Peyer's Patch Lymphocytes Research Articles

Lipid Compositions of Total Parenteral Nutrition Affect Gut Peyer's Patches and Morphology in Mice

IntroductionAlthough parenteral nutrition (PN) is the only option for providing adequate nutrition to patients who cannot tolerate oral ingestion, it severely impairs intestinal barrier function in terms of morphology and immunity. While addition of either soybean oil (SO) or fish oil (FO) to PN partially reverses these defects, the effects of the oil composition (FO/SO ratio) on morphology and gut-associated lymphoid tissues (GALT) have yet to be elucidated. We focused on the effects of the FO/SO ratio in PN on the number of lymphocytes in Peyer's patches, immunoglobulin A levels, and intestinal structures. MethodsMale ICR mice (n = 61) were randomized into five groups; oral nutrition (Chow, n = 14) and four groups receiving PN without oral nutrition. PN solutions contained fat emulsions with the following FO:SO ratios: 0:1 (SO, n = 12), 1:11.5 (11.5FSO, n = 17),1:2 (1:2FSO, n = 13) and 1:0 (FO, n = 5). All mice underwent jugular vein catheter insertion. The PN groups were given isocaloric and isonitrogenous nutritional support with 20% of total calories from fat emulsions with equivalent fat delivery in 11.9 g/kg/d. After 5 d of each feeding, Peyer's patches lymphocytes were isolated from the small intestine, counted and analyzed with flowcytometry for determination of their phenotypes (αβTCR+, γδTCR+, CD4+, CD8+ and B cells). Villus height and crypt depth of the jejunum and ileum were evaluated with hematoxylin-eosin staining. Immunoglobulin A levels in the intestinal washings were also determined. ResultsNumbers of total lymphocytes and B lymphocytes in PP were increased in the 1:2 FSO-PN but neither in the 1:11.5 FSO nor the FO group, as compared to the SO group. There were no marked differences among the groups in numbers neither of total T cells nor in any of T cell phenotypes determined. The 1:2 FSO group showed significantly greater villus height and crypt depth than the SO group. IgA levels did not differ significantly among the four PN groups. ConclusionsThe PN with 1:2 FSO (FO:SO = 1:2) maintained lymphocyte numbers in PP and intestinal villus morphology at levels nearly the same as those obtained with chow feeding. An appropriate ratio of FO to SO in PN is expected to prevent immunological impairment and morphological atrophy of the gut associated with lack of oral nutrition.

Chronic heat stress induces the disorder of gut transport and immune function associated with endoplasmic reticulum stress in growing pigs.

Although high temperatures influence gut health, data on underlying mechanisms remains scant. Using a pig model, this study performed a global analysis on how chronic heat stress affects the transport and immune function of the gut through transcriptome, proteome, microbial diversity and flow cytometry. A total of 27 pigs with similar body weights were assigned into 3 groups, control (Con) group (23 °C), chronic heat stressed (HS) group (33 °C), and pair-fed (PF) group, in a controlled environment for 21 days. Our results showed that pigs in the HS group had reduced growth performance and diminished height of ileal villi (P < 0.01). Transcriptome and proteome analyses demonstrated notable modification of expression of nutrients and ion transport-related transporters and gut mechanical barrier-related genes by chronic heart stress (P < 0.05), suggesting damage of transport functions and the gut barrier. Chronic heat stress-induced endoplasmic reticulum stress also increased the synthesis of misfolded proteins, leading to upregulation of misfolded protein degradation and synthesis, as well as vesicle transport disorder (P < 0.05). Energy supply processes were enhanced in the mitochondrion (P < 0.05) to maintain biological processes with high energy demands. Furthermore, chronic heat stress activated complement cascade response-related genes and proteins in the gut mucosa (P < 0.05). Our flow cytometry assays showed that the proportion of gut lymphocytes (CD4+ T cells, T cells, B cells in Peyer's patch lymphocytes and CD4+ CD25+ T cells in intraepithelial lymphocytes) were significantly altered in the HS group pigs (P < 0.05). In addition, the occurrence of gut microbial dysbiosis in the HS group pigs was characterized by increased potential pathogens (e.g., Asteroleplasma, Shuttleworthia, Mycoplasma) and suppression of beneficial bacteria (e.g., Coprococcus and Aeriscardovia), which are associated with gut immune function. Altogether, our data demonstrated that chronic heat stress induced gut transport and immune function disorder associated with endoplasmic reticulum stress in growing pigs.

Effects of Long-time Exposure to Lipopolysaccharide on Intestinal Lymph Node Immune Cells and Antibodies Level in Mice.

Endotoxin, widely present in the living environment of humans and animals, leads to endotoxemia during a short period. However, the long-term effects of endotoxin on immune function are unclear. To determine the importance of long-term endotoxin treatment on function of immune system. The mice were treated with different doses of lipopolysaccharide (LPS) for a month; the collected samples were then analyzed in terms of value changes in hematological parameters, lymphocyte subtypes, and immunoglobulins level. The number of monocytes (MONO) and neutrophils (NEU) in the three treatment groups was significantly lower than the control after 30 days. However, the proportion of CD8+ T lymphocytes showed a rising trend in the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) while the CD4+ T cell was reduced. At the same time, a decrease was observed in the percentage of CD19+CD38+ B lymphocytes. Interestingly, the change of lymphocytes in PPs was more significant than that in MLNs, suggesting that immune response in the PPs occurred before the MLNs. Consistent with the changes in B cells, the content of IgA and IgG showed a downward trend. Long-term exposure to low-dose endotoxin had little or no effect on the immune function of the body, suggesting that the endotoxin can be rapidly eliminated by the immune system. Nonetheless, the number of immune cells was reduced in the high-dose group. T- and B-lymphocytes were significantly reduced, resulting in a decrease in immunoglobulin level, and showing a significant immune suppression state.

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches.

Naïve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer's patches (PPs) or mesenteric lymph nodes, are where naïve lymphocytes sense antigens. Naïve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Effect of Lentinan on Peyer's patch structure and function in an immunosuppressed mouse model

Lentinan (LNT), a polysaccharide isolated from Lentinus edodes, has been shown to stimulate immune response. Despite its widespread use owing to its health benefits, epidemiologic and experimental studies that address the biological activities of LNT after oral administration to animals or humans are rare. In this study, the effects of LNT on the intestinal mucosal immune system of immunosuppressed mice were investigated. The number and size of the Peyer's patches (PPs), proliferation ability of PP lymphocytes, T and B lymphocyte percentage, and T-cell activation proportion, as well as the number of M-like cells in PPs, were determined. The antigen transfer ability of M-like cells and intestinal secretory immunoglobulin A (IgA) levels were also detected. After oral administration of LNT for 7 days, the number of PPs, lymphocytes in PPs, and the level of intestinal soluble IgA in immunosuppressed mice were increased. LNT maintained the percentage of T and B lymphocytes and upregulated the proportion of activated T cells in PPs. Furthermore, the number of M-like cells, which were differentiated from intestinal epithelial cells, and their antigen transfer ability were enhanced. These results indicate that orally administered LNT can improve the immune status of the intestinal mucosa in immunosuppressed mice.

Oral administration with chitosan hydrolytic products modulates mitogen-induced and antigen-specific immune responses in BALB/c mice.

The aim of this study was to investigate whether oral administration in BALB/c mice with chitosan hydrolytic products including chitosan hydrolysate, LMWC and a chitooligosaccharide mixture (oligomixture), modulates mitogen-induced and antigen-specific immune responses. A water-soluble chitosan hydrolysate was obtained via cellulase degradation of chitosan, and a LMWC and the oligomixture were separated from this hydrolysate. In non-immunized mice, both the chitosan hydrolysate and oligomixture significantly increased the phagocytic activity of peritoneal macrophages. Three chitosan hydrolytic products significantly increased the mitogen-induced proliferation of splenocytes and Peyer's patch (PP) lymphocytes. LMWC and oligomixture up-regulated IFN-γ secretion, and induced predominantly Th1 cytokine secretion in splenocytes. In antigen-specific immunity, similar effects of the chitosan hydrolytic products were observed on augmenting ovalbumin (OVA)-, as well as mitogen-, induced proliferation of splenocytes harvested from OVA-immunized mice. Interestingly, oligomixture was the most potent chitosan hydrolytic product to elicit OVA-specific IgM, IgG, and IgA production, while LMWC was the most potent one to elevate splenic IFN-γ production and IFN-γ/IL-4 (Th1/Th2) ratio. These results provide the distinct immunomodulatory properties of chitosan hydrolytic products in response to mitogens and specific antigen, paving the way for further development and application of dietary chitosan hydrolytic products against immune disorders and infection.

Influences of Short-Term Fasting and Carbohydrate Supplementation on Gut Immunity and Mucosal Morphology in Mice.

Preoperative carbohydrate (CHO) supplementation has been recommended in enhanced recovery after surgery protocols. However, the effects of CHO supplementation on gut and systemic immunity are not well understood. Mice (n = 60) were randomized to 1 of the following 5 groups: control (ad lib feeding), 12-hour fasting without CHO administration (fasting), and 12 hours of fasting with CHO administration at 2, 4, and 8 hours before sacrifice. Then, lymphocytes were isolated from gut-associated lymphoid tissue, that is, Peyer's patches, the intraepithelial space, and the lamina propria of the small intestine. These lymphocyte numbers and phenotypes were evaluated. IgA levels in respiratory and small-intestinal washings were determined by ELISA. Morphology, proliferation, and apoptosis of the intestinal epithelium were also evaluated histologically. Although there were no significant differences in IgA levels among the 5 groups, fasting decreased intraepithelial and lamina propria, but not Peyer's patches lymphocyte numbers. CHO at 2 hours prevented lymphocyte loss in intraepithelial, whereas CHO at 4 hours reversed lamina propria lymphocytes numbers. Percentages of lymphocyte phenotypes were similar in each site among the 5 groups. Fasting caused villous atrophy; however, CHO at 2 hours restored villous structure along with maintenance of epithelial cell proliferation rate. Only 12 hours of fasting causes marked gut-associated lymphoid tissue cell loss along with gut atrophy. However, CHO at 2 hours preserves gut immunity and morphology not completely but moderately.

Effects of semielemental diet containing whey peptides on Peyer's patch lymphocyte number, immunoglobulin A levels, and intestinal morphology in mice

BackgroundEnteral nutrition (EN) is the gold standard of nutritional therapy for critically ill or severely injured patients, because EN promotes gut and hepatic immunity, thereby preventing infectious complications as compared with parenteral nutrition. However, there are many EN formulas with different protein and fat contents. Their effects on gut-associated lymphoid tissue remain unclear. Recently, semielemental diets (SEDs) containing whey peptides as a nitrogen source have been found to be beneficial in patients with malabsorption or pancreatitis. Herein, we examined the influences of various dietary formulations on gut immunity to clarify the advantages of SEDs over elemental diets. MethodsForty-four male Institute of Cancer Research mice were randomized to four groups: chow (CH: n = 5), intragastric total parenteral nutrition (IG-TPN: n = 13), elemental diet (ED: n = 13), and SED (n = 13). The CH group received CH diet ad libitum, whereas the IG-TPN, ED (Elental, Ajinomoto, Japan), and SED (Peptino, Terumo, Japan) groups were given their respective diets for 5 day via gastrostomy. After 5 days, the mice were killed to obtain whole small intestines. Peyer's patch (PP) lymphocytes were harvested and counted. Their subpopulations were evaluated by flow cytometry. Immunoglobulin A (IgA) levels in intestinal and respiratory tract washings were measured with enzyme-linked immunosorbent assay. Villous height (VH) and crypt depth in the distal intestine were measured by light microscopy. ResultsSED increased the PP cell number and intestinal or respiratory IgA levels to those of CH mice, while ED partially restored these parameters. The IG-TPN group showed the lowest PP cell number and IgA levels among the four groups. VH was significantly greater in the CH than in the other groups. VH in the ED and SED groups also exceeded in the IG-TPN group, while being similar in these two groups. No significant crypt depth differences were observed among the four groups. ConclusionsSED administration can be recommended for patients unable tolerate complex enteral diets or a normal diet in terms of not only absorption and tolerability but also maintenance of gut immunity.

The silencing effect of miR-30a on ITGA4 gene expression in vitro: an approach for gene therapy.

Integrins are adhesion molecules which play crucial roles in cell-cell and cell-extracellular matrix interactions. Very late antigen-4 or α4β1 and lymphocyte Peyer’s patch adhesion molecule-1 or α4β7, are key factors in the invasion of tumor cells and metastasis. Based on the previous reports, integrin α4 (ITGA4) is overexpressed in some immune disorders and cancers. Thus, inhibition of ITGA4 could be a therapeutic strategy. In the present study, miR-30a was selected in order to suppress ITGA4 expression. The ITGA4 3' UTR was amplified, cloned in the Z2827-M67-(ITGA4) plasmid and named as Z2827-M67/3'UTR. HeLa cells were divided into five groups; (1) untreated without any transfection, (2) mock with Z2827-M67/3'UTR transfection and X-tremeGENE reagent, (3) negative control with Z2827-M67/3'UTR transfection alone, (4) test with miR-30a mimic and Z2827-M67/3'UTR transfection and (5) scramble with miR-30a scramble and Z2827-M67/3'UTR transfection. The MTT assay was performed to evaluate cell survival and cytotoxicity in each group. Real-time RT-PCR was applied for the ITGA4 expression analysis. The findings of this study showed that miR-30a downregulated ITGA4 expression and had no effect on the cell survival. Due to the silencing effect of miR-30a on the ITGA4 gene expression, this agent could be considered as a potential tool for cancer and immune disorders therapy.

A novel Lactobacillus plantarum strain P-8 activates beneficial immune response of broiler chickens

To investigate whether Lactobacillus plantarum P-8 may be used as an alternative to antibiotics in the broiler chicken diet, we compared P-8 and antibiotics for their immunobiotic properties and their effect on growth performance of broiler chickens in a 42-day trial. The results showed that P-8 provided similar benefits in weight gain, feed intake and feed efficiency as antibiotics did. Importantly, P-8 activated protective immune responses of the broilers while antibiotics lacked this effect. P-8 induced higher fecal secretory IgA (sIgA) levels on day 42 (P≤0.027) and IgA+ lymphocytes in the jejunum and Peyer's patches (PP) (P<0.001) compared to antibiotic treatment. Antibiotics reduced the IgA+ lymphocytes in jejunum and PP on day 42 compared to the control. P-8 increased CD3+ T cells in the small intestinal tissues in most test situations whereas antibiotics had fewer CD3+ cells in PP and cecal tonsil compared with the control broilers at the end of the trial. In addition, P-8 increased CD4+ T cells significantly in the intestinal tissues compared to both antibiotics and the control (P<0.0052). Both Th1 and Th2 cytokine expression were enhanced by P-8 on day 14, consistent with the clinical trial results showing probiotic benefits in diseases. Antibiotics up- and down-regulated interleukin (IL)-2, IL-4 and IL-10 transcripts in an age-dependent manner, and showed anti-inflammatory potential. These data indicate that P-8 may provide protective immune response to broilers while maintaining similar growth performance and may be a potential alternative to antibiotics supplemented in chicken feeds.

MON-PP065: Semi-Elemental Diet Maintains Peyer Patch Lymphocyte Number and Immunoglobulin a Levels in Mouse Small Intestine as well as a Normal Diet

MON-PP065: Semi-Elemental Diet Maintains Peyer Patch Lymphocyte Number and Immunoglobulin a Levels in Mouse Small Intestine as well as a Normal Diet

Parenteral glutamine supplementation in combination with enteral nutrition improves intestinal immunity in septic rats

ObjectivesThe gut-associated lymphoid tissue is continuously exposed to antigens in the gut lumen and becomes the first line of defense against enteric bacteria and associated toxin. The aim of this study was to investigate the effects of parenteral glutamine (GLN) supplementation in combination with enteral nutrition (EN) on intestinal mucosal immunity in septic rats by cecal ligation and puncture (CLP). MethodsMale Sprague-Dawley rats were randomly assigned into four groups: A sham CLP + EN + saline group (n = 10), a sham CLP + EN + GLN group (n = 10), a CLP + EN + saline group (n = 10), and a CLP + EN + GLN group (n = 10). At 2 h after CLP or sham CLP, all rats in each of the four groups received an identical enteral nutrition solution as their base formula. Then, the rats in the sham CLP + EN + GLN group and CLP + EN + GLN group were given 0.35 g GLN/kg body weight daily for 7 d, all at the same time, via a tail vein injection; whereas those in the sham CLP + EN + saline group and CLP + EN + saline group were daily administered isovolumic sterile 0.9% saline for comparison. All rats in each of the four groups were given 290 kcal/kg body wt/d for 7 d. At the end of the seventh day after the nutritional program was finished, all rats were euthanized and the entire intestine was collected. Total Peyer's patches (PP) cell yield was counted by a hemocytometer. The percentage of PP lymphocyte subsets was analyzed by flow cytometry. The number of intestinal lamina propria IgA plasma cells was determined by the immunohistochemistry technique. The intestinal immunoglobulin A (IgA) levels were assessed by ELISA. PP apoptosis was evaluated by terminal deoxyuridine nick-end labeling. ResultsThe results revealed total PP cell yield, the numbers of PP lymphocyte subsets, intestinal lamina propria IgA plasma cells, and intestinal IgA levels in the CLP + EN + GLN group were significantly increased when compared with the CLP + EN + saline group (P < 0.05). On the other hand, the number of TUNEL-stained cells within PPs in the CLP + EN + GLN group was markedly decreased as compared with the CLP + EN + saline group (P < 0.05). ConclusionThe results of this study show that parenteral glutamine supplementation in combination with enteral nutrition may attenuate PP apoptosis, increase PP cell yield and intestinal lamina propria IgA plasma cells, and subsequently improve intestinal mucosal immunity. Clinically, these results suggest therapeutic efforts at improving intestinal immunity may contribute to the prevention and treatment of sepsis.

Hypertrophy of infected Peyer's patches arises from global, interferon-receptor, and CD69-independent shutdown of lymphocyte egress.

Lymphoid organ hypertrophy is a hallmark of localized infection. During the inflammatory response, massive changes in lymphocyte recirculation and turnover boost lymphoid organ cellularity. Intriguingly, the exact nature of these changes remains undefined to date. Here, we report that hypertrophy of Salmonella-infected Peyer's patches (PPs) ensues from a global “shutdown” of lymphocyte egress, which traps recirculating lymphocytes in PPs. Surprisingly, infection-induced lymphocyte sequestration did not require previously proposed mediators of lymphoid organ shutdown including type I interferon receptor and CD69. In contrast, following T-cell receptor-mediated priming, CD69 was essential to selectively block CD4+ effector T-cell egress. Our findings segregate two distinct lymphocyte sequestration mechanisms, which differentially rely on intrinsic modulation of lymphocyte egress capacity and inflammation-induced changes in the lymphoid organ environment.

Differential immune modulation by deoxynivalenol (vomitoxin) in mice

Mycotoxin deoxynivalenol (DON), a secondary metabolite produced by Fusarium fungi, is a contaminant in wheat, barley, and corn worldwide. It has been suggested that DON exhibits toxicity in various organs. Due to the lack of immunotoxicity data for DON, we investigated the differential immunomodulatory effects of DON in mice. DON was orally administered to female BALB/c mice at a dose of 0, 0.5, or 2mg/kg body weight for 14 days and various immunotoxicity tests were performed with standard protocols. The population of CD19+ and CD11c+ cells in the spleen and mesenteric lymph node (MLN) and of F4/80+ cells in the spleen was significantly decreased in DON-treated mice, whereas the level of CD8+ and CD4+CD25+Foxp3+ cells in the spleen and CD4+ T cells in MLN was significantly increased. In intra-epithelial lymphocytes (IELs) of the small intestine, the population of CD4 + and CD19+ cells was increased but that of CD8+ cells was decreased. Levels of CD4 + and CD8+ cells were decreased in lamina propria lymphocytes (LPLs) of small intestine; however, the level of CD4+ and CD8+ lymphocytes was increased but that of CD19+ cells was decreased in Peyer's patches lymphocytes (PPLs). Normalized expression of TLR4 in spleen, TLR9 in PPs, and TLR2, TLR3 and TLR4 in MLNs was significantly decreased, whereas expression of TLR5 and TLR9 was increased in spleen. The concentration of IgA and IgE was decreased and increased, respectively, in serum; however, the mucosal IgA level was significantly increased in the duodenum. Levels of IFN-γ, IL-2, IL-4, and IL-6 were significantly increased in serum. Furthermore, DON induced apoptosis in spleen, MLNs, and PPs, and DON-induced apoptosis was promoted by increased expression of Bax and decreased expression of Bcl-2. The autophagy genes Atg5 and Beclin-1 were up-regulated in spleen but down-regulated in MLN. After priming of the RAW 264.7 macrophage cell line with different TLR ligands, DON exposure differentially modulated IL-1β, IL-10, and TNF-α production. These results indicate that DON can cause various immunomodulatory effects in mice, creating a milieu that might allow invasion by other microorganisms.

Cranberry Proanthocyanidins Improve Intestinal sIgA During Elemental Enteral Nutrition

Elemental enteral nutrition (EEN) decreases gut-associated lymphoid tissue (GALT) function, including fewer Peyer's patch lymphocytes and lower levels of the tissue T helper 2 (Th2) cytokines and mucosal transport protein polymeric immunoglobulin receptor (pIgR), leading to lower luminal secretory immunoglobulin A (sIgA) levels. Since we recently demonstrated that cranberry proanthocyanidins (PACs) maintain the Th2 cytokine interleukin (IL)-4 when added to EEN, we hypothesized the addition of PACs to EEN would normalize other GALT parameters and maintain luminal levels of sIgA. Institute of Cancer Research mice were randomized (12/group) to receive chow, EEN, or EEN + PACs (100 mg/kg body weight) for 5 days, starting 2 days after intragastric cannulation. Ileum tissue was collected to measure IL-4 by enzyme-linked immunosorbent assay, pIgR by Western blot, and phosphorylated STAT-6 by microarray. Intestinal wash fluid was collected to measure sIgA by Western blot. Compared with chow, EEN significantly decreased tissue IL-4, phosphorylated STAT-6, and pIgR. The addition of PACs to EEN prevented these alterations. Compared with chow, EEN resulted in significantly lower levels of luminal sIgA. The addition of PACs to EEN increased luminal sIgA levels compared with EEN alone. This study suggests the addition of PACs to EEN may support GALT function and maintain intestinal sIgA levels compared with EEN administration alone.

Prenatal Zinc Supplementation of Zinc-Adequate Rats Adversely Affects Immunity in Offspring

AbstractWe previously showed that zinc (Zn) supplementation of Zn-adequate dams induced immunosuppressive effects that persist in the offspring after weaning. We investigated whether the immunosuppressive effects were due to in utero exposure and/or mediated via milk using a cross-fostering design. Pregnant rats with adequate Zn nutriture were supplemented with either Zn (1.5 mg Zn in 10% sucrose) or placebo (10% sucrose) during pregnancy (3 times/wk).At postnatal d 3, 4 pups of Zn-supplemented dams (Zn-P) were exchanged with 4 of placebo-supplemented dams (P-Zn).The remaining pups continued with their biological mothers (Zn-Zn and P-P). Pups were orally immunized with dini trophenol ovalbumin-BSA and/or cholera toxin B subunit (CTB), and serum Zn concentrations and cellular and humoral responses were assessed. Pups of Zn-supplemented dams had higher serum Zn when fostered either by placebo- or Zn-supplemented dams compared to pups of placebo-supplemented dams (P < 0.01). Postnatal Zn exposure reduced the number of Peyer's patches in both the Zn-Zn and P-Zn groups (P < 0.01). Prenatal Zn exposure suppressed CTB- (P = 0.05)and BSA-specific proliferation response of Peyer's Patch lymphocytes (P = 0.07). Prenatal Zn exposure effects on the splenocyte cytokine response were differently influenced by fostering mothers Zn status. Antigen presenting cell (APC)activity of splenocytes was lower in the Zn-Zn group than in the P-P group (P < 0.08). In conclusion, prenatal Zn exposure increases serum Zn levels in pups and suppresses antigen-specific proliferation and antibody responses and APC function, whereas postnatal exposure may suppress the mucosal immune reservoir.

Influence of Adding Pyrroloquinoline Quinone to Parenteral Nutrition on Gut‐Associated Lymphoid Tissue

Experimental intravenous (IV) parenteral nutrition (PN) diminishes gut-associated lymphoid tissue (GALT) cell number and function. PN solution cannot maintain GALT at the same level as a normal diet, even when delivered intragastrically (IG). Previous studies demonstrated pyrroloquinoline quinone (PQQ)-deficient mice to be less immunologically responsive. Because standard (STD) PN solution lacks PQQ, PQQ supplementation may prevent PN-induced GALT changes. This study was designed to determine the influence of adding PQQ to PN on GALT. In experiment 1, mice (n = 32) were randomized to chow, IV-STD-PN, and IV-PQQ-PN groups. The chow group was fed chow with the same caloric content as PN. The IV-STD-PN group received STD-PN solution, whereas the IV-PQQ-PN group was given PQQ (3 mcg/d)-enriched PN by the IV route. After 5 days of feeding, lymphocytes were isolated from the Peyer's patch (PPs), intraepithelial space (IE), and lamina propria (LP) of the small intestine. GALT lymphocyte number and phenotype (αβTCR+, γδTCR+, CD4+, CD8+, B220+ cells) and intestinal immunoglobulin A (IgA) level were determined. In experiment 2, mice (n = 28) were randomized to IG-STD-PN or IG-PQQ-PN group. After IG nutrition supports, GALT mass and function were determined as in experiment 1. The IV-PQQ-PN group showed increased PP lymphocyte number and PP CD8+ cell number compared with the IV-STD PN group. The IG-PQQ-PN group had significantly greater PP lymphocyte number and PP CD4+ cell numbers than the IG-STD-PN group. Neither IV nor IG PQQ treatment raised IgA level. PQQ added to PN partly restores GALT mass, although its effects on GALT function remain unclear.

Effects of Adding Butyric Acid to PN on Gut‐Associated Lymphoid Tissue and Mucosal Immunoglobulin A Levels

Parenteral nutrition (PN) causes intestinal mucosal atrophy, gut-associated lymphoid tissue (GALT) atrophy and dysfunction, leading to impaired mucosal immunity and increased susceptibility to infectious complications. Therefore, new PN formulations are needed to maintain mucosal immunity. Short-chain fatty acids have been demonstrated to exert beneficial effects on the intestinal mucosa. We examined the effects of adding butyric acid to PN on GALT lymphocyte numbers, phenotypes, mucosal immunoglobulin A (IgA) levels, and intestinal morphology in mice. Male Institute of Cancer Research mice (n = 103) were randomized to receive either standard PN (S-PN), butyric acid-supplemented PN (Bu-PN), or ad libitum chow (control) groups. The mice were fed these respective diets for 5 days. In experiment 1, cells were isolated from Peyer's patches (PPs) to determine lymphocyte numbers and phenotypes (αβTCR(+), γδTCR(+), CD4(+), CD8(+), B220(+) cells). IgA levels in small intestinal washings were also measured. In experiment 2, IgA levels in respiratory tract (bronchoalveolar and nasal) washings were measured. In experiment 3, small intestinal morphology was evaluated. Lymphocyte yields from PPs and small intestinal, bronchoalveolar, and nasal washing IgA levels were all significantly lower in the S-PN group than in the control group. Bu-PN moderately, but significantly, restored PP lymphocyte numbers, as well as intestinal and bronchoalveolar IgA levels, as compared with S-PN. Villous height and crypt depth in the small intestine were significantly decreased in the S-PN group vs the control group, however Bu-PN restored intestinal morphology. A new PN formula containing butyric acid is feasible and would ameliorate PN-induced impairment of mucosal immunity.

Changes in Peyer’s Patch and Cecal Tonsil B Lymphocytes in Laying Hens Following Challenge with Salmonella enterica Serovar Enteritidis

Two trials were conducted to determine B cell changes in Peyer's Patches (PP) and Cecal Tonsils (CT) of specific-pathogen-free Single Comb White Leghorn hens challenged with Salmonella enterica Serovar Enteritidis (SE). Prior to challenge and then weekly post challenge, 4 or 3 hens in Trials 1 and 2, respectively, were sacrificed and their intestinal tracts excised. Cells were purified from proximal and distal PP along with both CT and then aliquots of cells were incubated with antibodies to chicken immunoglobulins IgM, IgG and IgA. The B cells expressing the different immunoglobulin isotopes were identified via flow cytometric analysis. B lymphocytes expressing IgM were most prevalent, representing 40-60% and 30-50% of CT and PP B cells, respectively, while 20-30% of CT and PP lymphocytes expressed IgA. Only a small percentage of CT and PP lymphocytes, <10%, expressed IgG. Significantly more IgM+ cells were detected in CT vs proximal and distal PP and proximal PP in trials 1 and 2, respectively. Significantly more IgA+ cells were observed in proximal PP vs distal PP and CT in trial 1 but not in trial 2. Following SE infection, these differences were no longer observed. For IgG+ cells, however, no significant differences between tissues were observed prior to challenge but significantly more IgG+ cells were observed in both PP vs CT at weeks 1 and 3 post challenge in trial 1 and week 1 post challenge in trial 2. These results indicate that B lymphocyte differences do occur in PP vs CT in adult chickens and these populations can change in response to stimuli such as intestinal infection.

Neutrophil Elastase Inhibitor Restores Gut Ischemia Reperfusion-Induced Impairment of Gut Immunity with Reduced Plasma Interleukin-6 Concentrations in Mice

Gut-associated lymphoid tissue (GALT) is regarded as central mucosa-associated lymphoid tissue that influences systemic mucosal immunity. Our previous study revealed that gut ischemia and reperfusion (I/R) reduces GALT lymphocyte numbers. Because gut hypoperfusion frequently occurs in trauma, shock, and surgery patients, establishment of a therapeutic method to preserve GALT mass after gut I/R may be important for the prevention of infections. We examined the effects of sivelestat sodium hydrate, a selective inhibitor of neutrophil elastase, on GALT mass and plasma cytokine concentrations in a murine gut I/R model. Seventy male ICR mice were randomized to the control (n = 34) or the sivelestat (n = 36) group. After intravenous cannulation of the animals, the superior mesenteric artery was occluded for 60 min. After reperfusion, physiologic saline or sivelestat 5 mg/kg hourly was infused for 24 h. Sixteen mice in the control and 22 in the sivelestat group were alive at 24 h. Twenty-six mice (n = 13 in each group) were chosen randomly for harvest of the small intestine. Lymphocytes from Peyer patches (PP), the intraepithelial space (IE), and the lamina propria (LP) were counted; and their phenotypes (αβT-cell receptor (TCR), γδTCR, CD4, CD8, B cell) were determined by flow cytometry. Cytokine concentrations (interleukin [IL]-6, IL-1β, IL-10) in the plasma and bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay. Sivelestat treatment did not improve survival but increased PP and IE lymphocyte numbers significantly and reduced the LP CD8(+) cell percentage and plasma IL-6 concentration compared with controls. There were no significant differences between the two groups in other cell phenotypes or cytokine concentrations. Sivelestat treatment after gut I/R may be useful for maintaining gut immunity and preventing systemic inflammatory responses.