Molecular Mechanisms of Lipopolysaccharide (LPS) Induced Inflammation in an Immortalized Ovine Luteal Endothelial Cell Line (OLENDO)

Escherichia coli (E. coli) is the most common Gram-negative bacterium causing infection of the uterus or mammary gland and is one of the major causes of infertility in livestock. In those animals affected by E. coli driven LPS-mediated infections, fertility problems occur in part due to disrupted follicular and luteal functionality. However, the molecular mechanisms by which LPS induces inflammation, and specifically, the role of LPS in the disruption of capillary morphogenesis and endothelial barrier function remain unclear. Here, we hypothesized that LPS may lead to alterations in luteal angiogenesis and vascular function by inducing inflammatory reactions in endothelial cells. Accordingly, OLENDO cells were treated with LPS followed by evaluation of the expression of selected representative proinflammatory cytokines: NF-kB, IL6, IL8, TNFα, and ICAM 1. While TNFα was not affected by treatment with LPS, transcripts of NF-kB, IL6, and IL8 were affected in a dosage-dependent manner. Additionally, the activity of TLR2 and TLR4 was blocked, resulting in suppression of the LPS-induced expression of ICAM 1, NF-kB, IL6, and IL8. Inhibition of the PKA or MAPK/ERK pathways suppressed the LPS-stimulated expression of NF-kB, IL6, and IL8, whereas blocking the PKC pathway had the opposite effect. Furthermore, LPS-induced phosphorylation of Erk1 and Erk2 was inhibited when the TLR4 or MAPK/ERK pathways were blocked. Finally, LPS seems to induce inflammatory processes in OLENDO cells via TLR2 and TLR4, utilizing different signaling pathways.


Introduction
Bacterial infections of reproductive organs in dairy cows are common worldwide. Gram-negative bacteria, in particular Escherichia coli (E. coli), are the main cause of clinical metritis and mastitis and reduce reproductive performance in livestock [1][2][3]. By activating a local or systemic inflammatory response, the lipopolysaccharide (LPS) endotoxin, an outer cell wall component of Gram-negative bacteria, frequently causes infertility or subfertility in affected animals [3][4][5][6]. Moreover, luteinizing hormone (LH) secretion and pulsatility are also affected by LPS in cows and ewes [7][8][9]. LPS accumulates in ovarian follicular fluid and induces a local inflammatory response in the ovaries of animals affected by uterine or mammary gland infection [10][11][12]. As a result, inhibited follicular growth, reflected in disturbed ovarian cyclic activity, and lowered intrafollicular and circulating levels of estradiol (E2) are observed [3][4][5][6]. This is corroborated by both in vivo and in vitro observations demonstrating an LPS-dependent decrease in E2 production in bovine granulosa cells [13]. In LH-stimulated theca cells of rats, and in the bovine ovary, LPS inhibits progesterone and androstenedione synthesis [11,14]. Furthermore, administration of LPS in Endothelial Cell Growth Supplement (ECGS) (all from Chemie Brunschwig AG, Basel, Switzerland). Cells were trypsinized and transferred into 6-well plates at a concentration of 2 × 10 6 cells per well and used for experiments 24 h later. Prior to the cell culture experiments, serum-containing medium was removed, and cells were rinsed with sterile phosphate-buffered saline (PBS) solution. A serum-free stimulation medium containing increasing concentrations of E. coli LPS (O55:B5; Sigma-Aldrich GmbH, Buchs, Switzerland) (1 ng/mL, 10 ng/mL, and 100 ng/mL) was then added to the wells. These dosages of E. coli LPS were derived from previously published reports in which mean concentrations of 176.1 ± 112 ng/mL LPS were detected in the follicular fluid of cows with clinical endometritis [4]. OLENDO cells were treated with a stimulation medium that contained specific inhibitors of PKA (H89, 25 µM), PKC (GF-109203X, 20 µM) and MAPK (UO126, 10 µM) activities (all reagents purchased from Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA), alone or in combination with 100 ng/mL E. coli LPS. These concentrations of specific inhibitors were chosen based on previously published reports [32,33]. Selective blockers of TLR receptors from Sigma-Aldrich Chemie GmbH were used, targeted against TLR1/TLR2 and TLR4. They were applied at increasing dosages (5 µM and 10 µM for TLR1/TLR2 and 1 nM, 10 nM, and 100 nM for TLR4) in stimulation medium over 6 h and ICAM 1 expression was assessed. This incubation period was chosen based on previous experiments showing the highest expression level of ICAM 1 in OLENDO cells upon stimulation with 100 ng/mL LPS [31]. The lowest effective dosages of TLR4 (TAK-242, 100 nM) and TLR1/TLR2 (CU-CPT22, 5 µM) inhibitors in OLENDO cells were determined in pilot experiments, based on the lowest levels of ICAM 1 expression after 6 h of stimulation. These dosages were then used for all stimulation experiments. Non-treated cells served as controls.

RNA Isolation, Reverse Transcription and Qualitative RT-PCR
TRIzol ® reagent (Invitrogen, Carlsbad, CA, USA) was used for extraction of total RNA from OLENDO cell samples. The isolation procedures were performed according to the manufacturer's instructions and our previously published protocols [34,35]. A NanoDrop 2000C ® spectrophotometer (Thermo Fisher Scientific AG, Reinach, Switzerland) was used to measure the quality and quantity of isolated total RNA. In order to remove genomic DNA contamination, DNase treatment was performed by using RQ1 RNase-free DNase (Promega, Duebendorf, Switzerland). Thereafter, RNA samples were reverse transcribed (RT) into cDNA following the manufacturer's instructions and utilizing random hexamers as primers (Applied Biosystems by Thermo Fisher, Carlsbad, CA, USA).
Following cDNA synthesis, a conventional hot start PCR reaction was performed using the GeneAmp Gold RNA PCR Kit (Applied Biosystems by Thermo Fischer) according to the manufacturer's protocol. The primers for TLR1 (Table 1) were designed using Primer Express Software ver. 2.0 (Applied Biosystems by Thermo Fischer). Primers, purchased from Microsynth (Balgach, Switzerland). The PCR reaction was run with the annealing temperature set at 60 • C. Following its separation on an ethidium bromide stained 2% agarose gel, the 317 bp amplicon of TLR1 was purified using the Qiaex II gel extraction system (Qiagen GmbH Hilden, Germany). The product was then subcloned into the pGEM-T vector (Promega) and transformed into XL1 Blue competent cells (Stratagene, La Jolla, CA, USA). Plasmid isolation and purification were performed using the Pure Yield Plasmid MidiPrep System (Promega), and isolated plasmids were then sent for commercial sequencing (Microsynth) on both strands using Sp6 and T7 promoters. Autoclaved water instead of RNA and the so-called RT minus controls were used as negative controls. Table 1. List of primers used for the conventional and semi-quantitative real time (TaqMan) RT-PCR.

Semi-Quantitative RT-PCR and Data Evaluation
Semi-quantitative Real-Time (TaqMan) PCR was carried out using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems) as previously described [34,35]. DNase treatment and RT were performed as described above. PCR reactions were run in duplicates in 96-well optical plates (Applied Biosystems) under the following conditions: denaturation at 95 • C for 10 min, followed by 40 cycles each of 95 • C for 15 s and 60 • C for 60 s. The sequences for forward and reverse primers and TaqMan ® probes labeled with 6-carboxyfluorescein (6-FAM) and 6-carboxytetramethylrhodamine (TAMRA) were designed using Primer Express Software v 2.0 (Applied Biosystems) and were bought from Microsynth. The list of self-designed primers and TaqMan ® probes is presented in Table 1. In order to determine the expression of ICAM 1, a commercially available ovine-specific TaqMan Gene Expression Assay was used, purchased from Applied Biosystems (Prod. No. Oa04658646_m1).
In order to normalize gene expression levels of the target genes, GAPDH and ACTB were used as reference genes. The sample with the lower expression level was used as a calibrator. Calculation of the relative gene expression of each target gene was performed using the comparative CT method (∆∆CT method) according to the ABI Prism 7500 (Applied Biosystems) manufacturer's protocol and as previously described [34,36].

Protein Preparation and Western Blot Analysis
Western blot analysis was performed as previously described [34,36]. Briefly, cells were washed with ice-cold PBS and harvested with NET-2 lysis buffer (50 mM Tris-HCl, PH 7.4, 300 mM NaCl, 0.05% NP-40) containing 10 µL/mL protease inhibitor cocktail (Sigma-Aldrich Chemie GmbH). Cell lysates were homogenized with a sonic distributor (Vibra-Cell, Newton, CT, USA) 75 Watt for 15 s on ice, then centrifuged at 10,000× g for 10 min at 4 • C to remove cell debris. The Bradford assay was used to measure the protein concentrations in the supernatants. Samples were solubilized in sample buffer (25 mmol/L Tris-Cl, pH 6.8, 1% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue) and equal amounts of proteins from the cell lysates (20-30 µg) were electrophoresed on 12% polyacrylamide gels (Bio-Rad Laboratories GmbH, Munich, Germany) at 120 V. Afterwards, proteins were blotted onto a methanol-activated polyvinylidene difluoride (PVDF) membrane (Bio-Rad) for 1 h at 100 V. The membranes were blocked with 5% low-fat dry milk in PBST (PBS/0.25% Tween-20) for 1 h at ambient temperature and incubated overnight at 4 • C with a primary antibody diluted in PBST (PBST/0.25% Tween 20) containing 2.5% skimmed milk. The primary antibodies used were rabbit polyclonal anti-human p44/42 MAPK (Erk1/2) (#9102; dilution 1:1000) and rabbit polyclonal anti-human phospho-p44/42 MAPK (Erk1/2) (#9101; dilution 1:1000), both purchased from Cell Signaling Technology, Inc., Beverly, MA, USA. The following day, after washing in PBST, membranes were incubated for 1 h with a specific horseradish peroxidase (HRP)-coupled secondary antibody at ambient temperature. Signals were developed with Immun-Star TM WesternC TM Chemiluminescent Kit substrate according to the manufacturer's protocol (Bio-Rad). The detection of the signals was performed using the ChemiDoc XRS+ System (Bio-Rad) and Image Lab Software (Bio-Rad). The optical density of bands was assessed for p44/42 MAPK (Erk1/2) and phospho-p44/42 MAPK (Erk1/2), using Image Lab Software (Bio-Rad). Membranes were re-probed with an anti-ACTB antibody and the values are presented as the ratio of the optical density of the target protein relative to that of ACTB. Representative immunoblots are shown.

Statistical Analysis
All cell culture experiments were repeated independently at least three times, using cells from different passages.
The Shapiro-Wilk test was performed to assess the normality of the distribution of results. Since the data obtained in our current study showed a normal distribution, to assess the effects of LPS treatment on the expression of TNFα, NF-kB, IL6, IL8, and ICAM 1, a global comparison was performed by applying parametric one-way analysis of variance (ANOVA) with the statistical software program GraphPad 3.06 (GraphPad Software, San Diego, CA, USA). In the case of p < 0.05, the Tukey-Kramer multiple comparisons post-test was performed. The data are presented as the mean ± standard deviation (SD). The level of significance was considered as p < 0.05.

LPS Treatment Induces Inflammatory Reaction in OLENDO Cells
The cells were treated with increasing concentrations of E. coli -derived LPS for 6 and 12 h and the gene expression of inflammation markers TNFα, NF-kB, IL6, and IL8 assessed ( Figure 1A-D). The respective mRNA was detectable in both treated and untreated OLENDO cells. While TNFα was not affected by treatment with LPS (p = 0.3 for 6 h and p = 0.9 for 12 h, Figure 1A), NF-kB (p < 0.0001 for 6 h and p < 0.002 for 12 h, Figure 1B), IL6 (p < 0.0001 for both 6 h and 12 h, Figure 1C) and IL8 (p < 0.0001 for both 6 h and 12 h, Figure 1D), were affected in a dosage-dependent manner. In greater detail ( Figure 1B), although 100 ng/mL LPS significantly stimulated NF-kB mRNA expression over a 6 h time course (p < 0.001), a lower dosage of LPS (10 ng/mL, p < 0.01) was effective in a 12 h stimulation experiment. The expression of IL6 ( Figure 1C) increased significantly over controls within 6 h, in response to both the lower (10 ng/mL) and higher (100 ng/mL) LPS concentrations (p < 0.001 for both). This effect was also apparent in cells incubated for 12 h, with the respective mRNA levels increasing gradually in response to all LPS dosages (p < 0.001 for 1, 10 and 100 ng/mL LPS), compared with the respective control. Similarly, mRNA levels of IL8 resembled that of IL6 and were significantly upregulated at 6 and 12 h in response to 10 ng/mL and 100 ng/mL LPS (p < 0.001 for both dosages) compared with the respective controls ( Figure 1D).
Vet. Sci. 2022, 9, 99 6 of 17 6 h, in response to both the lower (10 ng/mL) and higher (100 ng/mL) LPS concentra (p < 0.001 for both). This effect was also apparent in cells incubated for 12 h, wit respective mRNA levels increasing gradually in response to all LPS dosages (p < 0.00 1, 10 and 100 ng/mL LPS), compared with the respective control. Similarly, mRNA l of IL8 resembled that of IL6 and were significantly upregulated at 6 and 12 h in resp to 10 ng/mL and 100 ng/mL LPS (p < 0.001 for both dosages) compared with the respe controls ( Figure 1D).

OLENDO Cells Respond to E. coli LPS through TLR1/TL2 and TLR4 Pathways
The potential involvement of TLRs in the LPS-induced inflammatory reacti OLENDO cells was investigated by applying commercially available blockers of TL and TLR4 (Figures 2 and 3). While the presence of TLR2 and TLR4 in OLENDO cell been shown previously [31], here the basal availability of TLR1 in these cells was firmed by conventional PCR (Figure 2A), and its expression was stably mainta throughout passages. Next, cells were treated with LPS following the application o specific functional blockers of TLR1/TLR2 (CU-CPT22) or TLR4 (TAK-242), and the expression of proinflammatory cytokines ICAM 1, TNFα, NF-kB, IL6, and IL8 wa sessed.

OLENDO Cells Respond to E. coli LPS through TLR1/TL2 and TLR4 Pathways
The potential involvement of TLRs in the LPS-induced inflammatory reaction in OLENDO cells was investigated by applying commercially available blockers of TLR1/-2 and TLR4 (Figures 2 and 3). While the presence of TLR2 and TLR4 in OLENDO cells has been shown previously [31], here the basal availability of TLR1 in these cells was confirmed by conventional PCR (Figure 2A), and its expression was stably maintained throughout passages. Next, cells were treated with LPS following the application of the specific functional blockers of TLR1/TLR2 (CU-CPT22) or TLR4 (TAK-242), and the gene expression of proinflammatory cytokines ICAM 1, TNFα, NF-kB, IL6, and IL8 was assessed.
Apart from TNFα, the expression of all factors was significantly upregulated in response to LPS treatment. Similarly, both blockers significantly suppressed LPS-driven gene expression. In detail, whereas LPS induced the expression of ICAM 1 (p < 0.001), the TLR1/TLR2 blocker (CU-CPT22) significantly reduced its levels for both dosages tested ( Figure 2B).

Modulation of Functionality of Kinases (PKA, PKC, MAPKs) Alters the Expression of Inflammatory Markers in OLENDO Cells
The downstream signaling cascades in LPS treated OLENDO cells were assessed by modulating the activity of the kinases, PKA, PKC, and MAPK, via specific blockers, H89, UO126 and GF-109203X, respectively (Figures 4-6). The inflammatory response was Vet. Sci. 2022, 9, 99 9 of 17 evaluated by checking the gene expression levels of selected proinflammatory cytokines: TNFα, NF-kB, IL6, and IL8.
While TNFα was not affected by treatment with H89 (p = 0.8 for both 6 h and 12 h, Figure 4A), inhibition of the PKA pathway suppressed the LPS-stimulated expression of NF-kB (p < 0.01), IL6 (p < 0.001) and IL8 (p < 0.05) in a 6 h time course (Figure 4B-D). However, this effect of H89 was not apparent in cells incubated for 12 h (Figure 4B-D).
Similarly, blocking the MAPK/ERK pathway significantly suppressed the LPS-stimulated gene expression levels of NF-kB, IL6 and IL8. In greater detail, LPS induced mRNA expression of NF-kB (p < 0.05 for both 6 h and 12 h, respectively), IL6 (p < 0.05 for 6 h and p < 0.001 for 12 h, respectively), and IL8 (p < 0.001 for both 6 h and 12 h), but their expression was significantly decreased in response to UO126 ( Figure 5B-D).

Modulation of Functionality of Kinases (PKA, PKC, MAPKs) Alters the Expression o Inflammatory Markers in OLENDO Cells
The downstream signaling cascades in LPS treated OLENDO cells were asses modulating the activity of the kinases, PKA, PKC, and MAPK, via specific blocker UO126 and GF-109203X, respectively (Figures 4-6). The inflammatory response wa uated by checking the gene expression levels of selected proinflammatory cyt TNFα, NF-kB, IL6, and IL8.     In contrast to the effects observed in response to H89 and UO126 treatment, blocking of the PKC pathway significantly potentiated the LPS-stimulated gene expression levels of NF-kB, IL6 and IL8, as well as the expression of TNFα (Figure 6A-D). In detail, the LPSinduced gene expression levels of NF-kB (p < 0.05 for 6 h and p < 0.01 for 12 h, respectively) and IL6 (p < 0.001 for 6 h and p < 0.05 for 12 h, respectively) were significantly potentiated in response to GFX. However, this effect of GFX-109203X was apparent for IL8 only in cells incubated for 12 h, with the respective mRNA levels significantly upregulated in response to the dosage used (p < 0.05) ( Figure 6D). Figure 5. Effects of LPS treatment and inhibition of MAPK (UO126) activity on expression of TNF NF-kB, IL6 and IL8 in immortalized OLENDO cells. Expression of TNFα (A), NF-kB (B), IL6 (C), a IL8 (D) as determined by real-time (TaqMan) PCR. Inhibition of MAPK (UO126) activity was p formed as described in the Materials and Methods. A parametric one-way ANOVA was appli followed by the Tukey-Kramer multiple comparisons post-test. All numerical data are presented the means ± S.D. p-values < 0.05 were considered significant and are indicated.

LPS Induces Phosphorylation of p44/42 MAPK (Erk1/2) in OLENDO Cells
To examine the possible effect of LPS on MAPK (Erk1/2) activation in OLENDO cells, 100 ng/mL LPS was applied to the cells, and cell extracts collected at different time points were processed for western blotting to detect the expression and activation (i.e., phosphorylation) of MAPK (Erk1/2). As shown in Figure 7, activation of MAPK (pErk1/2) was seen 4 h after LPS treatment and remained stable for up to 6 h. However, this effect of LPS was attenuated for MAPK (pErk1/2) in the presence of 10 µM MAP kinase inhibitor (UO126) or 100 nM TLR4 blocker (TAK-242) in the stimulation medium. Interestingly, neither of the blockers nor LPS had a significant effect on the expression of total (unphosphorylated) MAPK (Erk1/2) (Figure 7). 1 Figure 7. Effects of LPS treatment and MAPK (UO126, 10 µM) and TLR4 (TAK-242, 100 nM) inhibitors on protein expression of p44/42 MAPK (Erk1/2) and phosphorylated p44/42 MAPK (Erk1/2) proteins for 0-6 h. ACTB expression (45 kDa) was used as a loading control. Representative immunoblots are shown. Lower panels represent densitometric values (standardized optical density; SOD) for Erk1 (p44), Erk2 (p42) and pErk1 (p44), pErk2 (p42) expression normalized against ACTB. A parametric one-way ANOVA was applied followed by the Tukey-Kramer multiple comparisons post-test. All numerical data are presented as the means ± S.D. p-values < 0.05 were considered significant: * indicates p < 0.01, ** indicates p < 0.001. The original western blotting figures in Figure 7 can be found in Figure S2

Discussion
Bacterial infections of the uterus or mammary gland may affect the structure and function of the CL by involving the functionality of the vascular bed. Although the expression of TLR2 and TLR4 have been found in luteal endothelial cells of different mammalian species, the impact of LPS on luteal angiogenesis and vasculogenesis in livestock is not well understood. Therefore, by using the previously established OLENDO cell line [31], this study was designed to examine the effects of LPS on the endothelial cell-mediated immune response. Building on the previous study that proved the presence of TLR2 and TLR4 in OLENDO cells, here the expression of TLR1 was also confirmed in OLENDO cells and remained stable throughout the passages. The expression of the three receptors in OLENDO cells suggests the involvement of luteal endothelial cells in recognition of PAMPs associated with Gram-negative and Gram-positive bacteria. While TLR4 is activated by LPS, TLR2, by forming a heterodimeric complex with TLR1, is involved in the recognition of microbial components of Gram-positive bacteria, such as lipopeptides, peptidoglycan, and lipoteichoic acids [19][20][21]. An LPS-induced inflammatory reaction via TLR4 has been demonstrated in human umbilical vein endothelial cells, lung microvascular endothelial cells, and coronary artery endothelial cells [37][38][39]. In line with this, treatment of OLENDO cells with LPS significantly increased expression of NF-kB, IL6, and IL8 mRNA in a dose-dependent manner within 6 h of stimulation. These effects were maintained in 12 h stimulation experiments, with increased response towards lower concentrations of LPS, implicating time-dependent effects. However, TNFα was not affected by treatment with LPS in OLENDO cells. Effects of LPS treatment on the expression of TNFα may vary depending on particular cell types. For example, in human umbilical vein endothelial cells (HUVEC) [40] and human adipocytes [41], in contrast to OLENDO cells and human airway leucocytes [42], LPS induced expression of TNFα. The regulatory mechanisms behind the differential action of LPS on expression of TNFα in different cell types remain unclear, but certainly deserve further investigations. Furthermore, in the present study, we were able to show that LPS-induced elevation of NF-kB and cytokines IL6 and IL8 is modulated by both TLR2 and TLR4 receptors. Accordingly, the suppression of TLR2 and TLR4 activity resulted in a significant reduction of their expression.
Additionally, in the present study with OLENDO cells, the effects of TLR1/2 and TLR4 receptors on LPS-induced ICAM 1 expression were assessed. ICAM 1 is an important adhesion molecule involved in the attachment and recruitment of leukocytes to the endothelium [30,43,44]. Its activation induces inflammation and vascular disruption, as shown e.g., in rat brain endothelial cells or human umbilical vein endothelial cells [30,43,44]. Similarly, recent results from our group showed that LPS disrupts in vitro capillary morphogenesis and endothelial barrier function, in association with increased ICAM 1 expression and altered gap junctional intercellular communication, mediated particularly by Cx43 in OLENDO cells [31]. Interestingly, as presented here, blocking of TLR1/2 or TLR4 receptors significantly decreased the expression of ICAM 1 in LPS-treated OLENDO cells in a dose-dependent manner. This strongly suggests the involvement of these receptors in LPS induced inflammatory processes and disruption of capillary morphogenesis and endothelial barrier function in luteal endothelial cells.
Moreover, MAPK/ERK regulated pathways are known to play important roles in the regulation of inflammation-associated cytokine and chemokine production, as shown e.g., in mouse splenocytes, human mesangial and proximal tubular cells and peripheral mononuclear cells [45][46][47]. For instance, activation of TLRs with PAMPs in bovine and human granulosa cells stimulates downstream cascades involving activation of mitogenactivated protein kinases (MAPK) and nuclear translocation of NF-kB [24,25]. In the present study, we were able to show that the functional suppression of the MAPK/ERK pathway diminishes the LPS-induced expression of proinflammatory cytokines in OLENDO cells. This is also consistent with observations made in other studies, in which exposure of immune cells to PAMPs activated the MAPK/ERK pathways and, thereby, production of inflammatory cytokines and chemokines [48]. Moreover, our data demonstrate that LPS induced activation of Erk1/2 phosphorylation was diminished by a MAPK/ERK pathways inhibitor (UO126) without affecting protein expression of ERK1/2 in OLENDO cells. Since, LPS induces phosphorylation of ERK 1/2 and production of pro-inflammatory cytokines such as IL6, and chemokines such as IL8 in OLENDO cells, this strongly implicates the importance of the MAPK/ERK signaling in LPS associated inflammatory effects in luteal endothelial cells. Additionally, in the present study, we were able to demonstrate that the LPS-driven phosphorylation of Erk1/2 is modulated by TLR4. The functional suppression of its activity diminished the LPS-induced phosphorylation of Erk1/2 in OLENDO cells. Therefore, the present study further emphasizes the previously shown interaction between TLR4 and MAPK/ERK pathways in different cell types, e.g., in human monocytes or bovine granulosa cells [10,49]. However, the possible involvement of TLR2 in LPS-mediated activation of MAPK/ERK pathways in OLENDO cells and other cell types also appears interesting and requires further investigation.
Although the importance of the MAPK/ERK cascade in LPS-mediated inflammation was investigated intensively in different cell types, the potential role of other kinases in this process remains to be elucidated. Some information derives from studies with human gingival fibroblast cells, mouse pituitary cells or human bladder epithelial cells and monocytes, in which blocking of the PKA pathway resulted in a decrease in LPS-induced production of cytokines such as IL6, and TNFα [50][51][52][53]. Here, by making use of OLENDO cells, we assessed the potential role of the PKA and PKC pathways in LPS-mediated inflammation in luteal endothelial cells. Similar to the situation observed in human and mouse models [50][51][52][53], we found a significant reduction of the LPS response in OLENDO cells following treatment with the PKA blocker H89. With this, we have shown for the first time the functional involvement of the PKA pathway in regulating LPS induced inflammation in luteal endothelial cells. Another protein kinase family member, PKC, was also implicated in regulating the LPS-and other TLR ligand-induced inflammatory processes in human monocytes, mouse macrophages, dendritic cells, and neutrophils [54][55][56][57]. The blocking of its function induces the production of inflammatory mediators such as TNFα and IL1B in human monocytes [56]. Accordingly, in our experiments, cells were treated with LPS alone or in combination with the PKC blocker and the expression of proinflammatory cytokines was assessed. Notably, the PKC blocker appeared to amplify the gene expression of LPS-induced proinflammatory cytokines. While these findings suggest a modulatory role of PKC in the LPS-inflammatory response in endothelial cells towards increasing inflammation, seemingly contrary to those effects mediated via PKA, it needs to be emphasized that our in vitro observations need further confirmation in vivo.

Conclusions
In conclusion, luteal endothelial cells, represented in our study by OLENDO cells, appear to be involved in the recognition of PAMPs associated with Gram-negative and Gram-positive bacteria in the CL through the expression of TLR1, TLR2 and TLR4. In particular, the TLR2 and TLR4 systems, by mediating the production of pro-inflammatory cytokines, appear to be important players mediating the adverse effects of LPS in CL. The underlying molecular mechanisms, interactions and communication between different signaling cascades need further study as they may reveal important mechanisms underlying the regulation of LPS-induced inflammation, resulting e.g., in disruption of luteal functionality and impaired ovarian cyclic activity in domestic mammalian species and thereby bearing great clinical relevance.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/vetsci9030099/s1. Figure S1: Original unedited image indicating the expression of TLR1 in OLENDO cells (317 bp) by qualitative PCR (M = marker, GAPDH as internal control) used in Figure 2A of the manuscript. Figure S2: Original unedited images indicating p44/42 MAPK (Erk1/2), phosphorylated p44/42 MAPK (Erk1/2) and ACTB for representative western blots used in Figure 7 of the manuscript.  Institutional Review Board Statement: Ethical approval is not applicable, because the research does not contain any studies with human or animal subjects.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.