered experimental and clinical studies are needed to further explicate the effects of localized inflammatory conditions and sub-clinical infections on distinct aspects of female reproductive efficiency in different reproductive phases and to compound our understanding regarding the underlying mechanisms that dictate the crosstalk between the immune system and infertility.

Animals and Ethical Approval
The trial was carried out at the Laboratory of Animal Reproduction and Embryo Engineering, Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Chengdu, Sichuan, China.All rabbits were provided with a similar feeding regime and environmental and sanitary conditions.The feeding was done as previously described [88] were fixed in 10% formaldehyde and kept at room temperature until further analysis.

RNA Isolation and cDNA Synthesis
The tissues were homogenized immediately in 1 ml TRzol Reagent (Ambion, USA) after collection, and total RNA were extracted according to the manufacturer's protocol.The quality of RNA was examined using 1.5% agarose gel electrophoresis, and the concentration of total RNA was determined with a NanoDrop ND-2000C (Thermo Scientific, USA).The purified RNA samples were reverse-transcribed using a PrimeScript ® RT Reagent Kit with gDNA Eraser (Takara, Japan) following the manufacturer's protocol.Briefly, a 10 µl reaction mixture (3 µl of RNase free water, 2 µl 5 × gDNA Eraser Buffer, 1 µl of gDNA Eraser, and 4 µl RNA) was placed at room temperature for 5 min to remove genomic DNA.Then, 4 µl of RNase free water, 4 µl of 5 × PrimeScript Buffer 2 (for real time PCR), 1 µl of PrimeScript RT Enzyme Mix 1, and 1 µl of RT Primer Mix were added to the reaction mix (total volume = 20 µl).The samples were incubated at 42 °C for 2 min, followed by heat inactivation at 85°C for 5 s on a programmable thermal controller PTC-100 (Bio-Rad, USA) for cDNA synthesis.

Quantitative Real-Time PCR
Quantitative real-time PCR (RT-qPCR) was performed using a CFX 96 Real-Time PCR Detection System (Bio-Rad, USA) in a 10 µl reaction mixture containing 3 µl dH2O, 5 µl 2 × SYBR Premix EX Taq II, 0.5 µl of each forward and reverse primer (primer sequences are provided in Table 1), and 1 µl cDNA.Following initial denaturation at 95 °C for 3 min, PCR was carried out with a thermal cycling protocol of 95 °C for 10 s and an annealing temperature of 60 °C (Table 1) for 30 s for 40 cycles.Specificity of the amplified products was verified by melting curve analysis.2-ΔΔCt method was used to calculate the expression of candidate genes.The GAPDH was used as an internal control.Each gene was analyzed in triplicate.
After rinsing with TBS with Tween 20 (TBST, 5 times in 5 min), the sections were incubated with peroxidase-labeled secondary antibody (goat anti-mouse IgG, 1:200, Boster, China) for 1 h at room temperature.After incubation, slides were rinsed with TBST and then exposed to an avidin biotin complex (ABC Kit, 1:100, Boster, China) for 1 h and rinsed again with TBST.To reduce variations in staining, tissue sections from all four groups were incubated together in a moist chamber during each immuno-histochemical procedure.The peroxidase activity sites were visualized using a DAB chromogenic substrate kit (TianGen, China).Sections were rinsed with distilled water and counterstained with Mayer's hematoxylin, washed in running tap water, and mounted in aqueous mounting agent (BBI Life Sciences, Canada).Specimen slides were randomly selected and observed under a light microscope (Nikon 90i, Japan).The representative images captured under high-power magnification (×200) using a digital camera (Nikon Dxm 1200, Japan) were obtained and processed in TIFF format.The positive areas of immune histochemical staining were analyzed using Image-Pro Plus 6 software (Media Cybernetics, Rockville, MD USA).Average optical density (AOD) was used to assess the expression intensity of IL-6 and IL-10 [89].AOD = Integrated optical density (IOD)/Area.Three sections were examined from each tissue per animal, and three representative visual fields randomly selected on a section were analyzed for IL-6 and IL-10 immunoreactivity.

Statistical Analysis
Data was analyzed using SPSS statistical software (version 23.0; SPSS Inc, USA).Variations among groups were analyzed using one-way analysis of variance (ANOVA).Student's t-test was used to compare differences between two independent samples (control group vs. LPS group or CsA group; LPS group vs. LPS + CsA group).A p-value of < 0.05 was considered to be statistically significant.

and 16 )
. Moreover, CsA treatment did not affect IL-10 expression following the LPS challenge (p > 0.05; Figures11h-15h and 16) but significantly increased the basal expression in the cervix (p < 0.01;

Posted: 13 November 2018 doi:10.20944/preprints201811.0289.v1
Twelve healthy and sexually mature female New Zealand white rabbits of uniform body weight (2.5 ± 0.1 kg) and age (3.5 months) were randomly divided into four groups: control, LPS, CsA, and LPS + CsA group (n = 3 each).The rabbits in the LPS group were given an intrauterine infusion of E.
. All experimental procedures were performed strictly in accordance with the regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, revised in June 2004) and approved by the Institutional Animal Care and Use Committee in the College of Animal Science and Technology, Sichuan Agricultural University, Sichuan, China (approval No. SCAUS20163636).Preprints (www.preprints.org)| NOT PEER-REVIEWED |

Table 1 .
Primer information and PCR conditions.