Expression study of pro-inflammatory cytokines and acute phase proteins in post-partum crossbred dairy cows with subclinical endometritis

Expression study of pro-inflammatory cytokines and acute phase proteins in post-partum crossbred dairy cows with subclinical endometritis. J. Vet. Anim. Sci.


Subclinical endometritis (SCE) is one of the causes of poor reproductive performance in dairy cows. The objective of the study was to evaluate the expression of pro-inflammatory cytokines [PICs; Interleukin -1 beta (IL-1β), Interleukin -6 (IL-6), and Tumor necrosis factor-alpha (TNF-α)] and Acute phase proteins [APPs; Serum Amyloid-A (SAA) and Haptoglobin (HP)] in blood and
uterine flushings of postpartum dairy cows with subclinical endometritis (SCE). Animals (n=49) were screened for SCE at 60 days postpartum (dpp) using the endometrial cytobrush cytology, and those with PMN cell percentage ≥ 18% were identified as SCE cows. The blood samples and uterine flushings from SCE (n=15) and the control group (n=15) were assessed for mRNA expressions of PICs and APPs using the Real-Time PCR method (RT-PCR). The PICs and APPs except TNF-α were non-significant (p>0.05) in blood samples whereas a significant difference (p<0.05) was found in uterine flushings. The PICs and APPs were significantly up-regulated in the uterine endometrium indicating that collection of uterine flushings at 60 dpp could be used as a diagnostic method for evaluating gene expressions of inflammatory mediators in dairy cows with SCE.
Keywords: Endometrial cytobrush, pro-inflammatory cytokines, acute phase proteins 450 Evaluation of Pro-inflammatory cytokines and Acute phase proteins ________________________________ Post-partum uterine infection is one of the most frequent causes of infertility in high-producing dairy cattle, which lowers the viability and sustainability of dairy farming. According to reports, 80-100 per cent of cows in dairy herds under intensive management have postpartum bacterial contamination of the uterine environment. Even while the immune system normally eradicates the majority of harmful microorganisms, 15 to 40 per cent of postpartum cows are reported to experience chronic illness (Sheldon et al., 2008). Subclinical endometritis (SCE) is an endometrial inflammatory disease with an increasing number of polymorphonuclear cells in the uterus. It has also been hypothesized that the subsequent onset of SCE may be influenced by an imbalance of pro-inflammatory/antiinflammatory cytokines in the first week postpartum (Sheldon et al., 2006).
Toll-like receptors (TLRs), which are most frequently found in endometrial cells and macrophages, identify the pathogen through pathogen-associated molecular patterns (PAMPs) and release PICs such as IL-1β, IL-6, and TNF-α (Herath et al., 2009). The synthesis of APPs from the liver or other tissues is stimulated by the release of PIC (mostly IL-1β, TNFα-and IL-6) at the site of injury. Acute phase proteins are non-specific innate immune components, which maintain biological process equilibrium and regulate microbial proliferation (Chan et al., 2010). In the present study, the expression of PICs (IL-1 β, IL-6, and TNF-α) and APPs (SAA and HP) in dairy cows with SCE was compared to control animals at 60 dpp.

Experimental animals
The present study was conducted at the Instructional Livestock Farm Complex (ILFC), College of Veterinary and Animal Sciences, Pookode and Livestock Research Station (LRS), Thiruvazhamkunnu. Crossbred dairy cows between 3 to 10 years of age were evaluated for the percentage of PMN cells to diagnose SCE at 60 dpp.

Diagnosis of subclinical endometritis
A total of 49 crossbred dairy cows without any post-partum complications were screened for the detection of SCE based on endometrial cytology. The cytobrush technique (Kasimanickam et al., 2005;Gayathri et al., 2020) was used for collecting endometrial samples from all postpartum dairy cows and a smear was prepared by rolling cytobrush over a clean glass slide. These slides were stained using modified Wright Giemsa and observed under 400 magnification of a light microscope and 200 nucleated cells were counted to estimate the percent of PMN cells. A total of 49 animals were screened, 15 postpartum dairy cows with more than 18 percent PMN cells were selected as positive for SCE (Group I) and 34 animals with less than 18 percent PMN cells were selected as control (Group II).

Sample collection
Blood (2 mL) was collected through jugular venepuncture into EDTA vials (Ultimate, Thrissur), immediately placed in ice and transported to the laboratory within 1 h of collection. The uterine flushings were collected by intrauterine infusion using 50mL phosphate buffer saline and then the fluid was aspirated from the uterus by negative pressure using a 10 mL syringe. Approximately 2 mL fluid was recovered from the uterus, collected in a microcentrifuge tube, placed on ice, and transported to the laboratory within 1 h of collection.

Collection of pelleted cells
Blood samples were centrifuged at 2050 g for 10 min to separate the buffy coat layer into a microcentrifuge tube and RBC cell lysis buffer (Merck, US) 0.5mL was added, incubated for 5 min and centrifuged at 5000rpm for 6 min at room temperature. The supernatant was removed completely and the pelleted cells were mixed with 0.75-1 mL TRIzol TM reagent (Thermo Fischer Scientific, Karnataka) and used for RNA isolation. The lavage collected was centrifuged at 5000 rpm for 6 min at room temperature. The pelleted cells were collected and mixed with 0.75-1mL of TRIzol reagent and used for RNA isolation.

RNA isolation and cDNA synthesis
The RNA was isolated from pelleted cells obtained from blood and uterine flushings using the TRIzol chloroform method. The isolated RNA was checked for its purity and quantity using a Nanodrop spectrophotometer (Thermo Scientific, USA). The quality of the RNA sample was assessed by electrophoresis using 1% agarose gel. It was then used for cDNA synthesis using a cDNA kit (Thermo Scientific, USA).

Quantitative Real-time PCR
The primers of genes for PIC except for TNF-α and APPs were designed using Primer quest and oligoanalyzer using the mRNA sequences downloaded from sequences available in NCBI Databases. Published primers sequences for amplifying TNF-α (Manimaran et al., 2019) were custom synthesized from Sigma Chemicals Co. (St. Louise, M.O., USA). The sequence of the primers and annealing temperature (Tm) used in the present investigation and the size of the PCR amplified products are detailed (Table 1).
The Pfaffl technique was used to compute the fold change of the target genes between the control and SCE groups, and Actin-beta was used as the housekeeping gene for the expression analysis using the following formula:

Statistical analysis
Prism 9 (GraphPad Software, Inc) was used to conduct an independent t test for the statistical analysis of gene expressions in RT-qPCR.

Results and discussion
Gene expressions of PICs and APPs vary according to the inflammatory conditions of the uterus. Average CT values are presented in Table 2. Average CT values of β-actin in blood were 26.58 and 25.10 in control and SCE group, respectively whereas in uterine washings it was 14.33 and 15.27, respectively (p= 0.4 and 0.3). There was no significant difference in the gene expression of β-actin noticed between control and SCE groups. The results showed the stability of β-actin expression in the present study in both blood and uterine washings and acknowledge the gene applicability as a reference (Fig.1). 452 Evaluation of Pro-inflammatory cytokines and Acute phase proteins ________________________________

Fig. 1. Melt curve of β-actin in tested samples
In the present study the mean ± SE of Ct values of IL-1β in the blood and uterine washings were 1.02 ± 0.03 and 38.78 ± 1.74, respectively. The fold change noticed for IL-1β in the blood and uterine washings were 1.31 and 41.5 (Table 3). The endometrial mRNA expression of IL-1β was found to be significant in uterine washings (p<0.05) and it was nonsignificant in blood (p>0.05). Peter et al. (2015) observed IL-1β was highly expressed in SCE as 15-fold and 20-fold at 24-30 and 45-51 dpp in the cytobrush samples from the uterus. The higher fold change noticed in this study might be due to different cut off levels taken for PMN cells per cent. Most of the studies conducted adopted the per cent of PMN cells between five to ten at 60 dpp and hence, the cytokines expression might not be enhanced much as reported by Ghasemi et al. (2012) and Fagundes et al. (2019). Genes of cytokines were highly expressed in the case of PMN cells above a certain threshold (Gabler et al., 2010;Galvao et al., 2011;Ghasemi et al., 2012;Fagundes et al., 2019). According to Roach et al. (2002), IL-1β induced the production of IL-8, which attracted neutrophils and monocytes to the pathogen. The higher number of neutrophils found in the present study might be due to increased expression of IL-1β.
The mean ± SE of IL-6 in blood and uterine washings were 0.94 ± 0.05 and 34.90 ± 1.01, respectively. The gene expression of IL-6 was found to be significant in uterine washings (p<0.05) and it was non-significant in the blood (p>0.05). The fold change observed for IL-6 were 34.54 and 0.99 in the uterine washings and blood, respectively (Table 3). The production of IL-6 was stimulated by various factors like LPS, IL-1β, and TNF-α. In the present study, IL-6 was up-regulated in the uterine washings might be due to the higher expression of IL-1β. The findings of this study were in harmony with Ghasemi et al. (2012) who perceived increased gene expression for IL-6 (30-fold) in the endometrial tissue collected by cytobrush technique with a threshold of more than 18 per cent PMN cells in SCE.
In the present study, TNF-α was upregulated in the blood and uterine washings of SCE cows (p<0.05), respectively. The mean ± SE of TNF-α expression in blood and uterine washings were 1.10 ± 0.14 and 6.63 ± 0.80

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in SCE compared to control animals. The fold change observed for TNF-α in the blood and uterine washings were 1.42 and 7.52 (Table 3). The expression of TNF-α noticed by Ghasemi et al. (2012) was 20-fold in the uterine washings that was higher compared to the present study (7.52-fold), which may be due to changes in the sample collection method, post-partum days, and techniques used for studying gene expression. Endometrial epithelial cells, which are the main targets for bacteria (Herath et al., 2009), could be collected using cytobrush technique however uterine washings were employed in the present study to examine gene expression. These could be the cause of comparatively low expression of TNF-α compared to other cytokines found in this study.
SAA and HP expression change was non-significant in the blood (p>0.05) and it was found to be significant in the uterine washings (p<0.05). The mean ± SE of SAA and HP in the blood were 1.01 ± 0.93 and 1.22 ± 0.18, respectively. The expression of SAA and HP in the blood was 0.93 and 1.68 folds. The mean ± SE of SAA and HP in the uterine washings of SCE animals were 1.94 ± 0.08 and 1.23 ± 0.09, respectively. The fold change noticed for SAA and HP were 1.24 and 1.72 in the uterine  (Table 3). In the present study, HP was significantly up-regulated in the uterine washings of SCE cows compared to SAA. In clinical endometritis, Manimaran et al. (2019) found that the fold change of HP (1.65-fold) was similar to our findings. The expression of SAA and HP was up-regulated in the present study might be due to the increased expression of PIC in the endometrium. Serum amyloid A was released in response to the expression of TNF-α and HP due to IL-6 (Tothova et al., 2014). Hence, increased expression of HP rather than SAA was seen in the current investigation. This could be because IL-6, an inflammatory mediator, is expressed at a higher level than TNF-α. Serum amyloid A may not constantly rise; rather, it only seems to do so in postpartum cows under the circumstances of the accompanying stress and acute uterine infection (Chan et al., 2010). Fig.2 depicts the expression profile in uterine washings.
conclusion Subclinical endometritis is often diagnosed late due to the absence of clinical signs and is commonly detected when AI turns out to be ineffective. In the present study, animals with SCE was diagnosed with more than eighteen per cent PMN cells at 60 dpp. This study showed that endometrial cytology using the cytobrush technique is an accurate diagnostic approach for identifying SCE at 60 dpp. The mRNA expression of PICs except TNF-α and APPs was found to be nonsignificant in blood whereas that of PICs and APPs (IL-1β, IL-6, TNF-α, SAA, and HP) were found to be significant in the uterine washings of post-partum dairy cows diagnosed with SCE. So, evaluating PICs and APPs in uterine washings could be done to detect SCE.

acknowledgement
The financial support provided by Kerala Veterinary and Animal Sciences University is acknowledged.