Weaning caused imbalanced T lymphocytes distribution and impaired intestinal immune barrier function in piglets

doi:10.21203/rs.3.rs-2368056/v1

A total of 40 piglets with similar body weights were selected in pairs at 21 days old and divided into the suckling group (SG: breastfed by their mothers) and weaning group (WG: weaned at 21 days old). Eight piglets from each group were randomly selected and sacrificed at 24 days (SG3 and WG3) and 28 days of age (SG7 and WG7). The growth performance, T lymphocyte subpopulations, the concentration of cytokines and immunoglobulins, and the expression of Notch2 signaling proteins were determined. The weaning caused a decrease in body weight (P < 0.01) and the ratio of CD3⁺CD4⁺/CD3⁺CD8⁺ T cells in thymus (P < 0.05). Compared to SG3, the concentration of secretory immunoglobulin A (sIgA) in jejunum was decreased, and that of interleukin 2 (IL-2) in serum and ileum, IL-1β and IL-2 in jejunum were upregulated (P < 0.01), while IL-10 in the small intestine was downregulated (P < 0.05) in WG3. Weaning downregulated gene expression of IL-4 and upregulated gene expression of IL-1β, IL-12, and interferon γ (IFN-γ) in small intestine (P < 0.05). Further, weaning downregulated protein expression of Notch2 and Hes1 but upregulated Jagged1 expression in small intestine of piglets (P < 0.05). In summary, weaning caused an imbalance in T lymphocytes distribution, thus impairing the intestinal immune function of piglets, which might be associated with the Notch2 signaling. Furthermore, the impairment of intestinal immune barrier function was more severe at 3 days post-weaning than that at the 7 days post-weaning in piglets.

weaning

intestine

immunity

pigs

Notch2

T lymphocytes

cytokines

The pressure of weaning is a severe challenge for the growth of piglets. Sudden changes in psychology, the environment, and feeding habits expose piglets to large immunological stress[1]. Due to this stress, the intestinal immunity of piglets is affected, making them more susceptible to the invasion of pathogenic microorganisms [2]. However, the underlying mechanisms of impaired intestinal immunity are not clear at present. We hypothesized that the decreased growth of weaning piglets might be associated with the impaired intestinal immune barrier function mediated by T lymphocytes and Notch2 signaling.

Weaning may adversely affect the intestinal immune system of piglets, thereby leading to intestinal inflammation and damage to the mucosal barrier functions [3]. T lymphocytes play a vital role in the regulation of the immune function of piglets. The decreased ratio of CD4⁺/CD8⁺ lymphocytes is an important indicator of immune deficiency [4]. The Notch2 signaling is critical for the control and development of T lymphocytes and blocking the Notch2 signaling pathway decreases T cell proliferation [5]. This study was conducted to investigate the effects of weaning on the growth performance, T lymphocyte subpopulations, concentrations of cytokines and immunoglobulins, and the expression of Notch2 signaling in the small intestine of piglets.

Jejunum and ileum are the main parts of the small intestine, which play a key role in the digestion and absorption of nutrients [6] [7]. Weaning causes shorter villi and higher crypt depth in jejunum and ileum thus resulting in impaired digestion and absorption capacity in piglets and may eventually cause diarrhea [8]. Therefore, special attention should be paid to the jejunum and ileum of weaning piglets, and was, therefore, studied to observe the different effects of weaning.

Different weaning times have different effects on the intestinal immunity of piglets. Early weaning during production results in a shorter reproductive cycle and higher annual productivity for sows [9]. Late weaning can improve the growth performance of piglets and reduce the occurrence of gastrointestinal diseases [10]. Colostrum contains more immunomodulatory substances than late breast milk, which may have a significant impact on the development of the immune system of piglets. However, breast milk cannot provide enough nutrients to meet the needs of the growing piglets [11]. The European Union (EU) banned the use of antibiotics as growth promoters in pigs and livestock production from 1 January 2006, which encouraged pig farms to delay weaning and reduce the stress of weaning for better health of piglets [12]. Weaning at 21 days of age was generally chosen in commercial pig production and in this study too, we have chosen to wean the piglets in the weaning group at 21 days.

Many researchers are also interested in the study of the recovery time of the piglets after weaning. A few studies have suggested that piglets recovered after 9 days, while some others reported that the piglets recovered at 20 days post-weaning [13] [14]. However, most of the studies suggested severe immune stress after weaning during the first week. Hence, we observed the changes in the body weight and the intestinal immune barrier function of piglets 3 days and 7 days post-weaning. The results of this study may guide the pig farming industry, especially for the management of piglets shortly after weaning.

Ethical Approval

The ethics committee of Yangzhou University has approved all animal experimentation procedures (SXXY 2015-0054).

Animal and experimental design

The feeding experiment was carried out at Yangzhou Susheng Ecological Agriculture Development Co., Ltd. in January 2017. A total of 20 pairs of healthy (Duroc×(Landrace×Yorkshire)) 21-day-old piglets with similar body weight were chosen from 8 sows. These sows were of similar body weight and similar parity (3-4 parities, half male and half female). Two piglets (one pair) from the same mother were divided into the suckling group and weaning group (WG), respectively. The piglets were reared in one confined house with controlled temperature and light. The piglets in the WG were randomly divided into two pens while the suckling piglets still lived with their mothers. The piglets in the WG had free access to feed and water, while the piglets in the SG had free access to water and the breast of their mothers.

All the piglets were vaccinated according to the farm routine before the experiment, and no vaccine was administered during the experiment. The daily management, disinfection, and epidemic prevention measures were performed according to the routine procedures of the pig farm. The feed for the weaning piglets was an in-house prepared meal with no zinc oxide or antimicrobial feed additives that may reduce the weaning stress. The nutrient contents of the diet (Table 1) for the weaning piglets were decided according to NRC (2012) [15]. The formula of the sow diet is given in Supplementary Table S1.

Sample collection and preparation

Eight piglets from each group were randomly selected and sacrificed at 24 and 28 days of age, respectively. Before sacrificing, each piglet was weighed, and the blood was collected from the posterior jugular vein. The blood samples were centrifuged at 3000 g for 10 minutes to extract serum, and the serum samples were aliquoted and kept at -80 °C until further analysis. The piglets were sacrificed and dissected after administering an intramuscular injection of sodium pentobarbital (50 mg/kg body weight) 2 hours after their final feeding. The intestines were separated, washed with PBS, and the intestinal segments were then cut open. After drying the water and other impurities on the surface with an absorbent paper, the intestinal mucosa was scraped with slides and placed in a 2 mL cryostorage tube before storing at -80℃ for further analysis [16] [17].

Body weight and organ indexes

All piglets were weighed at the beginning of experiment and on 3 days and 7 days post weaning (24th day and 28th day). The spleen and thymus of the piglets were separated and weighed. The organ index was determined by using the following formula: organ index = organ weight/body weight (g/kg).

Analysis of the activity of LDH

Serum samples stored at -80 °C were equilibrated to room temperature and the supernatant was collected after centrifugation at 3000 × g for 10 minutes. The mucosa samples from the jejunum and ileum were homogenized in normal saline in an ice-water bath at a 1:9 (w/v) ratio. The homogenate was centrifuged (3000× g for 10 minutes), and the supernatant was collected for lactate dehydrogenase (LDH) determination (A020-2, Nanjing Jiancheng Institute of Bioengineering, www.njjcbio.com) using a kit according to the manufacturer's instructions (Jiancheng Bioengineering Institute of Nanjing, Jiangsu, China).

Analysis of T lumphocyte hy flow cytometry

The porcine peripheral blood lymphocyte separator kit (P8770, Beijing Solebold Technology Co., Ltd, Beijing, China, https://www.solarbio.com/) was used to extract lymphocytes from 2.0 ml of fresh heparin sodium anticoagulant blood. To obtain a cell suspension, the particular operation stages were carried out in accordance with the manufacturer's instructions. FITC-CD3 (NO 559582), Alexa 647-CD4 (NO 561472) and PE-CD8 (NO 559584) antibodies (3 μl; 0.2 μg/μl) were added (Biosciences Company, USA, https://www.bdbiosciences.com), and the samples were kept at 4 °C in the dark overnight. On the second day, a FACSAria SORP flow cytometer was used to detect the number of T lymphocyte subsets in the blood (Beckman company, USA).

The spleen and thymus tissues were chopped into small pieces and put on a 300-mesh nylon net before being mashed with the core of a disposable syringe and 2-3 mL of saline added. The liquid under the net was collected in a sterilized dish, and the porcine Tissue Lymph Cell Separation Solution Kit (P6020, Beijing Soleibao Technology Co., Ltd., Beijing, China) was then used to extract the lymphocyte suspension. Next, FITC-conjugated anti-CD3, Alexa 647-conjugated anti-CD4 and PE-conjugated anti-CD8 antibodies (3 μl; 0.2 μg/μl) were added to the lymphocyte suspension and incubated overnight in the dark at 4 °C. On the second day, the numbers of T lymphocyte subsets in the spleen and thymus were evaluated.

Analysis of the content of cytokines and immunoglobulins by ELISA

An ELISA (Enzyme Linked Immunesorbent Assay) kit was used to measure the concentrations of immunoglobulin A (IgA, NO H108), immunoglobulin G (IgG, NO H106), interleukin-1 beta (IL-1β, NO H002), interleukin-2 (IL-2, NO H003), interleukin10 (IL-10, NO H009), interleukin-12 (IL-12, NO H010), Interferon-γ (IFN-γ, NO H025), tumour necrosis factor-alpha (TNF-α, NO H05 (Nanjing JianCheng Bioengineering Institute, Jiangsu, China) in the serum, jejunum and ileum according to the manufacturer's instructions (Nanjing JianCheng Bioengineering Institute, Jiangsu, China).

Quantitative real-time PCR analysis of relative mRNA expression

TRIzol was used to extract total RNA from mucosa of jejunum and ileum (Invitrogen, Shanghai, China). Following the manufacturer's instructions, RNA was utilized to generate complementary DNA (cDNA) using PrimeScript^TMRT reagent Kit with gDNA Eraser (TaKaRa Biotechnology Co. Ltd., Dalian, China). qRT-PCR was performed on cDNA using an Applied Biosystems 7500 Real-Time PCR System (Life Technologies, USA). To quantitatively analyze the target gene, the primers were diluted to 10 μM and β-actin was utilized as an internal reference. Primer-BLAST (http://www.ncbi.nlm.nih.gov) was used to create gene-specific primers. Suzhou Jinweizhi Biotechnology Co., Ltd. produced the primer sequences, which are presented in Table 2. (Jiangsu, China). The relative expression of the target gene was calculated using the comparative 2^-ΔΔCT approach [18].

Western blot analysis of relative protein expression

Protein was extracted from intestinal mucosal tissue using a total protein extraction kit (SunShinebio, Nanjing, China) as directed by the manufacturer. Protein concentrations were determined using the BCA technique (Thermo Scientific, Shanghai, China). Using % SDS–PAGE (SDS–polyacrylamide gel electrophoresis), equal quantities of protein were separated and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking for 1 h at room temperature [19], the membranes were incubated overnight with the following primary antibodies: anti-NOTCH2 (1:500, BioByt, Cambridge, UK), anti-DLL1 (1:500, BioByt, Cambridge, UK), anti-Jagged1 (1:800, Abcam, Cambridge, UK), anti-HES1 (1:500, LSBio, WA, USA), and anti-GAPDH (1:500, LSBio, WA, USA) (1:2000, Cell Signalling, MA, USA). After being thoroughly washed to remove nonspecific binding, the membranes were incubated for 1 hour at room temperature with a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG) (Boster Biological Technology Co., Ltd., USA). The immunoreactive bands were washed before being detected using an ECL western blotting detection method. For densitometric detection of band intensity, ImageJ software (Wayne Rasband, MD, USA) was utilized. The band density of each blot was standardized using the density of a reference sample and the GAPDH content.

Statistical analysis

Experimental data were analyzed by SPSS 23.0. Growth performance and organ weight data were analyzed by an independent t test. Other data were analyzed by two-factors analysis of variance with two fixed factors of time (T) and group (G). Independent t test was further conducted to investigated the difference between the SG and the WG on different time point post weaning. P values between 0.05 and 0.10 were identified as with a trend, and differences were identified as significant when P < 0.05. All data are shown as the mean ± standard error.

Growth performance and immune organ index

The results indicated that the body weight of piglets in the WG was lower than that in the SG at 3 days post-weaning and 7 days post-weaning (P < 0.01) (Figure 1A). There was no significant difference in the organ index of the spleen and thymus obtained from WG and SG (P > 0.05) (Figure 1B).

Lactate dehydrogenase (LDH) in the serum and small intestine

The concentration of LDH in the serum from the WG was increased as compared to SG, especially 3 days after weaning (P < 0.05) (Figure 2A). The levels of LDH in the jejunum and ileum were further upregulated at 7 days post-weaning (P < 0.05) (Figures 2B and 2C).

Number of T lymphocytes in the blood, thymus, and spleen

The number of CD3⁺ T cells in the blood was lower in the WG (P < 0.05) as compared to the SG, whereas those in the thymus and spleen were higher in the WG (P < 0.05). Furthermore, the number of CD3⁺CD4⁺ T cells in the thymus and the ratio of CD3⁺CD4⁺/CD3⁺CD8⁺T cells in the thymus and spleen of WG were lower than that in the SG (P < 0.05), especially 3 days post-weaning (Figures 3A-D).

Level of immunoglobulins and cytokines in the serum and intestine

There was no significant difference in the IgA and IgG concentrations in the serum between the SG and WG (P > 0.05) (Figure 4A). The sIgA concentration in the jejunum of WG3 was lower than that in the SG3 (P < 0.05) (Figure 4B). The concentrations of interleukin 2 (IL-2) in the serum and ileum, and that of IL-1β and IL-2 in the jejunum of WG3 were higher than that in SG3 (P < 0.01). Further, the concentration of IL-2 in the jejunum of WG7 was higher than that in SG7 (P < 0.01). In addition, the IL-10 concentrations in the small intestine of WG3 and jejunum of WG7 were lower than that in SG3 and SG7, respectively (P < 0.05) (Figures 4C-E).

Gene expression of cytokines in the small intestine

The gene expression of IL4 was downregulated in the jejunum, while that of IL-12 (P < 0.05) and interferon γ (IFN-γ) (P < 0.01) in the jejunum, and IL-1β in the ileum were upregulated in the WG in comparison with SG (P < 0.05). Further, the gene expression of IL-2 and IL-4 in the ileum of WG3 was higher than that in SG3 (P < 0.05). Considering the WG7 group, the gene expression of IL-4 was decreased, while that of IL-12 and IFN-γ in the jejunum and IL-1β, IL-2, and IFN-γ in the ileum were upregulated as compared to SG7 (P < 0.05) (Figures 5A and 5B).

Gene and protein expression of Notch2 signaling in the small intestine

The gene expression of Jagged1 in the ileum of WG3 was lower than that in SG3 (P < 0.01). Similarly, the gene expression of Notch2 and Jagged1 in the jejunum and Delta-like 1 (DLL1) in the ileum were downregulated (P < 0.05) (Figures 6A and 6B). Further, the protein expression of Hes1 and Notch2 in the jejunum was downregulated, while Jagged1 in the ileum was upregulated in WG when compared with SG (P < 0.05) (Figures 6C-F).

The immune system of piglets is not fully developed at weaning time. The immature adaptive immune system along with the change in the feed types and the living conditions make the piglets susceptible to the invasion of pathogenic microorganisms, and result in diarrhea and decreased growth [20]. However, the underlying mechanisms of these observations were not elucidated yet. We hypothesized that the diarrhea and decreased growth of weaning piglets might be associated with the T lymphocytes and Notch2 signaling caused impaired intestinal immune function. This study was conducted to investigate the effects of weaning on the growth performance and intestinal immune function of piglets. The results of this study may guide the pig raising industry, especially for the management of piglets shortly after weaning.

In this study, weaning caused a decrease in the body weight of piglets 3 days and 7 days post-weaning, which might be associated with the transition from breast milk to a solid diet and weaning stress. Previous studies also suggested that the piglets had a significant weight loss after weaning [6] [21] [22]. In addition, the body weight of the piglets was severely affected 3 days post-weaning, while it was better 7 days post-weaning. The piglets gradually adapted to the changes in the environment and diet 7 days after weaning, and the gastrointestinal digestive function gradually became matured driven by the adaptive immune system. A previous study had reported a similar trend wherein severe body weight loss was observed on day 3 after weaning and the piglets gradually regained weight 9 days after weaning [23]. Thus, these results indicated that the piglets experienced severe stress 3 days post-weaning, but the stress response was relieved 7 days post-weaning.

LDH catalyzes the mutual conversion of pyruvate and lactic acid and participates in the catabolism and anabolism of carbohydrates. During anaerobic glycolysis, ATP is produced when pyruvate is converted to lactic acid by LDH [24]. LDH is present in various tissues of animals and is an important indicator of the stress state [25]. Weaning causes stress reactions in piglets, including diarrhea, decreased feed intake, and increased LDH activity [6] [25]. The reduced feed intake of weaned pigs causes an increased LDH concentration in the serum [26]. In this study, weaning caused an increased content of LDH in the serum, especially 3 days after weaning. The stress state was severe up to 3 days of weaning while the level of LDH at 7 days post-weaning was alleviated, thereby confirming that the weaning stress may have a greater impact on young piglets on the first few days after weaning.

T lymphocytes play a vital role in the regulation of the immune function of piglets. Herein, we focused on the number of CD3⁺ T lymphocytes and its two major subsets, CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocytes. CD3⁺ T lymphocytes accounted for 30% ~ 70% of T lymphocytes. The other T lymphocytes, including CD4⁺CD45RA⁺, CD28⁺, CD38⁺, and CD95⁺ T lymphocytes, were not detected in the study [27]. The CD2⁺, CD3⁺, CD4⁺, CD8⁺, and other molecules on the surface of T lymphocytes are closely related to T lymphocyte recognition, antigen presentation, and immune function [28]. CD3⁺ T cells represent mature T lymphocytes and are associated with T cell receptor expression and signaling [29]. CD4⁺ T cells assist B cells to secrete antibodies and their activation can enhance the binding of TCR-CD3 complexes to MHC class II antigens and induce macrophages to produce a strong bactericidal effect [30]. CD8⁺ is the receptor of MHC class I molecules and an important marker of cytotoxic T cells. Its activation helps the cytotoxic T cells mediate the damage of target antigens by releasing perforin and granulation enzymes [31]. The major cause of illness in pigs is a decline in the number and function of CD4⁺ T cells among the T cell subsets [32]. In this study, weaning caused a reduction in CD3⁺CD4⁺ T cells in the thymus of piglets, especially 3 days post-weaning, which might explain the decreased ratio of CD4⁺/CD8⁺ lymphocytes to some extent. The decreased ratio of CD4⁺/CD8⁺ lymphocytes is an important indicator of immune deficiency and suggests that the piglets may be infected by pathogens [4]. The ratio of CD3⁺CD4⁺/CD3⁺CD8⁺ T cells in the thymus and spleen of WG3 was lower than that in the SG3, which suggested an immune deficiency in weaning piglets 3 days post-weaning, thereby emphasizing a need for strengthening the daily management during this period.

The intestine is the main point for the entry of pathogens. sIgA is the most abundant antibody in the intestine that defends the intestinal epithelium against harmful bacteria, modulates antigen capture, and aids in the immunological protection of the intestinal mucosa [33] [34] [35]. Breastfeeding has a long-term impact on future health and is an important physiological factor that affects the development of intestinal function and the immune system [36]. Many studies have shown that colostrum and breast milk contain high concentrations of sIgA, and its synthesis in animals after weaning mainly depends on their adaptive immune system [37]. The adaptive immune system of piglets is not fully developed at weaning time, and the level of sIgA in the small intestine decreases after weaning. The results of our study were in accordance with the previous studies [12] [13], where weaning caused a decreased concentration of sIgA in the small intestine 3 days post-weaning. The reason for the decreased IgA concentration in the intestine after weaning could be attributed to the reduced availability of the resources of IgA, colostrum and breast milk after weaning when the synthesis of sIgA mainly depends on the still weak adaptive immune system of animals. In addition, our study also showed that the concentration of sIgA in WG returned to the level of lactation 7 days after weaning, which may be due to the maturation of the immune system of piglets stimulated by weaning stress, which can promote the synthesis of intestinal sIgA in piglets. However, a previous study suggested that the concentration of IgA increased in the intestine 20 days after weaning [2]. The different results between the two studies may be due to the choice of different time points. The time points chosen in the earlier study were 4-, 20- and 40-days post-weaning and the IgA levels were not tested between 4 and 20 days post-weaning [2]. Thus, our results suggested that the intestinal immune barrier function of piglets was impaired after weaning, but after a period of adaptation, the synthesis of sIgA in the intestinal mucosa increased to a level like that of suckling piglets, which indicated that the immune barrier function of the intestine was recovered.

Cytokines play a key role in the regulation of intestinal immune function [34] [35]. Tumor necrosis factor alpha (TNF-α), IL-1β, and IFN-γ are proinflammatory cytokines that may impair intestinal epithelial barrier function and tight junction formation [38] [39]. However, some anti-inflammatory cytokines, such as IL-10, may downregulate the expression of pro-inflammatory cytokines (such as IL-6, TNF-α, and IL-1β), thus maintaining intestinal immune homeostasis [40] [41] [42]. IL-4 is a key regulator of the antibody-mediated immune response, influencing B cell proliferation, T cell growth and function, and immunoglobulin conversion [43]. Some studies have shown that weaning increases pro-inflammatory cytokines in the intestines of piglets, and the mRNA expression of IL-1β, IL-6, and TNF-α rises dramatically, which is connected to early inflammation and various intestinal disorders in piglets [44]. Furthermore, there are earlier reports demonstrating that the gene expression of TNF-α and IL-6 rises at 3 and 7 days after weaning and recovers to pre-weaning levels after 14 days. However, within 2 weeks after weaning at 21 days of age, no significant changes in the mRNA expression of inflammatory cytokines (TGF-β and IL-10) are observed in piglets [45]. In our study, weaning caused an increase in the level of IL-1β in the jejunum while decreasing the concentration of IL-10 in the jejunum and ileum. The increased concentration of pro-inflammatory cytokines and decreased concentration of anti-inflammatory cytokines suggested an impaired intestinal barrier function. In addition, 7 days after weaning, the gene expression of IL-4 in the jejunum was decreased. Our results were in accordance with a previous study, which also demonstrated a marked decrease in levels of IL-4 in mice lacking enteral feeding [46]. Further, the gene expression of IL-4 in the ileum was also upregulated, especially 3 days after weaning. In addition, the gene expression of IFN-γ in jejunum and IL-1β and IFN-γ in ileum were also upregulated at 7 days after weaning. Our results are consistent with a previous study, which demonstrated that the gene expression of cytokine increases sharply in the early acute weaning stage and the inflammatory response is obvious, while in the adaptation stage of weaning defense, the intestinal mucosal immune function recovers, and the gene expression of cytokines returns to the pre-weaning level [13]. IL-2 can induce T lymphocyte proliferation and differentiation, and its concentration directly reflects immune function [47]. Studies have shown that IL-2 can induce the pathologic opening of the intestinal tight junction barrier and increase intestinal epithelial permeability [48] [49]. In this study, the concentrations of IL-2 were upregulated in the serum and small intestine and the gene expression of IL-2 was also significantly increased in the ileum of weaned piglets, indicating a weaker intestinal barrier function in weaning piglets. In addition, both the anti-inflammatory and pro-inflammatory cytokines are secreted by Th1 cells and Th2 cells [50]. In the immune response system, the Th1/Th2 dynamic balance is an important factor in maintaining immune stability [51]. Once the balance is disturbed, changes in Th1 or Th2 cells can cause diseases. Hence, weaning causes an imbalance between anti-inflammatory and pro-inflammatory cytokines that may lead to impaired immune barrier function and destruction of the immune response dominated by the Th1/Th2 balance. However, in this study, the concentration of some cytokines in the intestine was not completely consistent with the gene expression of these cytokines. The reasons might be that the mRNA expression could not accurately reflect the quality and quantity of protein expression due to several processes involved before the mRNA is translated to the protein, including storage, transport, degradation, translation regulation, and post-translational processing [52].

The Notch2 signaling pathway affects the function of the intestinal immune system and plays a key role in the maintenance of intestinal immunological homeostasis [18]. The Notch2 signaling is critical for the control and development of T lymphocytes and blocking the Notch2 signaling pathway decreases T cell proliferation [5]. DLL1 mainly induces hematopoietic stem cells and thymic lymphocytes to differentiate into T cells and promotes the development of precursor T cells [53]. Jagged1 also promotes T cell development and activates the T cell response [54]. In this study, weaning caused the inhibition of the gene expression of Notch2, Jagged1, and DLL1 and the downregulation of the protein expression of Notch2 and Hes1 in the small intestine of piglets, which might explain the imbalanced T lymphocytes distribution and the variations in the concentration of cytokines in our previous results. However, the protein expression of Jagged1 in the ileum of weaning piglets was upregulated in this study, which is in line with the observation in a previous study where Jagged1 expression was upregulated in the intestine of mice with intestinal inflammatory injury [55]. The reason for the upregulated protein expression of Jagged1 in weaning piglets is not understood yet and needs further research. Nevertheless, our results suggest that the impaired immune barrier function in the intestine of weaning piglets might be associated with Notch2 signaling.

In conclusion, weaning caused decreased body weight, imbalanced T lymphocytes distribution, lowered concentration of sIgA and anti-inflammatory cytokines, and increased concentration of pro-inflammatory cytokines in piglets. The impairment of intestinal immune barrier function, which might be associated with the Notch2 signaling was more severe 3 days post-weaning than that at 7 days post-weaning in piglets.

Authors contribution

L.D., L.H.Y, and H. R. W. designed the research; L.D., L.H.Y, M.X.W., H.M.L., .Z.P., T.Q., Y.Y.Y., and H.R.W.. made the investigation; L.D., and M.X.W., made formal analysis and data curation; L.D., and M.X.W. wrote the original draft and had the funding acquisition; L.D., L.H.Y, and H. R.W., reviewed and edited the paper, and L.H.Y, had primary responsibility for the final manuscript. All authors read and approved the final manuscript.

Conflict of interest

There are no conflicts of interest to declare.

Funding

This work was supported by grants from the Special project of Science and Technology of North Jiangsu Province (grant numbers SZ-YC202101) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Availability of data and materials

The datasets analyzed in the present study are available from the corresponding author on reasonable request.

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Table 1 Composition and nutrient levels of basal diets (air-dry basis) (%）

Ingredient	Content	Nutrition levels¹	Content
Corn	60.50	DE（MJ/kg）	14.11
Fish meal	5.00	CP	20.21
Corn gluten meal	5.00	Ca	0.76
Soybean oil	1.00	AP	0.45
Soybean meal	24.00	Lys	1.25
Limestone	1.18	Met	0.43
CaHPO₄	1.30	Thr	0.71
L-Lys	0.60	Trp	0.16
Met	0.13
Thr	0.17
Ser	0.02
Choline chloride	0.10
NaCl	0.40
Premix²	0.60
Total	100.00

¹Nutrient levels were calculated values.

² The premix provided the following per kilogram of the diet: VA 6 000 IU, VD₃400 IU, VE 30 mg, VK₃2 mg, VB₁3.5mg, VB₂5.5 mg, VB₆3.5 mg, VB₁₂25.0μg, biotin 0.05 mg, folic acid 0.3 mg, D-pAntothenic acid 20 mg, niacin 20 mg, choline chloride 500 mg, Fe (as ferrous sulfate) 110 mg, Zn (as zinc sulfate) 100 mg, Cu (as copper sulfate) 20 mg, Mn (as manganese sulfate) 40 mg, Se (as sodium selenite) 0.30 mg, I (as potassium iodide) 0.40 mg.

Table 2 Primer parameters used in quantitative real-time PCR¹

Gene			Accession No.	Primer sequence（5’to 3’）	Amplicon size, bp
	Β-Actin		DQ845171.1	F AGGCCAACCGTGAGAAGATG	122
				R CATGACAATGCCAGTGGTGC
		IL-1β	NM_001302388	F GTGGCAGGACCTACACTCTTC	115
				R TTCCTTCAGAATGCCGTCCTC
		IL-2	FJ543109.1	F GGAGCCATTGCTGCTGGAT	116
				R ATTCTGTAGCCTGCTTGGGC
		IL-10	NM_214041	F GTGGCAGCCAGCATTAAGTC	103
				R AACTCTTCACTGGGCCGAAG
		IL-12	NM_214097.2	F CTCCCCCAAATCACATCCAATA	110
				R ATTCCCTCTCATTTCCTTGGGG
		TNF-α	NM_214022	F GCCCTTCCACCAACGTTTTC	97
				R CAAGGGCTCTTGATGGCAGA
		IFN-γ	NM_213948	F GGCCATTCAAAGGAGCATGGA	144
				R TCACTGATGGCTTTGCGCT
		Notch2	XM_021090690	F GCCCGGCAGGATGAATGATTAG	99
				R CCCGACATTGCAGTGCTTCT
		DLL1	XM_005659096	F ATCGCCACCGAGGTGTAAAG	103
				R CCTCTCTCAGCAGCATTCGT
		JAG1	XM_005672699	F TTTCAGGGCGACCTTGCATC	121
				R CCACACCACACCTTCGAGC
		Hes1	NM_001195231	F GTGAGTGCATGAACGAGGTG	118
				R GTCATGGCGTTGATCTGGGT

¹IL-1β: interleukin 1β; IL-2: interleukin 2; IL-10: interleukin 10; IL-12: interleukin 12; TNF-α: tumor necrosis factor α; IFN-γ: interferon-γ; Notch2: DLL1: Delta-like; JAG1: Jagge

No competing interests reported.

Supplementary.docx

Weaning caused imbalanced T lymphocytes distribution and impaired intestinal immune barrier function in piglets

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