Characterization and transcription of non-classical class I major histocompatibility complex (MHC) genes in buffaloesAbstract
The objective of the present study was to characterize non-classical class I major histocompatibility complex (MHC) genes in buffaloes and evaluate the expression of these genes in different tissue components of the placenta of buffaloes during pregnancy and in trophoblastic cells after stimulation using lipopolysaccharide (LPS). To do this, DNA was extracted from the blood of buffaloes and was subjected to PCR testing and sequencing of the genes NC3 and MICB. The RNA extracted from the placentome and intercotyledonary region of buffaloes in their first (n = 6), second (n = 6) and third (n = 6) trimesters of gestation was subjected to real-time PCR. Explants were created using the chorioallantoic membrane and two experimental groups were established: control and stimulated with LPS for four hours to evaluate the gene expression profile. Analysis on the sequences obtained showed that the genes NC3 and MICB of buffaloes were homologous with those of cattle, with high similarity in the analysis on the sequence variation pattern. The gene expression analysis showed that the genes assessed were transcribed at stages and in placental tissue that differed from what was seen in cattle. The transcription of these genes varied in the tissues studied, with greater transcription of MICB in the intercotyledonary region over the first third of gestation, while the genes studied in the placentome presented low rates of transcription. The trophoblastic cells of the chorioallantoic membrane stimulated with LPS for six hours did not present non-classic MFC-I transcription alterations. The present study therefore provides additional knowledge regarding the immune regulation of placental tissues of buffaloes.
Keywords:
buffaloes; MHC-Ib; gene expression; placenta; LPS
Resumo
O objetivo do presente estudo foi caracterizar genes não clássicos do complexo principal de histocompatibilidade (MHC) de classe I em búfalas e avaliar a expressão desses genes em diferentes componentes teciduais da placenta de búfalas durante a gestação e em células trofoblásticas após estimulação com lipopolissacarídeo (LPS). Para isso, o DNA foi extraído do sangue de búfalos e submetido a testes de PCR e sequenciamento dos genes NC3 e MICB. O RNA extraído do placentônio e região intercotiledonar de búfalas no primeiro (n = 6), segundo (n = 6) e terceiro (n = 6) trimestres de gestação foi submetido à PCR em tempo real. Explantes foram criados usando a membrana corioalantóide e dois grupos experimentais foram estabelecidos: controle e estimulado com LPS por quatro horas para avaliar o perfil de expressão gênica. A análise das sequências obtidas mostrou que os genes NC3 e MICB de búfalos foram homólogos aos de bovinos, com alta similaridade na análise do padrão de variação de sequência. A análise da expressão gênica mostrou que os genes avaliados foram transcritos em estágios e em tecido placentário diferentes do observado em bovinos. A transcrição desses genes variou nos tecidos estudados, com maior transcrição de MICB na região intercotiledonar ao longo do primeiro terço da gestação, enquanto os genes estudados no placentoma apresentaram baixas taxas de transcrição. As células trofoblásticas da membrana corioalantóide estimuladas com LPS por seis horas não apresentaram alterações não clássicas na transcrição da MFC-I. O presente estudo, portanto, fornece conhecimento adicional sobre a regulação imune de tecidos placentários de búfalos.
Palavras-chave:
búfalos; MHC-Ib; expressão gênica; LPS
1. Introduction
Buffaloes are important components of the livestock industry in many tropical countries, including Brazil, where they are used as working animals and milk and meat producers (Santos et al., 2016). Cattle and buffaloes are susceptible to a similar spectrum of infectious agents. However, buffaloes respond to pathogens in ways that differ from the responses of cattle (Davis et al., 2001). The variables involved in differential susceptibility to diseases need to be understood in order to resolve problems that are exclusive to buffaloes.
Despite considerable progress in defining the genome of buffaloes and in enhancements to selective breeding methods to improve performance (Michelizzi et al., 2010), progress regarding the healthcare of this species has been limited. This can be attributed to the limited information available regarding the mechanisms that regulate immune responses to pathogens.
The major histocompatibility complex (MHC) is one of the most extensively studied regions of the genome in cattle, especially because this region codes the most important proteins regarding the adaptative and innate immune response. The MHC gene function within the innate and adaptative immune response suggests that these genes are associated with both resistance and susceptibility to diseases (Kelley et al., 2005).
In buffaloes, the MHC genes are located in the short arm of chromosome 2 (BBU2p), which is homologous to the bovine chromosome 23 (Rodrigues Filho et al., 2008). The structure and organization of MHC genes in cattle and buffaloes are very similar, with three distinct gene classes recognized (I, II, and III) (Stafuzza et al., 2013). In comparison with other ruminant species, the MHC loci in buffaloes are not well characterized and are based mainly on the locations and molecular characterizations of MHC-II genes (Naskar et al., 2012, Stafuzza et al., 2016). However, few studies describing class I MHC genes in this species have been conducted (Patra et al., 2017).
Class I MHC genes and proteins are classified as either classical or non-classical. Classical MHC-I is expressed in most somatic cells and presents peptides derived from either the host or the intracellular pathogens in cytotoxic T lymphocytes. In contrast, non-classical MHC-I is either monomorphic or oligomorphic and presents limited cell expression (Davies, 2007). In humans, HLA-G is expressed at the maternal-fetal interface and, to a lower degree or conditionally, in other immune-privileged sites, such as the eyes, brain, prostrate and tumors (Carosella et al. 2008, Hunt and Langat 2009, Hunt et al., 2005). Among other functions, non-classical MHC-I can act as a binder to leukocyte receptors, including inhibitory receptors coded by natural killer complexes (Shiroishi et al., 2003, LeMaoult et al., 2004, Parham, 2005).
In addition to a physiological role, non-classical MHC-I is also associated with pathological conditions in humans, including viral and bacterial infections (García et al., 2011, Mir and Sharma (2013) and tumors (Groh et al., 2002, Salih et al., 2002, 2003, Doubrovina et al., 2003). In cattle, expression of non-classical MHC-I during infections caused by bovine papillomavirus (Araibi et al., 2006) and Brucella abortus (Santos et al., 2015) have been described.
Given the low genetic divergence between cattle and buffaloes, their non-classical MHC-I genes are believed to be homologous. However, they may present variation in expression during gestation, thus influencing the maintenance of pregnancy and placental defense mechanisms.
In this light, the objective of the present study was to characterize non-classical class I MHC genes in buffaloes and evaluate the expression of these genes in different tissue components of the placenta of buffaloes during pregnancy and in trophoblastic cells after stimulation using lipopolysaccharide (LPS).
2. Material and methods
2.1. Molecular characterization of non-classical MHC-I in buffaloes
2.1.1. Samples
Blood samples were taken from 15 non-pregnant buffaloes in slaughterhouses in the municipalities of São Luís, Cajari and Viana, state of Maranhão, Brazil, and on a farm on the island of Marajó, state of Pará. The samples were stored in isothermal boxes, under refrigeration, and were sent to the molecular pathology laboratory of State University of Maranhão. There, the material was kept at -20 °C until extraction of gDNA.
2.1.2. gDNA extraction, conventional PCR and optimization of PCR primers
DNA extraction from whole blood samples was carried out using the salt (NaCl) precipitation method (Medrano et al., 1990, modified). A total of 200 µL of TES (Tris HCl 10 mM pH 7.6; EDTA 1 mM; SDS 0.6%) and 5 µL of proteinase K (10 mg/mL) were added, followed by incubation for 2 hours at 55 °C. After incubation, 350 µL of saturated NaCl (5 M) was added, with vigorous shaking. The samples were centrifuged for 30 min at 13,000 xg. The supernatant was transferred to a new tube, in which twice the volume of absolute ethanol was added, followed by incubation at -20 °C for 2 hours. The tubes were shaken and then centrifuged for 30 min at 13,000 xg. The supernatant was discarded, 1 mL of 70% ethanol was added and the tubes were inverted several times. The material was then centrifuged for 5 min at 6,000 xg and the supernatant was discarded. Rinsing with 70% ethanol was repeated and, after discarding the supernatant, the tubes were kept open for 30 min to allow evaporation of residual ethanol. The DNA was rehydrated in 60 µL of TE (Tris HCl 10 mM; EDTA 0.1 mM) and was stored at -20°C until PCR was conducted.
The primers used are described in Table 1. They were developed for non-classical class I MHC (generic MHC-I, MICB, NC1 and NC3) in cattle (Birch et al., 2006; Shu et al., 2012).
To optimize annealing temperatures for the primers, PCR reactions were conducted along a temperature gradient, with the intention of obtaining a single PCR product containing buffalo DNA. To do this, a gradient thermal cycler (Veriti®; Life Technologies) was used, with a block of 96 samples distributed across 12 columns and 8 lines. The annealing temperatures ranged from 54 to 64 °C (54 °C, 56 °C, 58 °C, 60 °C, 62 °C and 64 °C).
PCR was conducted using the GoTaq® Colorless Master Mix kit (Promega), following the manufacturer’s recommendations. The final volume obtained in each reaction was 25 µL. The amplification conditions were 95 °C for 14.5 min, 38 cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 60 s, with a final extension at 72 °C for 3 min.
The PCR products were initially viewed by applying 5 μL of the amplified material to a 2% agarose gel, stained with ethidium bromide, and subjecting the sample to horizontal electrophoresis for 40 min at 90 V, using TBE 1X buffer. Bands were viewed under ultraviolet light and digital images were recorded using L-PIX Image EX equipment (Loccus Biotecnologia, Brazil). Once the desired amplification had been achieved, the PCR products were subjected to purification using the Wizard® SV Gel kit (PROMEGA, USA), followed by sequencing conducted by a specialized company.
2.1.3. Sequencing and data analyses
The sequencing was carried out through the terminal dideoxy method. Chromatograms were inspected and edited using the Chromas Lite 2.1.1 software (Technelysium Inc.), followed by alignment and new inspection using the MEGA 6 software (Tamura et al., 2013) through the MUSCLE algorithm (Edgar, 2004).
Sequences available in GenBank, with their respective accession numbers, were used to determine the differences between Bubalus bubalis and Bos taurus taurus in the genes studied, using the homologous genes MIC and BoLA of the bovine specie, as described in Table 2. The genetic distances and variability indexes within the species and between the species were calculated using MEGA 6, by applying the Kimura-2 parameter model (Kimura, 1968).
List of bovine homologous genes – MIC and BoLA – and GenBank accession numbers, used for alignment, as well as for the calculation of genetic distances and variability.
2.2. Non-classical MHC-I expression in buffalo placenta and stimulation using LPS
2.2.1. Samples
Fragments from the placentome and intercotyledonary region were collected from 18 buffaloes at different gestational stages (six from each trimester). The samples were obtained immediately after slaughter at slaughterhouses in the municipalities of Viana, Cajari and São Luís, state of Maranhão, Brazil. These were stored in an RNA stabilizer solution (RNAlater® Solution, Life Technologies) at -20 °C until RNA extraction. The gestational ages of the fetuses were estimated based on cephalo-coccygeal measurements (from the nape to the base of the tail) and were interpreted following the formula x = 2.5(y + 21) (Arthur et al., 1982), in which x is the variable “gestational age” and y represents apical-caudal length, thus estimating gestational age.
Chorioallantoic membranes were obtained from three intact uteruses during the second trimester of gestation, at these slaughterhouses in the state of Maranhão, Brazil, and were used for experimental stimulation with LPS, as described below.
The experimental protocol was approved by the ethics committee for animal experimentation of the State University of Maranhão (UEMA) (CETEA-UEMA, protocol no. 11/2014).
2.2.2. Chorioallantoic membrane cultures and stimulation using LPS
The intact uteruses, collected immediately after slaughter, were carefully treated with iodized alcohol, followed by dissection with exposure and aseptic removal of the chorioallantoic membrane (CAM). The CAM was kept for 20 min in RPMI 1640 sterile culture medium (Gibco, Invitrogen, CA, USA) containing antibiotic (10,000 U of penicillin and 10,000 g/mL of streptomycin; Gibco). The tissue samples were washed with PBS sterile solution to completely remove antibiotics and were then transferred to a container containing RPMI (Gibco). Small CAM fragments, measuring approximately 1.5 cm in diameter, were removed using sterile scissors. These fragments were placed on a culture plate with 6 wells containing RPMI supplemented with 4 mM glutamine, 1 mM pyruvate, 1 mM of non-essential amino acids and 10% cattle fetal serum (Gibco), and were stimulated in triplicate using LPS (500 ng/mL, E. coli lipopolysaccharide O111: B4, Sigma, L2630, 10 mg), which was dissolved in ultrapure water and kept at -20 °C. The tissue samples were maintained in a heated and humidified incubator at 37 °C with 5% CO2 for 6 hours. The CAM fragments of the control group (with no stimulation using LPS) were inoculated with a sterile and enriched RPMI 1640 medium, and were kept under the same conditions as described earlier.
2.2.3. RNA extraction, cDNA synthesis and quantitative RT-PCR
RNA was extracted using Trizol (Invitrogen; Carlsbad, CA, USA), following the manufacturer’s instructions. RNA purity and concentration were analyzed through spectrophotometry. RNA (1.5 μg) was retrotranscribed into cDNA using the Super Script III First-Strand Synthesis System commercial kit for RT-PCR (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions, for a final reaction volume of 20 μL. The quantitative real time PCR (qRT-PCR) was conducted using 2.5 μL of cDNA (approximately 500 µg/µL), 10 μM of primer pairs specific to each gene and 12.5 μL of SYBR Green qPCR SuperMix (Invitrogen), with a final volume of 25 μL per reaction. The parameters used for the RT-PCR were: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min, using a 7500 real-time PCR system thermal cycler (Applied Biosystems, USA). qPCR was conducted using specific primers for non-classical MHC-I, NC1, NC3 and MICB (Table 1), as described for cattle by Shu et al. (2012). The Ct values were normalized based on the expression of GAPDH and were analyzed using the comparative Ct method (2-ΔΔCt), as described by Livak and Schmittgen (2001). When appropriate, the transcription levels were indicated as the number of mRNA copies per 1,000 copies of GAPDH mRNA. RNA extracted from macrophages that were derived from peripheral blood monocytes (which were obtained from a mixed-breed bull of 3 years of age as previously described by Macêdo et al. (2013) was used as a positive control for amplification of class I MHC (Santos et al., 2015).
The cDNA obtained was also subjected to automatic sequencing and was compared with gDNA, as described in subitem 2.1.2.
2.2.4. Statistical analysis
All the normalized Ct values were logarithmically transformed and the data thus obtained were subjected to analysis of variance (ANOVA). The mean values of the groups encompassing each trimester were compared through the Student-Newman-Keuls test (SNK), while the mean values for the different placental tissues from each trimester were compared by applying Student’s t-test (GraphPad Prism 5.0, USA). Differences were considered significant when p < 0.05.
3. Results
3.1. Characterization of non-classical MHC-I in buffaloes
In the optimization of the annealing temperature of the PCR primers (generic MHC-I, MICB, NC1 and NC3) using a temperature gradient, only the genes NC3 and MICB presented amplified products, with fragments of approximately 300 bp and 200 bp (Figure 1). Temperatures of 60 °C (NC3) and 54 °C (MICB) were the most suitable for generating cleaner and more robust PCR products (Figure 2).
Specific amplification products from non-classical class 1 MHC (NC3 and MICB). 1 and 2: samples with amplified gene NC3; 3 and 4: samples with amplified gene MICB; NC: negative control; Kb: molecular weight marker.
2% agarose gel, stained with ethidium bromide, presenting PCR products (200 bp) with buffalo DNA, in a PCR experiment using a gradient of annealing temperatures (54 °C to 64 °C) with primers for the gene MICB. NC: negative control; Kb: molecular weight marker.
A fragment of 150 bp was obtained after alignment and editing of the sequences of the gene MICB (Acession number PQ561601; PQ561602; PQ561603; PQ561604). This fragment presented one polymorphic site for buffaloes, three sites for cattle and four polymorphic sites in comparing buffaloes and cattle. These data represent a genetic distance of 0.0005 for the gene MICB, between the species analyzed (Bubalus bubalis and Bos taurus taurus). Amplification of cDNA resulted in a sequence of 120 bp, thus indicating that there were no intronic regions for the fragment studied.
A fragment of 319 bp was obtained for the gene NC3, presenting variable sites among the buffalo sequences analyzed: 28 sites when comparing only cattle sequences, and 57 sites when comparing buffaloes with cattle. Regarding genetic distance, the sequences showed 0.002 among buffaloes, 0.046 among cattle and 0.166 between species. These differences between cattle and buffaloes were located especially in a region that presented two insertions. The first insertion consisted of one base pair and the second, 3 bp. The variation region and variable sites are represented in Table 3.
The fragment obtained using cDNA for NC3 presented 130 bp, corresponding to the second half of the gDNA sequenced region. The homologous area did not present any excision regions regarding RNA synthesis.
3.2. Expression of non-classical MHC-I in buffalo placenta
To validate the PCR protocol applied, non-classical class I MHC genes (MICB and NC3) were amplified using macrophages derived from monocytes of buffalo blood. These resulted in relative transcription levels corresponding to 15 and 9 copies of mRNA per 1,000 copies of GAPDH, respectively. To determine the expression of non-classical MHC-I in buffalo placenta during pregnancy, qRT-PCR analysis was conducted using total RNA extracted from the placentome and the intercotyledonary region to amplify the genes MICB and NC3.
The expression of MICB differed significantly in the first third of gestation between the different regions of the cattle placenta (p < 0.05), with higher expression levels found in the intercotyledonary region than in the placentome. In the former, MICB expression was greatest during the first trimester, with significant differences compared with the second and third trimesters (p < 0.005). In the placentome, this gene presented consistently low expression throughout the gestational period (Figure 3).
Non-classical class I MHC expression (MICB) in the placentome and intercotyledonary region of each gestational trimester (first, second and third thirds of gestation). Columns represent the arithmetic mean (n = 6) and standard error. * Indicates a statistically significant difference in the transcription of MICB in different placental tissues over the same trimester (p < 0.05); a, b: Different letters indicate statistical difference between the different gestational thirds (p < 0.005); Letters that are the same do not differ between each other significantly.
The NC3 gene presented comparable expression levels in both tissues, although these levels were low. There was no statistically significant difference among the gestational thirds (Figure 4).
Non-classical class I MHC expression (NC3) in the placentome and intercotyledonary region of each gestational trimester (first, second and third thirds of gestation). Columns represent the arithmetic mean (n = 6) and standard error.
To investigate whether the transcription of non-classical MHC-I in buffalo placenta is associated with the capacity of the placenta to respond to stimulation using LPS, the fragments from the second trimester grown in CAM were stimulated and the transcription of MICB and NC3 was assessed. The qRT-PCR results showed that the genes evaluated were not greatly sensitive to stimulation using LPS, with no significant alteration in RNAm expression noticeable (Figure 5).
Transcription of non-classical class I MHC genes (MICB and NC3) in buffalo trophoblastic cells grown in CAM for 6 hours after stimulation using LPS. The variations in the levels of transcription of each gene assessed were measured in relation to the non-infected control group. Columns represent the geometric mean and standard error (n = 3). No statistically significant difference was observed (p > 0.05).
4. Discussion
4.1. Characterization of non-classical MHC-I in buffaloes
This was the first study to characterize non-classical class I MHC genes in buffaloes. MHC genes in buffaloes had previously been characterized regarding classical MHC of classes I and II (Sakaram et al., 2010; Stafuzza et al., 2013, 2016; Patra et al., 2017). Primers named Bov 7 and 11 were used to amplify generic MHC-I (Birch et al., 2006), thus amplifying a sequence of classical MHC-I and non-classical alleles of locus NC1. Specific primers were used to amplify bovine non-classical MHC-I, NC1, NC3 and MICB (Shu et al., 2012). However, the generic MHC-I and the NC1 genes did not present any amplified product. In cattle, classical MHC class I genes with high polymorphisms have been described (Bhushan et al., 2007). In buffaloes, Patra et al. (2017) conducted characterization of exons 2 and 3 of classical class I MHC genes, through PCR-RFLP and sequencing, and observed high polymorphism among the sequences, which ranged from 713 to 715 bp, due to addition or deletion of bases. However, the levels of polymorphism in the genes evaluated in the present study are, so far, unknown. It can be suggested that failure to amplify a product may reflect polymorphism at a primer site, thus allowing the possibility that generic MHC-I and NC1 genes are present in all haplotypes of buffaloes.
Stafuzza et al. (2013) used high-resolution mapping to establish the general organization of the MHC region of the buffalo genome, with allocation of 53 markers from classes I, IIa, IIb and III. These authors built a comparative map of buffalo and cattle MHC and indicated homologous segments between the two species.
Genetic diversity indexes obtained through the present study showed that MICB and NC3 markers presented low variability within the population studied (Bubalus bubalis). The results found in the present study are similar to those described by Birch et al. (2006), who stated that non-classical MHC-I genes are well conserved in humans, rats and cattle. Non-classical class I MHC genes have been described in bird species (Drews et al., 2017; Zeng et al., 2016) and low rates of genetic diversity have been shown, as reported by Biedrzycka et al. (2017). This last author reported the occurrence of five different alleles with deletion of 3 bp in four cDNA samples from birds (Acrocephalus schoenobaenus).
Despite the small amplified fragment, the sequence obtained for the MICB gene was better conserved in buffaloes than in cattle, with a small genetic distance (0.0005) between the species analyzed (Bubalus bubalis and Bos taurus taurus). This demonstrates the similarity of this gene between the two species, and it justifies the use of primers described for cattle in buffalo samples. However, a more in-depth study is needed regarding this gene in buffaloes and its potential for variation in other regions of the buffalo genetic code.
On the other hand, the gene NC3 presented greater genetic distances among the buffalo studied (0.002), among the cattle sequences inserted (0.046) and in comparing buffaloes with cattle (0.166). Although the differences in the gene that were found between buffaloes and cattle were located in a specific region of the NC3 gene sequence with two insertions, the primers that were used annealed in conserved regions of the gene, thus validating their use for buffaloes.
Evaluation of genetic distances between and within populations is important because this information can be used to guide strategies regarding crossing, conservation, genetic resources and detection of genetically important diseases (Horin et al., 1999). Moreover, this also transforms all the available information on the populations assessed into a single number (Serrano et al., 2004).
The lower variability indexes found for MICB and NC3 in buffaloes, in comparison with cattle, may be an indication of possible natural selection in this region, with a tendency to fix alleles. For non-classical MCH genes, this situation has been investigated and it has been demonstrated that over the evolutionary process in humans, some alleles may temporarily increase in frequency as a response to the presence of specific pathogens (Hedrick, 2002). Moreover, the evolution of MHC-I has occurred differently among mammalian groups, which has allowed specific resistance to pathogens across different lineages, such as in bats (Ng et al., 2016). Thus, the present data suggest that there is a need to investigate the relationship between resistance to brucellosis and low variability of the genes MICB and NC3 in buffaloes.
4.2. Expression of non-classical MHC-I in buffalo placenta
In the present study, it was sought to understand how non-classical MHC-I molecules participated in maintenance of buffalo gestation and in pathological conditions. To do this, molecular techniques were used to assess the expression of these genes in different placental tissues over the course of pregnancy and after stimulation using LPS. The expression of these genes has been assessed previously for cattle placenta (Santos et al., 2015; Shu et al., 2012; Davies et al., 2000, 2006), but the present study was the first to evaluate the transcription of non-classical class I MHC in buffaloes in different regions of the placenta over the course of gestation.
The results obtained from the present study demonstrated that the MICB gene was expressed at high levels only during the first trimester in the intercotyledonary region of buffaloes. In mice, non-classical MHC-I is expressed in trophoblastic cells to inhibit activation of NK cells, thus causing lysis in fetal cells due to low expression of classical MHC molecules (Bulmer et al., 1984; Heemskerk et al., 2005).
The placentome showed extremely low levels of MICB and NC3 gene expression. Similar results were reported by Santos et al. (2015), who evaluated the expression of these genes in bovine placentomes and observed low levels of RNAm in the tissue studied. Low expression of genes relating to innate immunity has been reported in the placentome (Silva et al., 2012), thus demonstrating that this region tends to present an immunosuppression condition compatible with pregnancy.
Evaluation of MICB expression over the three gestational periods (1st, 2nd and 3rd trimesters) showed that there was high expression in the first trimester, followed by significantly lower expression in the second and third trimesters (p < 0.005). Similarly, Peng et al. (2011) and Shu et al. (2012) observed low MICB expression in cattle placental tissue that was collected during the peripartum.
Although MIC has an important effect on immune responses during human pregnancy, there are currently no functional data regarding buffaloes and cattle. Similarity in terms of genomic location, gene structure and limited profile expression among humans, cattle and buffaloes suggest that MIC has equivalent function among these species (Trowsdale, 1995; Birch et al., 2008; Santos et al., 2015). Since MIC acts as a binder for natural killer cells through a G2D receptor (NKG2D), it activates NK cells to eliminate cells that are either infected by pathogens or are neoplastic cells, while protecting normal ones from cell death (Mistry and O’Callaghan, 2007; Lanier, 2015).
The gene NC3 presented low expression at all stages of pregnancy in the intercotyledonary region of the buffalo in the present study, unlike what was described by Santos et al. (2015). The latter author observed a varying pattern of expression over the course of gestation among cattle, with high NC3 expression levels over the whole period, especially the second third. Shu et al. (2012) showed that there was high expression of this gene in bovine placental tissues over the last trimester of gestation. These authors suggested that this gene might act as an inhibitor of maternal immune response and fetal autoimmunity. Moreover, the differential transcription of classical genes is believed to probably act synergistically with non-classical NC3, as an immunological protection mechanism for the pregnant cow. This result highlights the differentiated behaviors of the gene NC3 between buffaloes and cattle.
Shi et al. (2018) analyzed the mRNA expression of transcription factors associated with MHC-I expression in placental trophoblastic cells (PTC) and in peripheral blood mononuclear cells (PBMC). In their study, they observed that the MHC-Ib expression in PTC was 4.5 times greater than what was found in PBMC, and that it was regulated through methylation of intragenic DNA of the gene IGF2 in cattle.
As measured through qRTPCR, the non-classical MHC-I expression remained at normal levels after exposure to LPS for 6 hours. The transcription level of mRNA in MICB and NC3 was not affected by this treatment. These findings are in agreement with investigations conducted by Tobian et al. (2003), who evaluated the expression of MHC-I and II in macrophages of mice stimulated with PAMPS, including LPS. These authors observed that the cell surface expression of MHC-I was unaltered after treatment with components of Mycobacterium tuberculosis (MTB) or other PAMPs that inhibit MHC-II expression.
Previous studies have shown that LPS increases the levels of inflammatory molecules, such as cytokines and chemokines, as a response to pathogenic agents that cause various infectious diseases in cattle (Sakemi et al., 2011; Fu et al., 2014). LPS is also crucial for activation of defenses against bacterial infections, through stimulating chemokines to trigger an inflammatory response (Moser et al., 2004; Schulz et al., 2002). These studies are also in agreement with results that have shown that LPS significantly enriches some biological processes, such as cell chemotaxis, regulation of inflammatory response, regulation of the immune system process, cell response to bacterial molecules and response to external stimulus. Cario et al. (2000) also stated that LPS induced a pro-inflammatory response, through activating different pathways in epithelial intestinal cells and regulating the expression of mRNA.
HLA-G (human leukocyte antigen G), a non-classical MHC-I molecule, is a binder for receptors that inhibit the natural killer complex (Shiroishi et al., 2003; LeMaoult et al., 2004; Parham, 2005). It probably acts similarly to the bovine leukocyte antigen (BoLA). Santos et al. (2015) evaluated the expression of non-classical MHC-I in trophoblastic cells of cattle after infection by B. abortus and observed increased expression of these genes. These authors stated that the increased expression of these genes inhibited the action of NK cells, thereby preventing an early innate immune response towards B. abortus. However, although B. abortus is a Gram-negative bacterium, this process is triggered by a type IV secretion system, coded by operon virB (Mol et al., 2014).
In conclusion, the present study demonstrated that there is homology between non-classical MHC-I genes in buffalo and cattle. The expression profile of these genes varies in different components of buffalo placental tissue during gestation. Buffalo chorioallantoic membranes stimulated with LPS did not show altered non-classical MHC-I expression, which could have implications in the pathogenic mechanisms of this PAMPS.
Acknowledgements
This study was financed by the Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) (UNIVERSAL-00710/14).
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