Piglet weight gain during the first two weeks of lactation influences the immune system development

Veterinary Immunology and Immunopathology 206 (2018) 25–34

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Piglet weight gain during the first two weeks of lactation influences the immune system development

T

Martin Lessarda, , Mylène Blaisa, Frédéric Beaudoina, Karine Deschenea, Luca Lo Versoa,b, Nathalie Bissonnettea, Karoline Lauzona, Frédéric Guayb ⁎

a b

Sherbrooke Research and Development Centre, Agriculture and Agri-Food Canada, 2000 College Street, Sherbrooke, QC, J1M 0C3, Canada Faculté des sciences de l’agriculture et de l’alimentation, Département des sciences animales, Université Laval, Québec, QC, G1V 0A6, Canada

ARTICLE INFO

ABSTRACT

Keywords: Lactation Immune system development Weight gain Piglet

The aim of this study was to investigate the effect of the piglet growth during the first week of life on ileal expression of genes and on development of the immune system. Eight litters adjusted to 12 piglets were used. Within each litter, the piglet that showed the lowest weight gain (LWG; n = 8) and the one that showed the highest weight gain (HWG; n = 8) in their first week of life were enrolled. Peripheral blood mononuclear cells (PBMC) were isolated on days 8 and 16 to characterize cellular population profiles and to assess ex-vivo secretion of interleukin-10 (IL-10), IL-6 and tumor necrosis factor-α (TNF-α). On day 16, piglets were euthanized and ileum samples were collected to extract RNA for microarray analysis and gene expression by qPCR. As expected, growth performance of LWG piglet was impaired compared to HWG piglets (P < 0.05). From day 8 to 16, the percentage of CD21+ B cells significantly increased in blood of heavier HWG piglets while the percentage remained constant in smaller LWG piglets (P weight x day = 0.01). For the CD4+CD8α− Th cells, a marked increase was observed in LWG piglets from 8 to 16 days of age (P = 0.002) whereas no significant change occurred in HWG piglets. Percentages of CD14+ monocytes and other MHC-II+ cells were respectively higher and lower on day 8 compared to day 16 for both groups of piglets (P < 0.01). On day 8, LPS-activated PBMC from LWG piglets produced less IL-6 compared to HWG piglets (P < 0.05). Microarray analysis of gene expression in piglets’ ileum tissue indicated that several genes involed in defense response and response to oxidative stress were modulated differently in LWG compared to HWG. Gene analysis by Q-PCR confirmed microarray results and revealed that IL-10, SOD1, NOS2, NOD2, TLR4, TLR9, CD40 and CD74 expressions were significantly decreased (P < 0.05) in LWG in comparison to HWG piglets, while MYD88 and NFkBiA showed a tendency to decrease (0.05 ≤ P < 0.07). These results suggest that birth weight and milk intake affect the growth performances and the development of immunity by modulating the expression of genes associated with immunity and oxidative stress in piglets’ intestinal tissue, and by affecting the leukocyte populations involved in innate and cell-mediated immunity in nursing piglets. Therefore, impaired development of immune system in LWG piglets might have an impact on their resistance to infections later in life.

1. Introduction Over the last few decades, the improvement of sow prolificacy through selection allowed to increase the number of piglets per litter but also led to increased heterogeneity of piglets' weight at birth and weight gain during lactation within litter (Milligan et al., 2002; Quesnel et al., 2008). As the proportion of low birth weight piglets has increased with selection for larger litters, this selection resulted in greater number of piglets with poor performances and increased losses before weaning due to higher risk of mortality and infections (Canario et al., 2010;

⁎

Damgaard et al., 2003). Since less vigorous and competitive low birth weight piglets ingest less colostrum and milk than normal and high birth weight littermates (Devillers et al., 2007, 2011), growth performance is greatly impairs in the former group (Beaulieu et al., 2010; Berard et al., 2008; Quiniou et al., 2002). At birth, piglets’ gut epithelium is immature anatomically, metabolically and immunologically. and the combination of genetic, microbial colonization and nutrition, including maternal milk, will condition the maturation processes. Indeed, various hormones and growth factors, such as insulin, insulin-like growth factors (IGF), epidermal

Corresponding author. E-mail address: [email protected] (M. Lessard).

https://doi.org/10.1016/j.vetimm.2018.11.005 Received 27 October 2017; Received in revised form 31 October 2018; Accepted 3 November 2018 0165-2427/ © 2018 Published by Elsevier B.V.

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Table 1 Description of antibodies used in flow cytometry. Targeted cells

Antibody specificity

Clone

Iso-Type

Fluorochrome

Dilution

Companies

memory T cells

CD3+ CD4+ CD8αlow+ CD3+ CD4+ CD8αCD3+ CD4CD8αhigh+ CD3+ γδ+ CD3CD21+ CD3CD4CD8αlow+ CD16+ CD172a+ MHC-II+ CD14+ MHC-II+

BB23-8E6-8C8 74-12-4 76-2-11 BB23-8E6-8C8 74-12-4 76-2-11 BB23-8E6-8C8 74-12-4 76-2-11 BB23-8E6-8C8 MAC320 BB23-8E6-8C8 BB6-11C9 BB23-8E6-8C8 74-12-4 76-2-11 FcG7 74-22-15A 1053h2-18-1 TÜK4 1053h2-18-1

IgG2a IgG2b IgG2a IgG2a IgG2b IgG2a IgG2a IgG2b IgG2a IgG2a IgG2a IgG2a IgG1 IgG2a IgG2b IgG2a IgG1 IgG2b IgG2a IgG2a IgG2a

FITC Horizon 450 APC-H7 FITC Horizon 450 APC-H7 FITC Horizon 450 APC-H7 FITC APC FITC PE FITC Horizon 450 APC-H7 Strep-V500 FITC PErCP-Cy5.5 Pacific blue PErCP-Cy5.5

3/400 1/500 3/1000 3/400 1/500 3/1000 3/400 1/500 3/1000 3/400 3/400 3/400 1/200 3/400 1/500 3/1000 1/200 1/200 2/1000 1/10 2/1000

Southern Biotech BD Biosciencea BD Biosciencea Southern Biotech BD Biosciencea BD Biosciencea Southern Biotech BD Biosciencea BD Biosciencea Southern Biotech BD Bioscience Southern Biotech Southern Biotech Southern Biotech BD Biosciencea BD Biosciencea BD Bioscienceb BD Bioscience BD Biosciencea AbD Serotec BD Biosciencea

T helper cytotoxic T cells γδ T cells B cells NK cells

Monocyte/ macrophage Other APC a b

custom service coupling. Primary antibody coupled to biotin.

Care guidelines on the care and use of farm animals in research, teaching and testing (Bailey, 2009) and were approved by the Institutional Animal Care Committee of Sherbrooke Research and Development Centre. All animals were cared for and slaughtered according to the Code of Practice for the Care and Handling of Pigs of the National Farm Animal Care Council (2014).

growth factor (EGF) and transforming growth factors (TGF), have been found in the mammary gland secretions of different species, including pigs (Xu, 1996). The concentrations of these bioactive compounds are usually high in colostrum, and decline rapidly in mature milk (Xu et al., 2002). Colostrum and milk also bring prebiotics and antimicrobial factors such as immunoglobulins, lactoferrin, lysozyme and oligosaccharides that contribute to control the growth and the establishment of different bacterial populations (Devillers and Lessard, 2007; Barile and Rastall, 2013; Mohanty et al., 2016). They also contains various immunodulatory factors that play an important role in protecting the piglets against infectious pathogens and are required for the development and maturation of piglet immune system (Cross and Gill, 2000; Hernandez and Guzman, 2003; Newburg, 2005; Turfkruyer and Verhasselt, 2015). Even if the piglet’s immune system is immature at birth, monocytes, B cells and different populations of T cells are present in blood and their number increase with age (Grierson et al., 2007; Talker et al., 2013). After birth, these cells and other population of immune cells have the potential to respond to antigenic and inflammatory agents, and can interact to develop specific immune response (Schwager and Schulze, 1997; Zelnickova et al., 2008). At the gut level, intestinal immunological structure of newborn piglets is poorly developed and very few lymphocytes are present in the epithelium and lamina propria (Stokes et al., 2004). Intestinal mucosal colonisation by CD4+, CD8+ and γδ + T lymphocyte populations begins around two weeks of age and their numbers will increase with age (Vega-Lopez et al., 1993; Rothkotter et al., 1999; Stokes et al., 2004). At that age, gene expression pathways involved in tissue development, functions and immunity are also activated in the small intestine and influenced by sow milk composition and consumption (Graugnard et al., 2015). As piglets’ growth performance during the lactation is highly correlated to colostrum and milk intake, this study aimed to evaluate the influence of piglets’ birth weight and growth performance in the first few weeks of life on the activation and the development of systemic and intestinal immunity by measuring immune cell populations, peripheral blood mononuclear cells (PBMC) functional properties and expression of genes involved in intestinal immune functions.

2.1. Animals and treatments A total of 8 Yorkshire × Landrace sows inseminated with certified semen from Duroc boars provided by the Centre d’insémination porcine du Québec (St-Lambert de Lauzon, Quebec, Canada) were used to provide piglets required to realize the project. Sows were housed in the Swine Complex facilities of Sherbrooke Research and Development Centre and were following herd feeding management program for the gestation and lactation periods. Two weeks before parturition, the sows were transferred in the maternity room. After delivery, litter size was adjusted to 12 piglets within the first two days after birth. The piglets were weighed at birth, at 7 and 16 days of age. According to their average daily gain (ADG) during the first week of life, one low weight gain (LWG) and one high weight gain (HWG) piglets were selected in each litter. Whole blood samples (sodium -EDTA) were taken on days 8 and 16 to characterize leukocyte populations by flow cytometry and to evaluate cytokine production of activated peripheral blood mononuclear cells (PBMC) by ELISA as described below. Piglets were euthanized at 16 days of age to perform intestinal samplings. Ileum slices were taken at 25 cm from the cecum and were immediately frozen in liquid nitrogen and stored at −80 °C until assays were performed to quantify gene expression. Ileum slices were also fixed in 4% paraformaldehyde solution for 24 h, then transfer in ethanol 70% solution until slices were embedding in paraffin blocks for histological analyses. 2.2. Characterization of leukocyte populations by flow cytometry Whole blood samples collected on days 8 and 16 were diluted in Hanks balanced salt solution and then layered on Ficoll-Paque PLUS (GE Healthcare Life Science, Mississauga, Ontario, Canada) to isolate peripheral blood mononuclear cells (PBMC). Isolated PBMC were labelled with antibodies directed against different cell surface antigens (Table 1) to characterize the percentage of different leukocyte populations including CD3−CD21+ B cells, CD3+CD4+CD8α− T helper

2. Materials and methods Experimental procedures followed the Canadian Council of Animal 26

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(Th) cells, CD3+CD4− CD8α+ T cytotoxic (Tc) cells, CD3−CD4− CD8α+CD16+ natural killer (NK) cells, CD3+ gamma-delta+ T cells (γδ T cells), CD14+ CD172a+ MHC-II+ monocyte/macrophage lineage and other MHC-II+ cells by flow cytometry as described previously (Lessard et al., 2009). Briefly, cell concentration was adjusted at 5 × 106 cells / ml in FACSflow solution (BD Biosciences, Mississauga, Ontario, Canada) containing 0.5% BSA as staining solution and 200 μl of each suspension were distributed in wells of 96-well round bottom microplates (VWR international, Mississauga, Ontario, Canada). After centrifugation at 300 g for 5 min at 4 °C, supernatants were discarded and 100 μl of titrated monoclonal antibodies diluted in staining solution were added in each well according to the immunophenotyping procedure described in Supplementary figures 1 and 2. For the staining of NK cells, the incubation of cells with the biotinylated anti-CD16 was followed by the addition of streptavidin conjugated to BD Horizon V500. Cells were incubated on ice in the dark for 20 min and then washed twice. Stained cells were suspended in 400 μl of FACSflow containing 0.5% formaldehyde and were analyzed with a calibrated BD FACSCanto II flow cytometer equipped with a conventional 3 laser configuration (4-2-2). From each sample, 10 000 events were recorded and the BD FACSDiva operating software (BD Biosciences, Mississauga, Ontario, Canada) was used for the acquisition and data analysis. Compensation between all fluorochromes was done using a pool of all cell samples with individual dye-antibody. The automatic calculation of compensation with FACSDiva software was used.

18 primer in a final volume of 20 μl, according to the supplier’s instructions (Thermo Fisher Scientific, Life technologies). The cDNA samples were diluted 1:15 in nuclease-free water and aliquots were stored at −20 °C prior to real-time PCR analysis. In order to exacerbate the effect of weight gain difference on gene expression, two piglets with very poor weight gain (< 0.5 kg) and three piglets with very high weight gain (> 1.5 kg) in the first week of life were selected and RNA samples were extracted from their ileum for microarray analysis. RNA samples from each groups were pooled and their final concentration were adjusted at 0.5 μg/μl. RNA integrity was assessed by Bioanalyser (Agilent technologies Canada inc., Mississauga, Ontario, Canada) and their RNA Integrity Number (RIN) was higher than 9.3. RNAs were hybridized on 44 K porcine chip (Agilent Technologies #026,440) by Genome Quebec team (Montréal, Canada). Data analysis, normalization, average difference and expression for each feature on the chip were done using Flexarray software version 1.6.1 (Genome Quebec, Montreal, Quebec, Canada). Gene expression levels that were significantly modulated more than 2 times (log2 > 1) were analysed according to Gene Ontology (GO) classification with Toppgene Suite (Transcriptome, ontology, phenotype, proteome and pharmacome annotation based gene list; http://toppgene.cchmc.org/). Raw data from the study were submitted to Gene Expression Omnibus database (GSE117082 study: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE30321) and can be viewed on the following link: https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE117082. Based on the analysis of microarray results, real-time PCR assays were performed with a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, USA) to validate the expression of genes associated with defense and immune response as well as oxidative stress. The PCR mixture was composed of the following: 5 μl Power SYBR Green Master Mix (Applied Biosystems), 2 μl of each gene specific primer set (IDT, Coralville, USA) at the indicated concentrations (Supplementary Table 1) and 3 μl of diluted 1:15 cDNA. Primers were selected using the following criteria, when possible: (1) both forward and reverse primers encompass two consecutive exons; and (2) no more than two guanines or cytosines within the last five nucleotides in the 3′ termini. The PCR cycling conditions were 10 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 45 s at 60 °C, and finally a dissociation curve. The relative expression level of target genes was calculated using ΔΔCt method (Pfaffl, 2001) with efficiency correction LinRegPCR (Ruijter et al., 2009). In order to confirm the specificity of the measured amplicons (i.e. the presence of only one amplicon), the melting curve was systematically analysed for all samples. Each run included a notemplate control in duplicate to detect DNA contamination of the reagents and each sample was tested in triplicate.

2.3. Stimulation of PBMCs and measurement of cytokines by ELISA Isolated PBMC at 8 and 16 days were plated in a 24 wells cell culture plate (VWR international, Mississauga, Ontario, Canada) at a concentration of 2.5 × 106 cells in a final volume of 1,5 ml of Roswell Park Memorial Institute (RPMI; Wisent Bioproduct, St-Bruno, Quebec, Canada) 1640 media supplemented with 7.5% fetal bovine serum (FBS; Wisent Bioproduct,) and Penicillin-Streptomycin antibiotic mixture containing 5000 units/mL of penicillin and 5000 μg/ml of streptomycin (Gibco Laboratories, Gaithersburg, MD, USA). The cells were stimulated with either 1.0 μg/ml concanavalin A (Con A), 50 ng/ml phorbol myristate acetate (PMA) or 500 ng/ml lipopolysaccharide (LPS) from E. coli 055:B5 (Sigma-Aldrich, Oakville, Ontario, Canada). Cells were incubated for 48 h at 37 °C in a 5% CO2 humidified incubator. At the end of the incubation period, supernatants from LPS activated PBMC were collected and used to measure interleukin-6 (IL-6) in, while tumor-necrosis factor-α (TNF-α) and IL-10 were respectively measured in supernatants from PMA or Con A activated PBMC. All ELISA were performed using Porcine DuoSet ELISA kit from R & D System (R&D System, MN, USA) according to manufacturer recommendations. All samples and controls were tested in duplicate.

2.5. Villus-crypt morphology in the ileum

2.4. Microarray and quantification of gene expression by real-time PCR

Ileal tissue was sampled (0·5 × 0·5 cm) at about 20 cm from the ileocaecal junction for assessment of villus-crypt morphology. Briefly, specimens were excised, rinsed in physiological saline, and fixed in phosphate-buffered formalin (10%, pH 7.6) for 48 h. Then they were rinsed with ethanol:water (3:1, vol:vol) and dehydrated before being embedded in paraffin. Serial sections of tissue (5 μm thick) were cut perpendicular to the axis of embedded intestine samples in paraffin using a microtome (HM330 rotation microtome, Heidelberg, Germany). The sections were fixed on glass slides (3 sections per slide). One slide per tissue sample was prepared and stained with hematoxylin-eosin. Villi and crypts were observed using an optical microscope (Nikon Eclipse E600 microscope, Nikon Canada, Mississauga, Ontario, Canada) equipped with a digital camera (Q-Imaging Exi Blue with RGB extern filter, QImaging Corporate Headquarters, Surrey, BC, Canada). ImageJ 1.46 r software (Wayne Rasband, National Institutes of Health, US) was used to measure villus height and crypt depth. Villus height was defined as the distance from villus base to tip, and crypt depth as the distance from villus base to lamina muscularis mucosae. Each slide was

Briefly, ileum slices that were conserved in -80 °C were homogenized in buffer RLT supplemented with β-mercaptoethanol (Qiagen, Toronto, Canada) using a KINEMATICA Polytron homogenizer (Thermo Fisher Scientific, Mississauga, ON). Total RNA was extracted with RNeasy Plus mini kit (Qiagen) following the manufacturer’s recommendations and was eluted in 50 μl of water. Contaminating DNA from RNA preparations was removed with DNA-free™ DNase Treatment and Removal Reagents kit according to manufacturer instructions (Thermo Fisher Scientific, Life technologies, Mississauga, ON). Total RNA was quantified using a NanoDrop spectrometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). Purity was assessed by determining the ratio of absorbance at 260 and 280 nm (A260/A280). All samples had a ratio between 1.9 and 2.1. The ratio of absorbance at 260 and 230 nm (A260/A230) was also measured and all samples had a ratio between 1.7 and 2.1. A 1 μg aliquot of total RNA was reversetranscribed with Superscript II reverse transcriptase using oligo (dT)1227

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examined without knowledge of origin for quantification. Mean values for villous height and crypt depth were determined for ten individual villi and crypts from each specimen, and individual means were calculated. 2.6. Statistical analysis Statistical analyses were conducted with SAS software (SAS Institute Inc., Cary, NC, USA). Before statistical analyses were performed, the Shapiro-Wilk test was used to evaluate the normality of residuals using the UNIVARIATE procedure. To evaluate the effects of piglet weight gain, blood leukocyte populations, PBMC cytokine production and intestinal gene expression, pairwise mean comparisons between LWG and HWG piglets were made using PROC MIXED procedure or paired Student′s t-test. Data on mRNA abundance were analyzed using ΔΔCt method comparing LWG to HWG piglets. Three different reference genes were analyzed in all cDNA samples, and the two most stable ones were selected for normalisation (ribosomal protein S18 (RPS18) and hypoxanthine-guanine phosphoribosyltransferase (HPRT). 3. Results 3.1. Growth performances of LWG and HWG piglets As expected, the growth of LWG piglets was impaired compared to that of HWG piglets (weight gain effect x time, P value < 0.0001). The average weight difference between the two groups increased with time from 0.26 kg at birth to 2.1 kg at 16 days of age and resulted in a significant reduced ADG in LWG piglets compared to HWG piglets (0.16 and 0.27 kg respectively) from birth to 16 days of age (P < 0.001). Fig. 1 shows the individual performance of selected LWG and HWG piglets within litters and highlights the four extremes in each group regardless of litter belonging, respectively identified as very LWG (VLWG) and very HWG (VHWG).

Fig. 2. Percentage of CD21+ B cells, CD4+CD8− Th cells, CD14+ monocytes and MHC-II+ cells in blood of LWG and HWG piglets in the first two weeks of life. Panel A show the results for all LWG and HWG animals (n = 8 for B and T cells and n = 6 for monocytes and other MHC-II cells) and panel B for the piglets making the lowest and highest weight gain (VLWG and VHWG) in the first two weeks of life (n = 4 for B and T cells and n = 3 for monocytes and other MHC-II cells).

3.2. Characterisation of blood leukocyte populations in LWG and HWG piglets, and cytokine production by activated PBMC Percentages of CD21+ B cells, CD4+CD8α− T helper cells, CD4−CD8α+ T cytotoxic cells, CD4+CD8α+ T cells, NK cells and subtype CD3+γδ T cells in piglet blood were not significantly affected by growth performances in the first two weeks of life when all LWG and HWG animals were included. However, when the analysis were performed with the four lowest LWG and four highest HWG piglets of each group, the data revealed that the percentages of CD21+ B cells and CD4+CD8α− Th cells were differently affected by the growth

performance in the first two weeks after birth (Fig. 2; P value of Weight gain x Day = 0.01 and 0.06 respectively). From day 8 to 16, the percentage of CD21+ B cells increased significantly in VHWG piglets (P = 0.003) while in VLWG piglets, the percentage remained the same. Regarding the CD4+CD8α− Th cells, a marked increase was observed in VLWG piglets from 8 to 16 days of age (P = 0.002) whereas no significant change occurred in HWG piglets. Analysis of CD172a+CD14+MHC-II+ monocytes and CD172a−MHCII + cells, including CD21+ B cells, indicated that the percentage of these populations were respectively lower and higher on day 8 compared to day 16 when all animals were considered (Day effect P value = 0.003 or < 0.001, respectively). However, when only the 4 extremes of each group were taken into account, the percentage of CD172a+CD14+MHCII+ monocytes in VHWG piglets decreased from days 8 to 16 (P = 0.08) while in VLWG piglets the percentage at 8 days of age was similar to percentages observed on day 16 in both VLWG and VHWG piglets (Weight gain x Day P value = 0.07) Supernatant concentration of IL-6, TNF-α and IL-10 respectively produced by LPS, PMA or Con A activated PBMC isolated from LWG and HWG piglets are shown in Fig. 3. Results revealed that production of IL-6 by LPS activated PBMC tended to be impaired in 8 days old LWG piglets compared to HWG piglets (P = 0.09) when all animals were included in the analysis. When only the four extremes of each group were considered (Fig. 3), production of IL-6 was markedly decreased in

Fig. 1. Individual performance of selected LWG and HWG piglets within each litter. * and ** identify the four extremes LWG and HWG piglets regardless of litter belonging. 28

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gain during the first week of life < 0.5 kg or > 1.5 kg respectively) were selected and used for this analysis. Results indicated that up to 357 probes from the array were differently expressed in LWG piglets compared to HWG piglets (173 upregulated and 184 downregulated more than 2 fold change), as shown in the Volcano plot of gene expression (Fig. 4). The list of known genes associated with each of the probes that were downregulated and upregulated in LWG piglets is shown in supplementary Tables 2 and 3 respectively, along with their fold induction. We further analysed the biological processes and molecular functions associated with these genes using ToppGene Suite. Interestingly, categories related to oxidative stress (antioxidant activity, response to oxidative stress, response to oxygen-containing compound, hydrogen peroxide metabolic process, etc), defense and inflammatory response, response to hormones, and transporter activity came out predominantly (Fig. 5, Tables 2 and 3). Taken together, these results suggest that piglets' early growth performances after birth affect the transcriptional activation of genes involved in oxidative metabolic stress and defense responses in the gut. Based on some of the microarray results obtained with RNA collected from the extreme LWG piglets and HWG piglets, along with differences observed between LWG and HWG piglets in some immune cell populations and cytokine production, further Q-PCR assays were performed to investigate the expression of different genes associated with immune processes in ileal tissue samples of all 16 experimental piglets (8 LWG and 8 HWG). Level of expression as measured by Q-PCR confirmed most observations that were obtained with the microarray analysis (Table 4). Results showed that the expression of IL10, LIPM, NOS2, SOD1, NOD2, TLR4, TLR9, CD40 and CD74 was significantly decreased (P < 0.05) in LWG in comparison with HWG piglets, while CA3, CD83, CLU, MYD88, and NFkBiA, presented a tendency to decrease (0.05 ≤ P < 0.07). Most of these genes are involved in defense response (GO:0006952) or response to oxidative stress (GO:0006979), while LIPM gene is mainly involved in lipid catabolic process (GO:0016042). 3.4. Villus-crypt morphology of the ileum and colon Villous height and crypt depth of the ileum were not affected by the growth performance of LWG and HWG piglets (Table 5). 4. Discussion The pork industry has achieved tremendous gains in litter size, through genetic selection and the introduction of hyperprolific dams into commercial production. However, increases in litter size have resulted in increased number of low-weight piglets at birth (Damgaard et al., 2003; Beaulieu et al., 2010; Canario et al., 2010), which are associated to poor growth performances and are more vulnerable to preweaning mortality (Milligan et al., 2001, 2002; Le Dividich et al., 2005). As reported in these studies, our results clearly demonstrated that the average daily weight gain of piglets born with low weight was impaired from birth in comparison with that of piglets born at higher weight (Morissette et al., 2017). Growth performance during the lactation period is highly correlated to colostrum and milk intake (Devillers et al., 2007; Milligan et al., 2002). Moreover, there are clear evidences in the literature that colostrum and milk intake influence gut development and maturation of the immune system in different animal species (Simmen et al., 1990; Xu et al., 2002; Nguyen et al., 2007; Salmon et al., 2009; Turfkruyer and Verhasselt, 2015). Such lactocrine signaling properties of colostrum and milk are believed to be critical for optimal neonatal developmental events and to enhance the capacity of neonates to sense, respond, and adapt to the circumstances into which they are born (Bartol et al., 2013). Little is known about the impact of piglets’ birth weight and growth performance in the first few weeks of life on the development of the

Fig. 3. Production of IL-6, TNF-α and IL-10 by respectively activated LPS, PMA and ConA PBMC isolated from HWG, LWG, very HWG (VHWG) and very LWG (VLWG) piglets at 8 and 16 days of age. Data are shown as the mean ± sem.

VLWG piglets compared to VHWG piglets at 8 days of age while at 16 days of age IL-6 production tended to be increased in VLWG compared to VHWG piglets (Weight gain x Day P value = 0.002). Production of IL-10 by Con A activated PBMC was not affected by the weight gain in the first two weeks of life whereas TNF-α produced by PMA activated PBMC tended to increase at 16 days of age in LWG compared to HWG piglets (P = 0.10). An increase of TNF-α production was observed in VLWG piglets compared to VHWG piglets (P value of Weight gain effect = 0.07) on day 16 when data analysis were performed with the four extremes of each group (Fig. 3). 3.3. Ileal gene expression in LWG and HWG piglets To determine whether lower birth weight and growth performances in the first two weeks of life were associated with a modulation of gene expression in the ileum of piglets, the analysis of LWG piglets' transcriptome was compared to the one of HWG piglets by microarray analysis. In order to emphasise the impact of weight differences, only piglets that had very low or very high growth performances (weight 29

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Fig. 4. Volcano plot of gene expression changes in intestinal tissue of LWG piglets compared to HWG piglets measured by microarray analysis. Probes representing genes associated with either response to hormones, response to oxygen-containing compound, lipid metabolic process or immune system are shown. Fold change (log2) are shown in x axe and pValue (log10) are shown in y axe. Table 2 List of genes up- or down-regulated in LWG piglets’ intestine compared to HWG piglets, according to their molecular function, as determined using ToppGene Suite. Molecular function

Downregulated in LWG

Upregulated in LWG

Transporter activity; GO:0005215

COX8A, COX2, FABP4, TF, CLCA4, GC, BAX, OSBP, ATP6V1C2, CYTB, CLVS2 SOD2, CAT ADIPOQ, CALCB CLCA4

COX1, KCND2, ATP1A1, FXYD3, HBA1, SLC22A2, BEST2, ANO5, CLCA1, SCNN1G GPX3, HP, HBA1 GAL, CGA, RETNLB ANO5, CLCA1

CLCA4

FXYD3, BEST2, ANO5, CLCA1

Antioxidant activity; GO:0016209 Hormone activity; GO:0005179 Intracellular calcium activated chloride channel activity; GO:0005229 Chloride channel activity; GO:0005254

immune system. Analysis of cell subset populations revealed that percentage of CD21+ B cells and CD4+CD8− Th cells respectively increased between 8 and 15 days of age in highest HWG and lowest LWG piglets. However, these differences in B and T lymphocytes between LWG and HWG piglets were not observed when the selection of LWG and HWG piglets was extended to a population showing weight gain closer to the average weight gain within the litter, which suggest that these differences are seen only in piglets with untypical weight gain. Indeed, weight gain difference between LWG and HWG piglets was not always as marked within litters, which could explain the inconsistency of results presented in this study between groups of piglets. Therefore, when the four poorest LWG and highest HWG piglets were considered regardless of the litter belonging, the effects of birth weight and weight

gain were exacerbated, hence showing a lack of immune maturation in the VLWG. However, changes observed in HWG piglets on immune cell populations mentioned above are in agreement with previous study indicating that CD21+ B cells markedly increased in the first few week of live while the percentage of CD4+CD8− Th cells slightly decreased (Juul-Madsen et al., 2010), which shows that the LWG piglets population is the one showing impaired immune response development. Previous studies have shown that CD4+ T cells and CD21+ B cells are present in blood of the newborn piglet and gradually increase with age (Borghetti et al., 2006; Talker et al., 2013) However, no difference was reported by Talker et al. (2013) in the number of CD4+ T cells between low and high body weight piglets at weaning. These results contrast with those presented in the present study. Discrepancy could 30

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Table 3 List of genes up- or down-regulated more than 2 times in LWG piglets’ intestine compared to HWG piglets, according to the biological processes they are involved in, as determined using ToppGene Suite. BIOLOGICAL PROCESS

Downregulated in LWG

Upregulated in LWG

Response to oxidative stress; GO:0006979 Response to hormone; GO:0009725

ADIPOQ, CA3, ND1, SOD2, CAT, F3, GPX3 ADIPOQ, LCT, NR0B1, GC, BAX, CAT, ATP6V1C2, CYTB, F3 ADIPOQ, LCT, NR0B1, GC, BAX, CAT, F3 ADIPOQ, COX8A, COX2, SOD2, BAX, CAT, CYTB, MT-ND4 LCT, FABP4, TF, SOD2, BAX, CAT, CYTB, F3 COX8A, SOD2, BAX, CAT, CYTB, MT-ND4

MSRA, COX1, ARG1, CCL5, HP, HBA1, AREG HSD11B2, PDK4, ATP1A1, ARG1, GAL, CCL5, CGA, ACTA1, ADRA1B, AREG, RETN, APOBEC1 HSD11B2, ATP1A1, ARG1, GAL, CCL5, ACTA1, ADRA1B, AREG MT-ND2, COX1, ADRA1B, PGM1

ADIPOQ, CA3, LCT, ND1, SOD2, GC, BAX, CAT, ATP6V1C2, CYTB, F3 SOD2, CAT COX8A, SOD2, BAX, CAT, CYTB, MT-ND4 ADIPOQ, CAT, ATP6V1C2, CYTB ADIPOQ, AGO2, LCT, NR0B1, GC, GSTM2, Cd83, BAX, CAT, LUM, F3 SOD2, GSTM2, CAT SAA2 SOD2, GSTM2, CAT ADIPOQ, SAA2, LYZ, FABP4, C8G, CALCB, F3 SOD2, CAT, F3 ADIPOQ, CA3, LCT, GC, BAX, CAT, F3 ADIPOQ, CAT, ATP6V1C2, CYTB ADIPOQ, CAT, ATP6V1C2 ADIPOQ, LCT, SOD2, BAX, CAT SOD2, GSTM2, BAX, CAT ADIPOQ, COX8A, SAA2, LYZ, FABP4, C8G, CALCB, OAS2, F3, Cd83 LYZ, C8G COX8A, COX2, SOD2, CYTB, MT-ND4

HSD11B2, GPX3, PDK4, ATP1A1, ARG1, GAL, CCL5, HP, CLEC7A, HBA1, ADRA1B, AREG, RETN, APOBEC1 GPX3, HP, HBA1 MT-ND2, COX1 HSD11B2, PDK4, ARG1, GAL, CCL5, AREG, RETN, APOBEC1 HSD11B2, ATP1A1, ARG1, GAL, CCL5, ACTA1, ADRA1B, AREG

Response to steroid hormone; GO:0048545 Generation of precursor metabolites and energy; GO:0006091 Response to inorganic substance; GO:0010035 Energy derivation by oxidation of organic compounds; GO:0015980 Response to oxygen-containing compound; GO:1901700 Hydrogen peroxide metabolic process; GO:0042743 Cellular respiration; GO:0045333 Response to peptide hormone; GO:0043434 Response to organic cyclic compound; GO:0014070 Cellular detoxification; GO:1990748 Acute-phase response; GO:0006953 Detoxification; GO:0098754 Inflammatory response; GO:0006954 Response to hydrogen peroxide; GO:0042542 Response to alcohol; GO:0097305 Response to peptide; GO:1901652 Response to insulin; GO:0032868 Response to drug; GO:0042493 Response to toxic substance; GO:0009636 Defense response; GO:0006952 Cytolysis; GO:0019835 Electron transport chain; GO:0022900

be related to piglets that were selected to evaluate the weight effect on T cells and to the day of performing assays. Also, the fact that very low weight piglets at birth are often discarded or lost from experimental groups because of their increased vulnerability to health issues and preweaning death might make that population under-represented at weaning in studies. In the present study, differences in the percentage of cells were observed when body weight gain differences in the first two weeks of life were amplified by selecting the lowest and highest performing piglets. It is also known that the number of different cell populations increases with age which can affect their relative percentage within blood leukocytes. A recent study also reveals that T and B lymphocytes are markedly reduced in immuno-deficient piglets compared to normal littermates over the first 21 days after birth (Ewen et al., 2014). Reduction of CD4 + T cells in suckling piglets have also been reported in blood, spleen and mesenteric lymph nodes of piglets infected with Isospora suis after birth while the number of CD21 + B cells was not affected (Worliczek et al., 2010). These studies provide evidences that the status of lymphocyte cell populations can be modulated in early life of piglets and support our results on B and T cells. However, it is important to take into account the age of piglets at sampling when interpreting results on ontogeny of leukocyte populations in blood and other tissues in suckling piglets. We also observed that the percentage of CD172a+CD14+ MHCII+ monocytes decreased from day 8 to 16 in HWG piglets compared to LWG piglets while CD3−MHC-II+ lymphocytes including CD21+ cells were increased. These results are in agreement with previous data indicating that CD172a+ monocytes decrease and MHCII+ lymphocyte populations increase during the lactation period (Juul-Madsen et al., 2010). Interestingly, the percentage of CD172a+CD14+ MHCII+ monocytes in the lowest LWG piglets was not as high as in HWG piglets at 8 days of age and remained unchanged during the same period. As these cells play important functions in innate immunity, presentation of antigens and activation of the immune system, these results suggest that the development of the immune system could be greatly affected in

ARG1, HP, HBA1, ACTA1, AREG, APOBEC1 MT-ND2, COX1, ADRA1B, PGM1

GPX3, HP, HBA1 REG3G, CCL5, HP, ITIH4 GPX3, HP, HBA1 REG3G, CCL23, GAL, CCL5, HP, CLEC7A, ITIH4, CCR3 ARG1, HP, HBA1, AREG ARG1, CCL5, AREG, APOBEC1 HSD11B2, PDK4, ARG1, GAL, CCL5, AREG, RETN, APOBEC1 HSD11B2, PDK4, GAL, CCL5, RETN, APOBEC1 HSD11B2, ATP1A1, ARG1, GAL, CCL5, SLC22A2, APOBEC1 GPX3, ARG1, CCL5, HP, HBA1 REG3G, PDK4, CCL23, GZMB, FCN2, GAL, CCL5, HP, CLEC7A, ITIH4, CCR3, APOBEC1, SFTPD, BPI GZMB, GZMH MT-ND2

LWG piglets by such effect on abundancy of different cell populations in the first few weeks of life. Additionally, the production of IL-6 and TNF-α by activated PBMC was differently modulated in extreme LWG compared to HWG piglets at 8 and 16 days of age. While IL-6 production in response to LPS was significantly lower in VLWG at day 8, the ability of PBMCs to cope with LPS was restored by day 16. These results could be explained by the decreased percentage of circulating CD14+ monocytes at day 8 in VLGW compared to VHGW. On the other hand, production of TNF-α after PMA stimulation was increased in VLWG piglets compared to VHWG at both days 8 and 16 indicating that broadly activated PBMC appeared to have a higher potential to react to stimulation in VLWG. Although both cytokines are known to be pro-inflammatory, they have distinct production profile depending on the cytokine environment and inflammatory stimuli to which epithelial and immune cells are exposed (Hunter and Jones, 2015). Indeed, the development of adaptive immune responses can be modulated by IL-6 which can govern the proliferation, survival and commitment of T and B cells and modulates their effector cytokine production. For instance, IL-6 in presence of TGF-β is a key driver of IL-17-secreting CD4+ or CD8+ T cells and plays an important role in lineage commitment of TH17 subset of helper T cells (Zhou et al., 2007; Hunter and Jones, 2015). However in presence of TNF-α, IL-6 is mostly considered as a pro-inflammatory marker and has frequently been recognized in the context of metabolic inflammation (Mauer et al., 2015; Möller and Villiger, 2006). Such modulation in the production of IL-6 and TNF-α by PBMC in the first two weeks of life of VLWG piglets may therefore have important impact on activation of the immune system and suggest that VLWG piglets may be prone of developing more severe inflammatory status than HWG piglets. Further analysis was then performed to characterize ileal gene expression in LWG and HWG piglets. The most interesting finding was the observation that there was a marked difference in the expression of several genes in LWG compared to HWG piglets after two weeks of lactation. Based on the gene ontology (GO) classification according to 31

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genes involved in production of nitric oxide and reactive oxygen species and lipid metabolism such as NOS2, SOD2 and LIPM were also downregulated in LWG compared to HWG piglets. As these genes play an important role in the activation and regulation of innate and adaptive immunity (Bogdan, 2015; Lochner et al., 2015; Nathan and Cunningham-Bussel, 2013), these results strongly suggest that defense response is a major biological process that is markedly affected in LWG compared to HWG piglets. Furthermore, results indicated that expression level of CD40, a costimulatory protein found on antigen presenting cells (APC) and playing an important role in the regulation of innate and acquired immune responses (Quezada et al., 2004; Van Kooten and Banchereau, 2000), CD74, a transmembrane glycoprotein expressed on APC which acts mainly as an MHC class II chaperone and play important roles in antigen presentation pathway (Su et al., 2017) and CD83, a marker for dendritic cells whom function primarily lies in the regulation of T- and B-lymphocyte maturation and in regulation of dendritic cell inflammatory response (Bates et al., 2015; Breloer and Fleischer, 2008), were also reduced in LWG. These results suggest that intestinal APC were less abundant in LWG piglets than HWG piglets as expression level of CD40, CD74 and CD83 is associated with the presence of antigen presenting cells. Among professional APC, activated-B cells are also considered as an important population that can directly present antigen to naïve CD8+ T cells to induce the generation of potent effectors able to secrete cytokines and control infections (Mathieu et al., 2012). In piglets, CD21+ activated B cells have been shown to also express MHCII+ cells and to have the potential of presenting antigens to T cells (Sinkora and Butler., 2016). Therefore, this observation on the expression of APC intestinal markers could be related to presence in the ileum of CD21+ B cells which cell population in PBMC of extreme LWG piglets showed to have a reduced relative number. However, further studies need to be done to better understand the impact of birth weight and growth performance in the first few weeks of life on leukocyte ontogeny in blood and gut associated lymphoid tissue. The microarray results also clearly indicated that several genes involved in response to hormones (ADIPOQ, LCT, NR0B1, BAX, CAT, ATP6V1C2, CYTB, PDK4, ATP1A1, GAL, CCL5, CGA, ACTA1, ADRA1B, AREG, RETN), cellular respiration (COX8A, BAX, CYTB, FABP4, MTND4, MT-ND2, COX1) and transporter activity (COX8A, COX2, FABP4, TF, CLCA, BAX, CYTB, COX1, KCND2, ATP1A1, HBA1, SLC22A2) were modulated by piglets' weight and growth performances in the first two weeks of life. Because these genes play important roles in the regulation of various metabolic pathways, these results provide new avenues of research to investigate and understand the interactions between genes involved in different biological processes in early stages of life and their long term influence on the development and health of pigs. Recent results obtained in our laboratory indicate that the establishment of neonatal gut microbial populations in LWG and HWG piglets during the neonatal period has been shown to be modulated in the first two weeks of life as well (Morissette et al., 2017). Previous studies realized in neonatal pigs indicated that housing environment also influence early-life intestinal exposure of piglets to bacterial populations and have important impact on the ileal immune transcriptome (Mulder et al., 2009, 2011; Lewis et al., 2012). Indeed, they observed that the expression of several genes involved in activation of innate immunity were up-regulated in pigs reared in indoor housed animal compared to outdoor-housed animals at 5 and 28 days of age. Moreover, several other genes involved in response to hormone, development and cytoskeleton remodeling were also modulated by housing environment. The lack of innate and pro-inflammatory gene expression in outdoor-housed animals has been considered to be indicative of a more homeostatic mucosal immune system development in these animals. Altogether, this data and the ones presented in the present study strongly suggest that development of intestinal innate immunity in piglets is not only modulated by sow and piglet housing conditions but also by piglet rearing condition during lactation which also appeared to play an

Fig. 5. Classification of genes increased or decreased more than 2 times in LWG piglets’ intestines according to A) the biological processes they are involved in or B) their molecular functions using ToppGene Suite.

biological processes and molecular functions, the expression of several genes involved in oxidative stress response (GO:0006979), acute phase response (GO:0006953), inflammatory reaction (GO:0006954), defense response (GO:0006952), hormone response (GO:0009725), hormone activity (GO:0005179) and cellular respiration (GO:0045333) was differently modulated in two week old LWG compared to HWG piglets. Since the microarray data clearly showed that important genes involved in the immune response were modulated in LWG piglets, we further investigated the expression of various genes involved in intestinal immunity and function by Q-PCR analysis. Among these genes, NOD1, NOD2, TLR4, TLR9, MYD88 and NFkB which are involved in recognition of pathogen-associated molecular patterns, signalisation pathways and in maintenance of gut homeostasis and activation of innate immunity (Sanderson and Walker, 2007; Warner and Núñez, 2013), were downregulated in LWG piglets. The expression of other 32

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Table 4 Fold change of intestinal gene expression in LWG piglets compared to HWG piglets as measured by microarray analysis and validated by Q-PCR. Gene name

gene symbol

Carbonic anhydrase III Cluster of differentiation 40 Cluster of differentiation 74 CD83 antigen-like Clusterin Ficolon-2 Interleukin 10 Lipase family member M Myeloid differentiation primary response 88 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha Nucleotide-binding oligomerization domain containing protein 2 Nitric oxide synthase 2 (inducible) Superoxide dismutase 1 Toll-like receptor 4 Toll-like receptor 9 a b

Microarraya

Q-PCRb

Fold change log 2

P-value

Fold change log 2

SEM

P-value

CA3 CD40 CD74 CD83 CLU FCN2 IL10 LIPM MyD88 NFkbiA NOD2

−4,98 −0,40 −0,44 −1,30 −0,96 0,80 −0,53 −2,75 −1,11 −0,37 −0,31

0,000 0,069 0,080 0,045 0,038 0,003 0,033 0,003 0,091 0,067 0,530

−2,18 −0,29 −0,49 −0,29 −0,40 0,50 −0,32 −2,25 −0,38 −0,36 −0,60

0,71 0,12 0,19 0,12 0,16 0,22 0,13 0,92 0,12 0,14 0,19

0,055 0,002 0,047 0,053 0,063 0,061 0,009 0,031 0,054 0,066 0,012

NOS2 SOD1 TLR4 TLR9

−0,48 −0,17 −0,21 −0,56

0,004 0,049 0,053 0,043

−1,09 −0,15 −0,32 −0,64

0,33 0,11 0,12 0,31

0,008 0,006 0,042 0,030

Microarray analysis was performed on pooled RNA samples from the two lowest weight gain and the three highest weight gain piglets. Q-PCR were performed on all LWG and HWG animals (n = 8).

could lead to altered resistance to diseases and poor pre and postweaning performances. The results also support that maternal lactocrine signaling is playing an important role in regulation of various biological processes supporting neonatal development. However, the mechanisms by which the short and long-term development of piglet’s immune response is modulated by body weight at birth and milk intake in the first few weeks of life remain to be elucidated. Future studies should address the longitudinal effect of growth performance during lactation on immune and microbiota outcomes in post-weaning piglets.

Table 5 Intestinal villous height and crypt depth in LWG and HWG piglets during the first two weeks of life. Site

HWG

LWG

SEM max

P-value

Ileum crypt depth (μm) Ileum villous height (μm)

0.124 0.478

0.128 0.524

0.006 0.039

0.414 0.414

important role in the early establishment of diverse and stable intestinal bacterial populations. Our findings are supported by recent studies indicating that development of neonatal immune system cannot be completed without the presence of maternal milk factors such as growth and immunomodulatory factors because they have direct impact on the maturation of the neonatal immune system and the regulation of their functions (Turfkruyer and Verhasselt, 2015). As mentioned above, colostrum and milk from different animal species including human, bovine and swine, contain innate immune effectors, such as antibodies, cytokines, and cells, reducing exposure to antigens, modulating the response to these antigens, and enhancing development and maturation of the immature immune system (He et al., 2016). They also contain glycoconjugates that modulate expression of immune signaling genes, repress inflammation at the mucosal surface, alter leukocyte function, and modulate cytokine and TLR expression in intestine epithelial cells (Pacheco et al., 2015). Recent data obtained in our laboratory using the intestinal epithelial cell line IPEC-J2 are in agreement with these studies as we observed that the addition of bovine colostrum whey to IPEC-J2 culture improve healing and reduce activation of NFkB and inflammatory immune response genes induced by killed enterotoxigenic Escherichia coli and Salmonella enterica (Blais et al., 2014, 2015). Finally, several studies performed during the neonatal period in human also indicate that the risk of infection and inflammatory response are reduced in neonates fed human milk (He et al., 2016).

Conflict of interest statement There was no conflict of interest. Authors’ contributions M. Lessard, F. Beaudoin and F. Guay contributed to the conception and design of the experiment. Acquisition, analysis and interpretation of cytometry and PBMC cytokine data were performed by F. Beaudoin, Luca Lo Verso, K. Lauzon and M. Lessard. Analysis and interpretation of microarray data and gene expression by qPCR were performed by M. Blais K. Deschene, K. Lauzon, and N. Bissonnette. All authors have made substantial contributions in writing and revising the final version of the submitted manuscript. Acknowledgements This work was financially supported by Agriculture and Agri-Food Canada and the Canadian Swine Cluster Research Program 1. The authors wish to thank the staff of the Swine Complex under the supervision of Mélanie Turcotte and Steve Méthot for helpful discussions on the experimental design and statistics. Appendix A. Supplementary data

5. Conclusion

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.vetimm.2018.11.005.

In conclusion, the development of mucosal and systemic innate and adaptive immune responses in piglets was influenced by growth performance in the first two weeks of life. Given that growth performance in the first two weeks of live is mainly linked to colostrum and milk consumption, our results infer that early life development and maturation of the immune system in LWG piglets are not following normal processes due to exaggerated responses to proinflammatory stimuli, and

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