Expression profile of Toll-like Receptors Pathway Genes in Chicken Erythrocytes Infected with Mycoplasma synoviae

Toll-like receptors (TLRs) are one of the important immunes signaling pathways that participate in the activation of host immune response after detecting microbial pattern molecules. Despite important expression profile TLRs genes in chicken erythrocytes, studies have been lacking. This study investigated the expression profile of TLRs pathway immune gene in chicken erythrocytes in response to Mycoplasma synoviae . The purpose of the current in-vitro study was to determine the chicken erythrocytes interaction with M. synoviae using a transmission electron microscope (TEM). The mRNA gene expression of TLR1, 2, 3, 4, 5, 7, 15, MHC I, II, and MyD88 in M. synoviae infected chicken erythrocytes was determined using quantitative real-time PCR (qRT-PCR) at four different time intervals (0, 2, 6 and 10 h) post-infection and compared to uninfected controls. The mRNA expression of TLRs such as TLR1, 2, 3, 15 , and MHC II were significantly upregulated at 6 and 10 h post-infection in infected chicken erythrocytes. However, significantly upregulated expression of TLR5 and MHC I were noted at 2, 6, and 10 h while TLR4 and MyD88 mRNA expression was also significantly upregulated but at different time intervals. This study provides the first evidence of upregulated expression of TLR signaling pathway genes in M. synoviae infected chicken erythrocytes. These results provide new insights on M. synoviae infection resistance mechanisms and the role of TLR signaling immune genes in the control of the host immune response.


INTRODUCTION
T he erythrocyte is the most abundant cell subset in blood circulation and functions mainly in the exchange and transportation of gases. Erythrocytes are the key in bactericidal cells, which perform pathogens clearance in the bloodstream. In certain species, such as avies, fishes and reptiles mature erythrocytes are nucleated and transcriptionally active . As a result, these erythrocytes contribute to other features of homeostasis such as immune system modulation (Morera and Mackenzie, 2011). Similarly, numerous studies showed that in immunology, an important role is played by chicken erythrocytes Niu et al., 2019). Furthermore, in erythrocytes, numerous toll-like receptors (TLRs) transcripts (TLR2,3,4,5,and 7) were expressed constitutively (Paolucci et al., 2013).
Mycoplasma synoviae is a significant pathogen of domestic poultry that leads to huge economic losses in the poultry industry (Kleven, 2008;Umar et al., 2017). Infection mainly occurs as a subclinical causing respiratory and systemic disease, autoimmune disorders, and infectious synovitis in chickens (Kleven et al., 2003). Mycoplasmas have been reported in many studies as active players in host-pathogen interactions leading to alterations O n l i n e

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in cell death patterns (Obara and Harasawa, 2010). It has been reported that several immune genes are modulated in response to M. synoviae infection in chicken macrophages (Lavrič et al., 2007(Lavrič et al., , 2008. More recently, nonphagocytic cell invasion capability has been found in M. gallisepticum and M. synoviae as another way of host defense evasion and persistence in the host (Much et al., 2002). TLRs, the pattern recognition receptors (PRRs) key family, play an important role in host defense against invading pathogens by prompt innate recognition and inflammatory responses (Akira and Takeda, 2004;Kopp and Medzhitov, 1999). The PRRs play an essential role in the rapid initiation of the host's immune responses and the genetic identification of an invading microbe (Medzhitov et al., 1997;Rock et al., 1998) via recognition of pathogenassociated molecular patterns. TLRs have emerged as a main constituent of the vertebrate PRR repertoire. Upon activation, TLRs induce the expression of an extensive range of effector and immune regulatory molecules (Remer et al., 2003;Thoma-Uszynski et al., 2001) and immune cell types maturation Banchereau et al., 1998;Brightbill et al., 2000;Hertz et al., 2001;Supajatura et al., 2001). Poultry TLRs are mainly located on the cells surface and cytoplasm (Brownlie, 2011), with different distributions, and thus perform different functions. Erythrocytes are one of the most numerous cells in the body, and there are TLRs expressed in the erythrocytes, TLRs are distributed inside and outside the cells. So TLRs play an irreplaceable role in the innate immune response of birds. TLRs have become the focus of biomedical research because these small molecule proteins can be used as one of the primary factors of the host's immune activation (Fitzgerald and Kagan, 2020).
Our earlier studies have also reported that in immunity against thiram induced TD chickens, or virus-infected erythrocytes, the main role had been played by chicken erythrocytes, which may also have a role in apoptosis, prostaglandin pathway especially Niu et al., 2019;Jia et al., 2018;Wang et al., 2018;Qadir et al., 2020a, b). Besides, chicken erythrocytes express TLRs and possess immune-related functions Jia et al., 2018). The current study examined the possibility that the expression of many immune-related genes in chickens erythrocytes respond to M. synoviae which may induce an inflammatory response. Little is known about TLR signaling immune-related genes that expressed in M. synoviae infected chicken's erythrocytes and how M. synoviae affects their genes expressions. Therefore, in the current study the interaction between M. synoviae and erythrocytes was determined. Furthermore, the results revealed the effect of M. synoviae strain on the mRNA expression of TLR signaling immune-related genes in chicken erythrocytes.

Erythrocyte collection
Blood was obtained from specific pathogen-free (SPF) chickens purchased from Longkol Company (Taigu, Shanxi). A total of 4 mL of fresh venous blood from the pterygoid vein of adult SPF chicken was drawn and mixed. To the 4 mL Histopaque-1119 solution (Sigma-Aldrich, Oakville, ON), the diluted blood was carefully added following centrifugation at 2000 r/min for 20 min. Consequently, the leukocytes and platelets were removed from the supernatant. Later procedures were done as described previously by Kabanova et al. (2009). Moreover, pure erythrocytes were obtained by a procedure as described by Niu et al. (2018).

Treatment of mycoplasma
A total of 8 mL of FM-4 mycoplasma culture medium was taken during the log phase (the medium has just turned yellow). The concentration of mycoplasma was about 1×10 6~1 ×10 7 / mL, followed by centrifugation at 12000 r/ min for 15 min, and the supernatant was discarded. After washing twice with PBS, the sample was recentrifuged at 12000 r/min for 10 min. Lastly, cells were then cultured in 98% Dulbecco's Modified Eagle Medium (DMEM) (Solarbio, Beijing, China), added with 2% fetal bovine serum (FBS) and 2% chicken serum (Longkol, Shanxi, China).

Experimental infection of chicken erythrocytes (CER)
Total 50 μL of erythrocytes were obtained from SPF chicken and drawn in sixteen 2 mL centrifuge tube containing a cell maintenance solution and distributed into four groups i.e., 0, 2, 6, and 10 h. The experiment was performed in an Animal Biosafety Level 2 Laboratory. To an experimental group, 100 μL of M. synoviae was added into all the four experimental groups with the addition of 900 μL of DMEM to make the final volume 1050 µL. To another four groups M. synoviae was not added and was designated as control group. The cells were then cultured at 37°C in 5% CO 2 incubator, and each of the respective group was taken out at 0, 2, 6, and 10 h, respectively after centrifugation for 10 min at 2000 r/min. Cell supernatant was discarded and cells were washed three times with PBS and stored in a refrigerator at −80°C upcoming experiments.

Transmission electron microscope (TEM) for interaction between M. synoviae and erythrocytes
Erythrocytes were isolated at 2000 r/min for 10 min and washed 3 times with phosphate buffer, fixed in osmium tetroxide for 2 h at room temperature, and prestained in acetabarbitone for 10 min. After dehydration through the graded ethanol series, the samples were embedded O n l i n e

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in Spurr's resin. Sections were prepared and then stained with uranyl acetate and lead citrate. Finally, the samples were sent to Shanxi Medical University for testing where, the ultrastructure was observed in JEM-1011 (JEOL Ltd., Tokyo, Japan) TEM.
Extraction of RNA, cDNA synthesis and qRT-PCR From both experimental and control groups at 0, 2, 6, and 10 h fresh blood samples were obtained. From obtained erythrocytes of each group total RNA was isolated by RNAiso Plus (9109; Takara Bio Inc., Dalian, China) according to the instructions of the manufacturer. The PrimeScript RT reagent Kit (RR047A; Takara Bio Inc., Dalian, China) was used to reverse transcribe RNA into cDNA, by following the instructions of the manufacturer's recommended protocol. The cDNA samples were then stored at −20°C after dilution at 1:10 in RNase-free water. For the expression analysis of TLR family genes, qRT-PCR was performed using kit of TaKaRa SYBR Premix Ex TaqTM II (RR820A; Takara Bio Inc., Dalian, China) by the QuantStudio™ 6 (Applied Biosystems, America). Primer designing of TLR family genes was done by using Primer Express 3.0 (Applied Biosystems, Foster City, CA, USA) according to NCBI gene coding sequences and manufactured by Shanghai Generay Biotech Co., Ltd. (Shanghai, China). Primer sequences, annealing temperature for this experiment along with accession numbers are shown in Table I. Thermal cycling parameters used for qRT-PCR were previously described by Niu et al. (2018). The expression profile of TLR family genes relative to the housekeeping gene 18S rRNA were calculated by the QuantStudio™ 6 Flex Real-Time PCR System Software (Applied Biosystems, USA).

Statistical analysis
The real time PCR data was calculated using 2 −ΔΔCt method. The data obtained between control and experiment groups for each time point post-infection was employed

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to Two-way ANOVA followed by Tukey's Multiple Comparison test to perform statistical analysis. All graphs were accomplished using GraphPad Prism 5. Significant differences were measured once the P value was P < 0.05, P < 0.01 or P ≤ 0.001.

Interaction between M. synoviae and erythrocytes
The interaction of M. synoviae and erythrocytes is shown in Figure 1B by having endosome. While, noninfected (control) erythrocytes was distributed uniform cytoplasm as shown in control section of Figure 1A.

Expression of TLR pathway genes in chicken erythrocytes infected with M. synoviae
The relative mRNA expression levels of TLRs signaling pathway genes in chicken erythrocytes following infection with M. synoviae were analyzed and presented in Figure 2 to investigate the degree of effect on gene expression in chicken erythrocytes after interaction with M. synoviae. This study determined that the expression levels of TLRs signaling pathway genes were significantly varied at different time intervals such as 0, 2, 6, and 10 h. The qRT-PCR results indicated that relative mRNA expression of TLR4, 5, 7, and MHC I were significantly upregulated at 0 h (P < 0.05), compared to the control group erythrocytes. Moreover, at 2 h, the expression of TLR4, MyD88, MHC I and MHC II were significantly upregulated (P < 0.05). Relative mRNA expression of TLR1, 2, 3, 5, 7, 15, MHC I, and MHC II were significantly up-regulated (P < 0.05) compared to a control group at 6 h. Furthermore, the expression of TLR1, 2, 3, 7 and MHC II in infected erythrocytes at 10 h were also significantly upregulated (P < 0.05) while TLR4 expression was significantly downregulated (P < 0.05) at 10 h compared with the control group.

DISCUSSION
M. synoviae infection emerges to cause considerable financial loses to worldwide poultry producers. M. synoviae infection controls numerous immune genes in chicken macrophages (Lavrič et al., 2007(Lavrič et al., , 2008 and apoptotic genes in chicken chondrocytes (Dušanić et al., 2012). Recent studies indicate that certain M. gallisepticum strains may invade cells, such as chicken erythrocytes and embryonic fibroblasts (Winner et al., 2000;Vogl et al., 2008). Therefore in the current study, we revealed that chicken erythrocytes have an important role in immunity.
The current study results proposed that transcripts for the TLR1, 2, 3, 4, 5, 15, MHC I, MHC II and MyD88, were constitutively expressed in M. synoviae infected chicken erythrocytes. Therefore, this result offers a novel perspective for poultry health because; targeting such TLRs for therapeutic purposes can be one of the ways to defend chickens from infection of mycoplasmas, particularly, M. synoviae. Based on expressed TLRs repertoire, erythrocytes possibly can respond to both bacterial and viral pathogens. Interestingly, the repertoire of TLRs expressed in erythrocytes is identical to that of various kinds of leukocytes (Iqbal et al., 2005).
It has reported from current study that chicken erythrocytes infected with M. synoviae strain respond to the TLRs pathway genes when compared with an uninfected group. This study first reported that mRNA expression of TLRs and other immune-related genes show upregulation at a certain time interval when chicken erythrocytes were infected with M. synoviae strain. In the current study, it was observed the upregulation of TLR1 and 2 at 6 and 10 h indicating that infection severity was enhanced with the O n l i n e

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Toll-like Receptors Pathway Genes Expression in Mycoplasma synoviae Infected Erythrocytes 5 Fig. 2. The expression pattern toll-like receptors pathway genes in chicken erythrocytes on 0, 2, 6, and 10 h. This experimental study group was as follows: the control group (Con, erythrocytes treated with PBS, n = 3) and the experiment group (Exp, erythrocytes infected with M. synoviae, n = 3). Expression levels of TLR pathway genes were relatively calculated to that of 18S rRNA the housekeeping genes via qRT-PCR. SEM (standard error of mean) represented, by error bars. Bars with asterisks indicate a significant (*P < 0.05, **P < 0.01, ***P ≤ 0.001) Up or downregulated relative mRNA expression when compared to uninfected control group.
passage of time which may be due to decreased immunity level of birds so, infection was more severe in later hour. Therefore, such results suggested that M. synoviae have an influential effect on gene expression of TLRs, as in many cases at certain time interval genes expression was significantly upregulated. Previously related to current study similar kind of studies was carried on for the observation of TLR genes expression in chicken in response to Clostridium perfringin infection (Lu et al., 2009), Salmonella and Campylobacter infection (Meade et al., 2009). Therefore previous studies are also in agreement with our study results in regard to upregulation of TLR1 and TLR2 in response to bacterial infections in chickens (Lu et al., 2009). Thus, the observed upregulation of TLR1 and TLR2 may be a direct consequence of initial TLR4 activation followed by self-downregulation (Higgs et al., 2006). TLR3 plays a key role in infected host immune response. The TLR3 role in identifying dsRNA, NF-κB pathway activation and induction of type I IFN production in chicken has been described (Schwarz et al., 2007;Karpala et al., 2008). In this study, the significant upregulation of TLR3 at 6 and 10 h in infected chicken erythrocyte was detected. Interestingly, in previous studies there were no evidences regarding mRNA expression of TLR3 in chicken erythrocytes in response to bacterial infections. Therefore, the altered expression of TLR3 in the current study has been linked to increased responsiveness to bacterial infection. Furthermore, such results suggest that chicken TLR3 is constitutively expressed in infected chicken erythrocytes and may contribute to the innate immune response induction against bacterial infections in in-vitro.
Upon gene expression profile of TLR4 and TLR7 in chicken erythrocytes, results revealed that their mRNA expression was significantly upregulated at 0 and 2 h, post-infection showing that severity of infection is higher at early time interval and decreased with passage of time. These results were in agreement with data reported about upregulation of TLR7 in chicken (Yilmaz et al., 2005). Whereas, significant differential expression of TLR4 helps to detect the capability of different microorganisms' growth and entry in different chicken tissues, therefore an expression of TLR4 activates intracellular signaling via the adaptor MyD88 (O'Neill, 2006). Moreover, in the chicken spleen, the mRNA expression of TLR4 and TLR7 was also reported upregulated in response to bacterial infection (Clostridium perfringen) (Lu et al., 2009), and suggested that TLR4 and TLR7 plays an important role in innate immune response to M. synoviae infection. Thus upregulation indicates that chicken erythrocytes meets criteria necessary to be considered an immunological organ as has been suggested for chicken erythrocytes in general.
TLR5 is activated via bacterial flagellins and is highly conserved in vertebrate species that play an important role in the first-line defense against bacterial pathogens and in immune homeostasis (Faber et al., 2018). The Appropriate function of TLR5 is, thus, essential for the timely immune response activation during many pathogenic bacterial infections (Iqbal et al., 2005). It was determined in current study that in response to M. synoviae strain infection the mRNA expression of TLR5 was significantly upregulated at 0, 2, and 6 h while not significant at 10 h post-infection revealing that severity of infection decreased at later stage of infection. Previously it was reported that TLR5is highly expressed in the spleen, tonsils, lung, kidney, intestine, heart, testis, liver, and immune cells (Iqbal et al., 2005a;Leveque et al., 2003). Therefore, the upregulated expression in chicken erythrocytes may reveal that TLR5 was involved in response to M. synoviae infection. TLR15 is unique in avian species, and its exact function is currently unidentified. In the current study, we examined that TLR15 expression was significantly upregulated in chicken erythrocytes infected with M. synoviae strain at 6 and 10 h post-infection, which was consistent with the previous studies results of TLR15expression in the chickens shown upregulation in response to M. synoviae induced infection at an early stage of infection (Oven et al., 2013). Previously, upregulated expression of TLR15 was reported after stimulation of cells with live and heatkilled Gram-positive and Gram-negative bacteria, usually isolated from chickens, but not with equine specific pathogen Rhodococcus equi, therefore indicating that TLR15 might respond specifically to avian pathogens (Nerren et al., 2010), furthermore, it was also reported previously that TLR15 was highly expressed in the bursa of Fabricius and bone marrow (Higgs et al., 2006). Therefore it is revealed that the erythrocytes have a vital role in mediating responses to M. synoviae infection. Similar to the report of Ciraci et al. (2011), the upregulation of both TLR15 and MyD88 expression in the chicken erythrocytes suggested that the response of TLR15 to M. synoviae may operates in a MyD88-dependent manner.
This study result also revealed the significant upregulation of MyD88 mRNA gene expression in chicken erythrocytes in response to M. synoviae strain. Previously it was reported that MyD88 gene was constitutively expressed on almost all tissues (Hardiman et al., 1997). Likewise, it was shown previously that in the spleen and thymus MyD88 gene was expressed higher, which is consistent with results of mouse and humans (Hardiman et al., 1996(Hardiman et al., , 1997. Previous studies also suggested an important role of MyD88 gene expression in NF-κB activation in-vitro in the chicken innate immune response to bacterial infections (Qiu et al., 2008). Recently it was also stated that all TLR3 employ the MyD88 dependent pathway (O'Neill, 2006). The significant upregulation of MyD88 suggested that this adaptor molecule plays an important role in the TLR signaling pathway and induces an innate immune response to M. synoviae infection.
In current study the MHC mRNA expression in chicken erythrocytes as observed was significantly increased when M. synoviae infected erythrocytes were compared with the uninfected control group which suggests that MHC I and II gene expression has been influenced by bacterial infections. Similar studies were reported formerly that associations of different MHC haplotypes in the chicken are responsible for the actions of vaccination and disease challenges (Briles et al., 1983;Bacon and Witter, 1995). Furthermore, to several microorganisms including bacterial (Joiner et al., 2005) and viral (Bacon et al., 2004;Boonyanuwat et al., 2006) the chicken MHC genes play an important role in disease resistance and susceptibility to bacterial infectious agents.

CONCLUSION
This in-vitro study provided the first evidence that M. synoviae interacts with chicken erythrocytes which can also constitutively express several different TLRs and other immune-related genes. The expression of such TLRs mediated pathway genes can be upregulated in response to M. synoviae infection in chicken erythrocytes at the early phase of infection at different time intervals. Future studies may be planned at exploring the further role of erythrocyte in TLR mediated response in chicken.

Statement of conflict of interest
The authors declared that there is no conflict of interest regarding the publication of this article.