Quercetin Promotes the Expression of Genes Involved in Phagocytosis in Bovine Neutrophils

Corresponding Author: Phongsakorn Chuammitri Department of Veterinary Biosciences and Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand E-mail:phongsakorn@gmail.com Abstract: This study was conducted to explore the effect of Quercetin (QH) on phagocytosis of bovine neutrophils. Neutrophils were isolated from fifteen multiparous Holstein cows. We in vitro treated neutrophils with PBS or 50 μM of Quercetin (QH) or Cytochalasin B (CytB), as phagocytosis inhibitor, prior to monitoring phagocytosis of Escherichia coli by flow cytometry and microscopic examination. Additionally, the expressions of CORO1A, CYBA (gp91 phox ), LAMP1, RAB7A, RAC1 and PAK1 mRNA were analyzed by real-time PCR. In the time-course experiment, treated neutrophils were allowed to co-cultured with live bacteria for 30, 60 and 90 min before measuring gene expressions. The expression levels of IL-1 β and TNF genes in order to demonstrate the antiinflammatory property of quercetin were assessed by conventional RTPCR. The results of flow cytometry and microscopic examination showed that the percentage of neutrophils performing phagocytosis were significantly higher in QH group in comparison with other groups. We reported here that mRNA expressions of CORO1A, CYBA, LAMP1, RAB7A, RAC1 and PAK1 genes involved in phagocytosis, were significantly up-regulated in QH group. As expected, CytB group had profound down-regulation of genes with one exception in PAK1. From our observations, the expression levels of phagocytic process genes in the QH group were optimum at 60 min and started to decline at 90 min of incubation. The data also indicated that quercetin inhibited inflammation by reducing the expressions of both IL-1β and TNF. In conclusion, the information in our experiments conclude that quercetin has the ability to boost the expression of genes involved in phagocytosis while reduces the expression of proinflammatory genes.


Introduction
The rapid and efficient innate defense mechanisms to resist microbial invasion may contribute to the overall health and well-being of human and animals. In innate immunity, neutrophils are actively phagocytic cells performing host defense through a variety of effector functions, i.e., Reactive Oxygen Species (ROS) generation, phagocytosis, degranulation and Neutrophil Extracellular Traps (NETs) release (Amulic et al., 2012;Brinkmann et al., 2004;Chuammitri et al., 2015). Neutrophil phagocytosis is a process, in which a large particle, such as bacteria, immune complex or apoptotic cell is being intake into this cell (Esmann et al., 2010;Grinberg et al., 2008). Innate immune cells; macrophages, neutrophils and dendritic cells (DCs) possess this effector function. Phagocytosis of human Polymorphonuclear leukocytes (PMNs) was assisted by the Fc receptor and Complement Receptors (CRs) led to the expression of many involving genes in cytokine production and signal transduction (Kobayashi et al., 2002). The gene expression patterns differ when phagocytosis is examined during the 90 min-interval as the genes were either induced or repressed (Kobayashi et al., 2002). The available information stated that gene expression and cell signaling pathways involved in phagocytosis, AMP-activated Protein Kinase (AMPK), may result in anti-inflammatory in phagocytosis of E. coli, as well as, the changes in cytoskeletal reorganization (Bae et al., 2011). The AMPK signaling also stimulated PAK1/2 genes and moreover the RAC1 gene that helped reduce the expression of other genes involved in the inflammatory process, such as TNF-α in neutrophils via the induction of Toll Like Receptor 4 (TLR4) (Bae et al., 2011). The microarray study of human PMN gene expression during bacterial Phagocytosis revealed the global changes of many key gene transcripts (Kobayashi et al., 2003). The genes in associated with the inflammatory response and apoptosis regulators i.e., IL-1α, IL-1β, IL-1ε, IL-1RN, IL-8, IL-10, IL-12β, IL-15, TNFα, Vascular Endothelial Growth Factor (VEGF), OncostatinM (OSM), IL-6, GROβ and GROγ were up-regulation as shown and reviewed by (Kennedy and DeLeo, 2009;Kobayashi et al., 2003;. At present, the role of herbs and natural substances classified as one option to help strengthen the immune system of cattle. One of spotlight herbal phenolic substances is quercetin, which is found in many edible plants, fruits and vegetables (Erlund, 2004;Formica et al., 1995). The bioavailability studies of quercetin and its metabolites; aglycone and rutin, in cows demonstrated that quercetin can be absorbed via digestive tracts and eventually entered the plasma (Berger et al., 2012;Gohlke et al., 2013;Maciej et al., 2015). Several in vivo studies of quercetin in neonatal calves, periparturient and lactating cows, in the aspects of bovine health and production, have been recently reported (Gohlke et al., 2013;Maciej et al., 2015;Stoldt et al., 2015); however, another point of view remains scarce in bovine innate immunity.
Many published works have emphasized that quercetin also exerts its effects by promoting and acting as an antioxidant and anti-inflammation (Boots et al., 2008;Cho et al., 2003;Chuammitri et al., 2017;De Marchi et al., 2009;Nair et al., 2006). In fact, quercetin affects the mRNA gene expressions and associated proteins in the innate immune cells, such as the reduction of the IL-1β mRNA level during inflammation was documented (Valerio et al., 2009). We have previously demonstrated the in vitro effects of quercetin on bovine neutrophil phagocytosis of Escherichia coli (Chuammitri et al., 2015), but in great detail study of how gene cascades in bovine neutrophil phagocytic processes were regulated due to proposed quercetin effects is incompletely elucidated at molecular levels. To our knowledge, little is known about the action of quercetin on the dynamic of gene expressions during bacterial phagocytosis of bovine neutrophils. To continue examining the immunomodulatory action of anticipating quercetin effects, this study aims to investigate how quercetin alters the expressions of key phagocytosis genes; CORO1A, CYBA (gp91 phox ), LAMP1, RAB7A, RAC1 and PAK1 during bacterial phagocytosis.

Ethical Approval
The animal experimental procedure was approved to be conducted under the Animal Care and Use Ethics Committee (FVM-ACUC Ref. No: S5/2557).

Animals, Blood Collection and Bovine Neutrophil Isolation
Healthy, multiparous Holstein cows during nonlactating period were used as blood donors (Jinghui et al., 2014;Martinez et al., 2014). A total number of 15 cows in 3 independent experiments, 5 cows each, were recruited in the study. All dairy cows were housed at local dairy farms of Mae On district and Mae Hia Agricultural Research, Demonstration and Training Center. Whole blood was collected by jugular venipuncture into a sterile syringe containing 1× ACD solution. Neutrophil isolation protocol was previously described (Chuammitri et al., 2015). Finally, cell density was adjusted to approximately 2×10 6 cells per ml in RPMI 1640 medium supplemented with 1% FBS.

Quercetin
Stock Quercetin solution at 5 mM was prepared by dissolving dry quercetin powder in 95% ethanol and filter sterile. Working solution at 50 µM concentrations was freshly prepared on the day of the experiment, protected from light and stored at room temperature until use. The 50 µM of quercetin showed no in vitro cytotoxicity to bovine neutrophils as previous report (Chuammitri et al., 2015).

Escherichia coli Propagation and Opsonization
Escherichia coli (E. coli) was isolated from clinical mastitis cow. The bacteria were freshly prepared by colony picking method and propagation in Luria-Bertani Broth (LB broth, Caisson Laboratories, North Logan, UT, USA).The bacterial number was adjusted to approximately 10 8 CFU/ml. Live E. coli was opsonized with 10% heat-inactivated normal bovine serum for 20 min at 37°C prior to use in the experiment.

Fluorescent Staining of Bacteria
Fluorescent E. coli was prepared by growing bacteria to log phase, heat killed (70°C, 60 min), washed with PBS and centrifuged (12,000 rpm, 5 min). Bacterial DNA pellet (10 8 CFU/ml) was stained with 5% Propidium Iodide (PI) in PBS and incubated at room temperature in the dark for 60 min (Keogh et al., 2011).
PI labeled E. coli was adjusted to 10 6 CFU/ml with PBS and stored at 4°C until use. Fluorescent E. coli was opsonized with 10% heat-inactivated normal bovine serum for 20 min at 37°C before using in phagocytosis assay.

Enumeration of Phagocytosis of E. coli by Bovine Neutrophils under Light Microscope
Neutrophils (2.5×10 4 neutrophils) were seeded into duplicate wells of a 96-well, flat cell culture plate. Cells were treated as previously described in phagocytosis assay. Next, live, opsonized fluorescent E. coli at MOI of 10 were then added to the cells, centrifuged and incubated for 45 min. Cytospin slides were prepared from an aliquot of neutrophils after phagocytosis and seeded onto circular coverlips (15 mm diameter) that were placed into a 24-well plate. Slides were fixed with absolute ethanol, stained with Dip Quick and examined with a Zeiss Axio Scope A1 (Carl Zeiss, Thornwood, NY, USA). Percentage of phagocytosis with different treatments were manually counted from 5 random fields (100 cells per field) at 10× objective by three well-trained observers in a blinded technique (Yan et al., 2012).

Gene Expression Studies by Quantitative Real-Time Reverse Transcription (qRT-PCR) and
Semi-Quantitative RT-PCR Neutrophils (1×10 6 cells) were seeded into duplicate wells of a 24-well cell culture plate. Then, cells were treated with PBS, quercetin, or CytB for 30 min before co-cultured with 10 MOI of opsonized E. coli in serum-free RPMI-1640 medium for another 45 min. After incubation, cells from two wells were combined, washed and spun. Neutrophil pellet was resuspended in RNAlater. In the time-course experiment, the assay setting was nearly identical to the above stated procedures, but with some modifications. In particular, neutrophils were allowed to co-cultured with opsonized E. coli for 30, 60 and 90 min at 37°C, 5% CO 2 , respectively.

RNA Extraction and Complementary DNA (cDNA) Synthesis
Total RNA was extracted and purified by NucleoSpin RNA (Macherey-Nagel, Bethlehem, PA, USA) according to the provided protocols. Complementary DNA (cDNA) was reverse-transcribed from a starting amount of 2 µg of total RNA by Tetro cDNA Synthesis Kit (Bioline, Taunton, MA, USA) and stored at -20°C until further use.

Primers for qRT-PCR and Semi-Quantitative RT-PCR
Primers for real-time PCR; CORO1A, CYBA, LAMP1, RAB7A, RAC1, PAK1 and ACTB as endogenous control (Shirasuna et al., 2015) were designed using Primer3 Plus. Primers for IL1B, TNF and GAPDH were previously reported (Chuammitri et al., 2015). The PCR primers were synthesized by Macrogen, Seoul, Korea. The sequence information of oligonucleotide primers used in this study is indicated in Table 1.

Quantitative Real-Time Reverse Transcription PCR (qRT-PCR)
The qRT-PCR was performed to investigate the effect of quercetin on phagocytosis related gene expressions in bovine neutrophils. The quantification of mRNAs was performed in triplicate on ABI 7300 Real-Time PCR System equipped with SDS Software v1.4 (Life Technologies). The qRT-PCR was performed on 100 ng cDNA using SensiFAST SYBR Hi-ROX Kit (Bioline) as per manufacture's instruction. Subsequently, specificity was confirmed by dissociation curve analysis (T m ). Correct product sizes were also determined by 2% agarose gel (Chuammitri et al., 2017). Analysis of relative gene expression was calculated from the C t of the gene of interest (target) and ACTB. The expression levels (fold difference) were reported using 2 -∆∆Ct method (Livak and Schmittgen, 2001).

Semi-Quantitative Reverse Transcription PCR (RT-PCR)
Semi-quantitative RT-PCR was performed to investigate the effect of quercetin on IL1B and TNF, as pro-inflammatory cytokine gene expressions in bovine neutrophils after phagocytosis and time-course induction of phagocytosis. PCR reactions were performed using MyTaq HS Red Mix (Bioline). The annealing temperature at 59°C (IL1B and GAPDH) or 57°C (TNF) was set. The amplified PCR products were electrophoresed in 1.5% agarose gel. Gel band intensities were quantified by Gel Analyzer Options in ImageJ version 1.46r (Chuammitri et al., 2015). Expression levels are represented relative to GAPDH expression.

Heat Map of Gene Expressions
Gene expression patterns from the average expression level of each gene presented in the form of a heat map of an average linkage method with Euclidean distance. Heat map was generated by Genesis. 1.7.6 (Sturn et al., 2002).

Data Analysis
The data were first screened for potential outlier by robust Regression and Outlier Removal (ROUT) methods. The normality test was done by the D'Agostino-Pearson omnibus test prior to performing the unpaired twotailed Student t test, one-way ANOVA or Kruskal-Wallis Test and two-way ANOVA. The statistical analysis was considered significant when p<0.05. The results are reported as mean with standard error (mean ± SE). Information obtained from the statistical analyses is represented as graphs generated by GraphPad Prism (GraphPad software, San Diego, CA, USA).

Neutrophil Phagocytosis of E. coli by Flow Cytometry
The ability of neutrophil phagocytosis under the supplementation of quercetin and phagocytosis inhibitor can be reflected by the amounts of fluorescently labelled bacteria being ingested or Median Fluorescence Intensity (MFI) as determined by flow cytometry. Neutrophils treated with quercetin was found to have the highest MFI when compared with PBS and CytB group ( Fig. 1B

Phagocytosis under Light Microscope
The mean percentages of phagocytosis as depicted in Fig

Gene Expressions by Real-Time PCR
We have previously reported that bovine neutrophils responded well for phagocytosis when supplemented with 50 µM quercetin (Chuammitri et al., 2015). Here we further explored the effects of quercetin on the levels of gene expressions during and at the end of phagocytosis of E. coli. The results suggested that the expression of genes involved in that process after neutrophils has been supplemented with PBS, quercetin and CytB for 30 min and cells were allowed to intake opsonized bacteria for another 45 min. The measurable gene expression after 45 min-interval on real-time PCR showed the expression of all tested genes; CORO1A (p<0.0001), CYBA (p <0.0001), LAMP1 (p = 0.0002), RAB7A (p = 0.0001), RAC1 (p<0.0001), PAK1 (p = 0.4464), in the quercetin group had higher levels than that of either control (PBS) or CytB supplemented groups. Although the expression level of PAK1 in the quercetin group was higher when compared to the control group, but the expression of this gene in CytB has increased as well, Fig. 3A. Unsupervised hierarchical clustering confirmed that the gene expression profile of quercetin-treated neutrophils helps increase the phagocytic activity Fig. 3B.

Time-Course of Gene Expressions
As reported earlier in this section that quercetin enhanced genes the expression of the phagocytic process at the determining time (45 min), we asked that the dynamic of genes involved in that process may depend on contact time with quercetin treatment. Neutrophils were fortified with PBS or quercetin for 30, 60 and 90 min of allotted time. The expression of all studied genes after receiving quercetin have changed dramatically in quercetin as measured by real-time PCR Fig. 4. The data show that cells in the quercetin group had higher expression levels of CORO1A, LAMP1, RAB7A and RAC1 compared to the control group at 30 and 60 min of phagocytosis with the highest levels at 60-min Fig. 4A. The expression levels of aforementioned genes in the quercetin group were declined significantly in 90-min time. The gene expression data of CYBA and PAK1 from cell treated with quercetin demonstrated the different patterns. The maximum expression of these two genes was found to be significantly increased at 30 min of incubation, but the levels were gradually decreased at 60 and 90 min-time, respectively Fig. 4A. A heat map of gene expression in quercetin groups at 30 to 60 min-time also depicted the increased in phagocytic activity, but later declined at 90 min-time Fig. 4B.

Pro-Inflammatory Gene Expressions
Effect of quercetin to reduce pro-inflammatory gene expressions in neutrophils being phagocytose E. coli was measured by reverse-transcription PCR. The expression of IL1B and TNF revealed a significant reduction of both IL1B (p = 0.0004) and TNF (p = 0.026) by quercetin compared with control Fig. 5.

Discussion
Bovine neutrophils ingest bacterial pathogens by a process known as phagocytosis and the ingested microbes are destroyed by the combination of armors equipped within these cells. The present work has emphasized bovine neutrophil phagocytosis of E. coli under quercetin supplementation. Our results demonstrate that quercetin mitigates the expression levels of genes associated with inflammation, including IL-1β and TNF. Those results are consistent with the report that quercetin lowers the level of proinflammatory gene expression as a consequence of oxidative stress and different stimuli (Chuammitri et al., 2015;Valerio et al., 2009). Accordingly, the antiinflammatory action of quercetin on TNF-α is dosedependent manner (Nair et al., 2006).
Based on the results of recent publication by Chuammitri et al. (2017), it could be considered that quercetin might exert its anti-inflammatory effects of mitigating of either proinflammatory genes (IL1B, IL6, TNF) or microRNAs (MIR24-2, MIR146A and MIR181C). Our results in a reduction of IL1β and TNF expressions by action of quercetin agreed with aforementioned study. Even though the two experiment settings used quercetin at 50 µM final concentration, the course of inflammatory processes might originate from diverse pathways. In fact, both bacterial cells (in the current study) and cell wall component (LPS) have one thing in common to initiate inflammatory signals, nevertheless, quercetin could possibly lessen proinflammatory gene expressions via inhibition of key transcription factors (Boesch-Saadatmandi et al., 2011). These phenomena could emphasize the important property of quercetin in specifically targeted of transcription factors.
Fundamentally, the transcription factor, NF-κB has played a pivotal role in mediation of genes related to the inflammatory process, such as IL-1β, IL-6 or iNOS (Bliss et al., 2005;Kobayashi et al., 2002). Furthermore, the increased expressions of many genes involved in phagocytosis are observed during the first few hours after induction (Kobayashi et al., 2002). This phenomenon may be controlled by the transcription factor NF-κB. Many reports have mentioned that quercetin exerts its modulatory effects by reduction of pro-inflammatory genes through NF-κB inactivation (Boesch-Saadatmandi et al., 2011;Cho et al., 2003;Nair et al., 2006). From previous studies, quercetin inhibits the production of many proinflammatory cytokines, including IL-1β and TNF, the findings from the present work suggest that quercetin may be able to modulate the immune response via the NF-κB pathway.
In the experimental mouse model, supplementation of quercetin and exploratory studies on mRNA expressions of IL-6, C-Reactive Protein (CRP), Monocyte Chemoattractant Protein 1 (MCP-1) and Acyloxyacyl Hydrolase (AOAH) were irrelevant between the amount of quercetin intake and the expression levels of genes (Boesch-Saadatmandi et al., 2012). This study intends to demonstrate the effects of quercetin on both the influence of phagocytosis and phagosome formation. In order to select the representative genes involved in the process of phagocytosis to be monitored, we have used the KEGG pathway analysis as a searching tool. We have focused on CORO1A (coronin), CYBA (gp91 phox ), LAMP1, PAK1, RAB7A and RAC1, which are illustrated as our candidate genes. The effects of quercetin have securely confirmed our prediction that quercetin is truly induced those genes during the phagocytosis in bovine neutrophils of live E. coli bacteria.
Many published works indicate the actual quercetin effects on gene expression are linked to the exposure time (Cho et al., 2003;Liu et al., 2005). Our results also show that most gene expression levels increase from 30 to 60 min of the phagocytic period by quercetin actions.
Thus, our results are consistent with a report in the macrophage cells (J774.A1) in which the changes in expression levels of gene transcription and related proteins (Hoffmann et al., 2010). In that particular study, the increase production of gene transcriptions and protein productions i.e., Lamp1/2, Rab7 were intensified during 30 to 60 min of incubation (Hoffmann et al., 2010;Zhang et al., 2005). In this study, the data provide the information that the expression of CORO1A, LAMP, RAB7A and RAC1 are all obviously increased during phagocytosis. Therefore, it may be concluded that quercetin might affect both the initial and terminal stage of phagocytosis of bovine neutrophils. In addition to the above-mentioned genes, there were some known correlations of activated genes in phagocytosis; Cdc42, Rac1 and Rho, that potentially triggering and may have been associated with exposure time (Zhang et al., 2005). The F-actin polymerization, as part of internalization and the formation of the phagosome, is controlled by PAK gene. This structure could be disrupted by cytochalasin B treatment in which actin polymerization are seized (Bao et al., 2012;Berends et al., 2010;Zhang et al., 2005). In this study, when bovine neutrophils were applied with cytochalasin B, the phagocytic activities of this cell were retarded as measurement by flow cytometry, microscopy or even with the gene expression profiling. The observed data are in accordance with earlier studies (Bao et al., 2012;Berends et al., 2010).
The engaging genes of phagocytosis in quercetin treated neutrophils tend to maximize most of its expression levels at 60-min interval. After reaching the maximum, it begins to decline after 90 min. This observation is inconsistent with previous study (Kobayashi et al., 2002). In that report, the expression studies on receptor-mediated phagocytosis of human PMNs were reached the highest levels in 90 min-time point and began to decline. Our experimental setting and that of previous report are not identical, but the expression levels of our studied genes during 60 mintime period may have shared some important aspects with that study (Kobayashi et al., 2002). Although, our studies were not focused on gene expression that involved in ROS molecules or respiratory burst, one of our studied genes in NADPH oxidase (CYBA) dictates a partial involvement in phagosome maturation. Our data show that the expression of CYBA was apparently increased during the first 30 min of phagocytosis Fig. 4A.
The increasing global concerns over the use of antimicrobial drugs in farmed animals urge us to search for a potential herbal bioactive agent like quercetin, as a feasibility of alternatives to synthesized drugs. From immunological stand point, diets rich in flavonoids, specifically quercetin, have positive health benefits, i.e., reduce infection risk, affect immunity and inflammation. Additionally, solid evidence also suggests that quercetin ultimately benefits in promoting growth and performance and improved feed efficiency in livestock (Gohlke et al., 2013;Maciej et al., 2015;Stoldt et al., 2015).
We speculate that quercetin, in some extents, plays an important role in the regulations at many cellular and molecular levels of bovine neutrophils. Our data herein raises a possibility of the beneficial outcomes of quercetin related to enhancement of bovine neutrophil phagocytosis in combating with pathogenic bacteria. Furthermore, the data in the present study suggest that quercetin further diminishes some proinflammatory cytokines. Our observations are only a starting point for more future research which could result in broadening knowledge of quercetin in bovine neutrophil biology.

Conclusion
This study provides useful information regarding the use of plant derived flavonoid, quercetin, in two important aspects on bovine neutrophils and could be extended to other innate cell types. Our first finding demonstrates that the use of quercetin during the onset of bacterial infections may enhance the ability of neutrophils to phagocytose invading bacteria. The second, supplementation of quercetin would aid in the anti-inflammation property by attenuating the inflammatory mediators as a consequence of the neutrophil functional response to bacteria. The information in our experiments is worth concluded that quercetin has the ability to boost the expression of genes involved in phagocytosis of bovine neutrophils while suppresses the expression of pro-inflammatory genes.
Kanruethai Wongsawan: Conceive and design the study, read and approve the final manuscript.
Phongsakorn Chuammitri: Conceive and design the study, execute the study and involve in data acquisition, as well as, analyze and interpret the data. Draft and revise the manuscript. Read and approve the final manuscript.

Disclosure Statement
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.