Interactions between gut-associated lymphoid tissue and colonization levels of indigenous, segmented, filamentous bacteria in the small intestine of mice.

Unlike most other indigenous bacteria, segmented filamentous bacteria (SFB) are potent activators of the mucosal immune system. SFB are strongly anchored to the epithelial cells of the small intestine where they have a preference for mucosal lymphoid epithelium. Since SFB are only present in high numbers shortly after weaning, it was investigated whether an SFB-induced immune reaction results in the removal of these bacteria from the small intestine. A correlation was found between age and colonization levels in the small intestines of SFB monoassociated Swiss mice. Five-week-old athymic BALB/c (nu/nu) mice showed lower colonization levels than their heterozygous littermates, but the opposite was found at the age of 12 weeks. However, SFB inoculation of germfree Swiss mice resulted in higher colonization levels in 5-week-old mice when compared with 4-month-old mice. We conclude that SFB colonization levels in the small intestine are likely influenced by the activity of the mucosal immune system. However, an additional age-dependent factor that modulates SFB colonization levels cannot be excluded.     

Colonization and distribution of segmented filamentous bacteria (SFB) in chicken gastrointestinal tract and their relationship with host immunity

Uncultivable segmented filamentous bacteria (SFB) reside in the gastrointestinal (GI) tract of mammals and can boost the host immunity. Immunoglobulin A (IgA) from mother's milk has been previously shown to be a key factor in regulating SFB colonization. Because neonatal chicken cannot acquire IgA from maternal milk, they are a good model to examine the role of IgA in SFB colonization. Here, we used the fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) to monitor the colonization and distribution of SFB in chickens aged from 2-day-old to 6-week-old. Early SFB colonization, which primarily occurred in the ileal mucosa (< 13 days old), was IgA independent. From the age of 17-42 days, there was an increase in IgA in the gut mucosa, which was correlated with a decrease in SFB. To examine the effect of probiotics and immunosuppression on SFB colonization, we treated the chickens by feeding them Lactobacillus delbrueckii or giving them a subcutaneous injection of cyclophosphamide (CTX). Feeding lactobacilli at birth rendered SFB colonization occurring 4 days earlier, while CTX treatment increases the SFB colonization through reducing the other non-SFB bacteria. Altogether, our data suggest that early colonization of SFB in chicken occurs independently of IgA and the population of SFB in the GI tract of chicken may be manipulated from birth via probiotic or CTX treatment.     

Development of intestinal microbiota in mice and its possible interaction with the evolution of luminal IgA in the intestine.

The development of the intestinal microbiota and the evolution of the fecal IgA in mice were analyzed from 18 to 40 days old by PCR temperature gradient gel electrophoresis (TGGE) and ELISA, respectively. There were two events for the diversification of the intestinal microbiota from suckling to maturity. The first change occurred between days 21 and 22 after birth, when the diversity of the intestinal microbiota showed a remarkable increase at this time. The second change occurred from days 27 to 30 after birth, and the increase in the diversity of the intestinal microbiota ceased. The amount of fecal IgA decreased from days 18 to 20, remained low until day 22, on day 23, it recovered and then continued to increase. This study suggests that there are possible interactions between the development of intestinal microbiota and the evolution of intestinal secretion of IgA in mice, the same as in rats, although the second change in mice intestinal microbiota occurred a few days later than in rats. The decline in maternal IgA supply as the suckling period proceeded presumably allowed the bacterial colonization. As a consequence of this increase in bacterial colonization, the secretion of the self-SIgA was accelerated in the pups.     

A gastrointestinal nematode in pregnant and lactating mice alters maternal and neonatal microbiomes

The maternal microbiome is understood to be the principal source of the neonatal microbiome but the consequences of intestinal nematodes on pregnant and lactating mothers and implications for the neonatal microbiome are unknown. Using pregnant CD1 mice infected with Heligmosomoides bakeri, we investigated the microbiomes in maternal tissues (intestine, vagina, and milk) and in the neonatal stomach using MiSeq sequencing of bacterial 16S rRNA genes. Our first hypothesis was that maternal nematode infection altered the maternal intestinal, vaginal, and milk microbiomes and associated metabolic pathways. Maternal nematode infection was associated with increased beta-diversity and abundance of fermenting bacteria as well as Lactobacillus in the maternal caecum 2 days after parturition, together with down-regulated carbohydrate, amino acid and vitamin biosynthesis pathways. Maternal nematode infection did not alter the vaginal or milk microbiomes. Our second hypothesis was that maternal infection would shape colonization of the neonatal microbiome. Although the pup stomach microbiome was similar to that of the maternal vaginal microbiome, pups of infected dams had higher beta-diversity at day 2, and a dramatic expansion in the abundance of Lactobacillus between days 2 and 7 compared with pups nursing uninfected dams. Our third hypothesis that maternal nematode infection altered the composition of neonatal microbiomes was confirmed as we observed up-regulation of several putatively beneficial microbial pathways associated with synthesis of essential and branched-chain amino acids, vitamins, and short-chain fatty acids. We believe this is the first study to show that a nematode living in the maternal intestine is associated with altered composition and function of the neonatal microbiome.     

Different degree of ileal colonization by segmented, filamentous bacteria in two strains of mice.

Segmented, filamentous bacteria (SFBs) are autochthonous, apathogenic inhabitants of the ileum of various animal species. Outbred Swiss (Cpb:SE) mice have significantly higher degrees of SFB colonization than do inbred BALB/c mice. The present studies were carried out to identify determinants of this strain difference. In a cross-fostering experiment it was shown that SFB colonization of the pups is determined by the strain of the pups themselves rather than by the strain of the nursing dam. Thus, maternal effects may not be involved in SFB colonization. In a cross-infecting experiment using germ-free and SFB-positive animals of the two mouse strains, it was found that ileal SFB colonization is determined by host characteristics rather than by origin of the SFBs. Thus, SFBs that are specific for a given mouse strain may not exist in the two strains of mice. It is concluded that the mouse strain difference in SFB colonization is determined by host characteristics, which probably have a genetic basis.     

Heligmosomoides polygrus (equals nematospiroides dubius): humoral and intestinal immunologic responses to infection in mice

Measurements of serum IgG₁, IgG_2a, IgM, and IgA levels and antibody titers in these immunoglobulin classes were made at intervals after initial infection and challenge infection of mice immunized by two or three previous infections. Identical measurements were made on the content of the small intestine in mice which had been exposed to the same infection schedule. Sections of small intestine taken after initial infection and challenge infection were examined by the fluorescent antibody technique for changes in populations of immunoglobulin-containing cells and by routine histologic procedures for histopathologic changes.In serum, only IgG₁ was consistently increased after initial infection, and antibody in IgG₁ was detected within the first 2 wk of infection. In immunized animals, only IgG₁ and antibody of this class always responded to challenge infection, although antibody in other immunoglobulin classes was detected.IgA concentration of the intestinal content did not differ significantly after initial infection or challenge infection of immunized mice. Immunized mice had about twice the IgG₁ concentration in intestinal content as singly infected animals. No intestinal antibody was detected after initial infection; only IgG₁ antibody was detected in the intestinal content of immunized and challenged mice.Cell infiltrates in the intestinal mucosa and submucosa of immunized animals contained numerous IgG₁-containing cells. Mast cells and globular leukocytes were observed in the intestine of immunized animals.     

Spatial and temporal colonization dynamics of segmented filamentous bacteria is influenced by gender,age and experimental infection with Helicobacter hepaticus in Swiss Webster mice

In this study, we examined colonization dynamics of segmented filamentous bacteria (SFB) in intestine of Swiss Webster (SW) mice infected with Helicobacter hepaticus (Hh). At 8 weeks post-inoculation with Hh (WPI), cecal and colonic SFB levels in the control males were significantly lower compared to those at 16 WPI. Hh infection in both genders did not alter SFB levels in the jejunum and ileum, but increased SFB levels in the cecum and colon of males compared to the controls (P < 0.05) at 8 WPI. At 16 WPI, the Hh-infected females contained lower levels of SFB in the jejunum, cecum and colon compared to the female controls. Irrespective of gender, aging and Hh infection, the Il-17A mRNA levels decreased from the small intestine to the cecum and then to the colon, whereas the Foxp3 mRNA levels were comparable in these intestinal regions. There were significant differences in Il-17A mRNA levels in the ileum (P < 0.05, R² = 0.31), with females having greater Il-17A mRNA levels than males, and higher SFB colonization levels related to more Il-17A mRNA. These results indicate that aging and gender play an important role in colonization dynamics of intestinal SFB and ileal SFB-associated Th17 response.     

Caloric restriction reduces IgA levels and modifies cytokine mRNA expression in mouse small intestine

The aim of this study was to determine the effect of caloric restriction (CR) in mouse small intestine on the production and secretion of immunoglobulin (Ig) A, the population of lymphocytes in the lamina propria, and the expression of cytokines that mediate and regulate innate and adaptive immunity. One group of young Balb/c mice was fed ad libitum, while the CR group was fed ad libitum and fasted on alternate days. When mice were six months old, IgA levels in the proximal small intestine were quantified by enzyme-linked immunosorbent assay, while the number of IgA containing cells, CD4⁺ T cells and CD8⁺ T cells in the duodenal mucosa was determined by immunohistochemistry. Furthermore, the expression of several intestinal cytokines, the genes for α-chain IgA, and the polymeric Ig receptor (pIgR) were analyzed by real-time polymerase chain reaction. CR decreased the levels of IgA in the intestine, apparently a consequence of a reduced number of IgA⁺ cells in the lamina propria that decrease the production and secretion of this Ig, and a reduced secretion of S-IgA into the bile, which in turn discharges into the proximal intestine. Contrarily, CR increased the expression of genes for α-chain IgA, and the pIgR, indicating that transport of IgA was not a key factor in the decrease of this Ig. Additionally, CR modified the expression of genes for tumor necrosis factor-α, interferon-γ, tumor growth factor-β, interleukin (IL)-2 and IL-10, all of which regulate the synthesis of IgA and pIgR, the inflammatory response, and the immune response in the intestine.     

Influence of thyroxine and propylthiouracil administration on the intake of maternal milk in sucklings of the laboratory rat

Young laboratory rats received a daily subcutaneous injection of thyroxine (T4) and propylthiouracil (PTU) in saline, from birth until the period of spontaneous weaning. The rate of growth and the time of eye opening as well as the spontaneous weaning was assessed. The intake of maternal milk was followed directly, i.e. by the transport of 85Sr in maternal milk from the maternal body to the sucklings. In the young treated with T4 the eyes opened earlier than in the young treated with PTU. The body mass differed in both groups between the 4th and 20th day of life (the T4 pups were heavier). The 85Sr transfer in maternal milk was higher in the PTU young from the 22nd day of life. The PTU sucklings weaned spontaneously one week later than those receiving T4. A hypothesis has been proposed on the primary significance of maturation of oral mechanisms of sucking for spontaneous weaning.     

Variations in enzyme activity in stomach and pancreatic tissue and digesta in piglets around weaning

A study was performed to investigate the effect of weaning at 4 weeks of age on the activity of digestive enzymes in the stomach and pancreatic tissue and in digesta from 3 days prior to weaning to 9 days postweaning in 64 piglets. In stomach tissue the activity of pepsin and gastric lipase was determined. Pepsin activity declined abruptly after weaning but 5 days postweaning the weaning level was regained and in the gastric contents no change in pepsin activity was observed. Weaning did not influence the activity of gastric lipase. The activity of eight enzymes and a cofactor was measured in pancreatic tissue. The effect of weaning on the enzyme activity was highly significant for all enzymes except elastase. The activity of all enzymes remained at the weaning level during day 1-2 postweaning followed by a reduction of the activity. The activity of trypsin, carboxypeptidase A, amylase and lipase exhibited minimum activity 5 days postweaning. Trypsin activity increased to the preweaning level on day 7-9 whereas the activity of the others increased but did not reach the preweaning level. The activity of chymotrypsin, carboxypeptidase B and carboxyl ester hydrolase decreased during the entire experimental period. In digesta no effect of weaning was observed on the activity of amylase and trypsin. The activity of chymotrypsin was reduced after weaning in the proximal third of the small intestine and lipase and carboxyl ester hydrolase activity was reduced in the middle and distal parts of the small intestine after weaning. The present study shows that the activities of the digestive enzymes in the pancreatic tissue are affected by weaning. Even though the pancreatic secretion cannot be judged from these results they show that the enzymes respond differently to weaning. In general the activity of the digestive enzymes in pancreatic tissue is low on day 5 postweaning which in interaction with other factors may increase the risk of developing postweaning diarrhoea.     

Effect of Weaning on Small Intestinal Structure and Function in the Piglet

Fifty-four piglets were selected from 12 litters weaned at 17 (Treatment 1), 21 (Treatment 2), 28 (Treatment 3) and 35 (Treatment 4) days old, respectively, to determine the effect of weaning age on small intestinal villus morphology, immunology and histochemistry. From proximal duodenum, proximal jejunum, distal jejunum and middle ileum, intestinal samples with three replicates (piglets) in each treatment were taken at 18, 22, 28 and 36; 22, 28, 36 and 43; 28, 36, 43, and 50; and 18, 22, 28, 36, 43 and 50d of age in Treatment 1, 2, 3 and 4, respectively. This was equivalent to 12h, 3d, 1 week, 2 week postweaning in Treatment 1; 12h, 1 week, 2 week, 3 week postweaning in Treatment 2 and 3, and all the same age in Treatment 4 as in Treatment 1, 2, 3, respectively. The results showed that villous height of duodenum and proximal jejunum decreased significantly in Treatment 1 and 3. Crypt depth in the duodenum, proximal jejunum and ileum also decreased significantly in Treatment 1. Date had significant effect on villous height of the duodenum, distal jejunum and ileum with the shortest on day 29 and crypt depth of all positions increased with piglet age except the crypt depth in proximal jejunum decreased on day 50. Weaning age and day of age had significant effects on intraepithelial lymphocyte (IEL) number and goblet cell (GC) number at all positions of small intestinal mucosa in piglets. The number of IEL at all segments of small intestinal mucosa in Treatment 3 increased significantly compared to those in other treatments, but IEL number at all locations of small intestinal mucosa in Treatment 2 decreased significantly compared to those in other treatments. The number of GC in small intestinal mucosa increased significantly in early-weaned (< day 21) piglets. It appears that providing fluid milk replacer for a few days postweaning could dramatically reduce the negative impact of weaning on villous morphology and digestive and absorptive function, especially in pigs weaned prior to 3 week of age. Finally, as weaning age was reduced, GC had a greater role in intestinal duct protection.     

Variations in enzyme activity in stomach and pancreatic tissue and digesta in piglets around weaning

A study was performed to investigate the effect of weaning at 4 weeks of age on the activity of digestive enzymes in the stomach and pancreatic tissue and in digesta from 3 days prior to weaning to 9 days postweaning in 64 piglets. In stomach tissue the activity of pepsin and gastric lipase was determined. Pepsin activity declined abruptly after weaning but 5 days postweaning the weaning level was regained and in the gastric contents no change in pepsin activity was observed. Weaning did not influence the activity of gastric lipase. The activity of eight enzymes and a cofactor was measured in pancreatic tissue. The effect of weaning on the enzyme activity was highly significant for all enzymes except elastase. The activity of all enzymes remained at the weaning level during day 1–2 postweaning followed by a reduction of the activity. The activity of trypsin, carboxypeptidase A, amylase and lipase exhibited minimum activity 5 days postweaning. Trypsin activity increased to the preweaning level on day 7–9 whereas the activity of the others increased but did not reach the preweaning level. The activity of chymotrypsin, carboxypeptidase B and carboxyl ester hydrolase decreased during the entire experimental period. In digesta no effect of weaning was observed on the activity of amylase and trypsin. The activity of chymotrypsin was reduced after weaning in the proximal third of the small intestine and lipase and carboxyl ester hydrolase activity was reduced in the middle and distal parts of the small intestine after weaning. The present study shows that the activities of the digestive enzymes in the pancreatic tissue are affected by weaning. Even though the pancreatic secretion cannot be judged from these results they show that the enzymes respond differently to weaning. In general the activity of the digestive enzymes in pancreatic tissue is low on day 5 postweaning which in interaction with other factors may increase the risk of developing postweaning diarrhoea.     

Biphasic assembly of the murine intestinal microbiota during early development

The birth canal provides mammals with a primary maternal inoculum, which develops into distinctive body site-specific microbial communities post-natally. We characterized the distal gut microbiota from birth to weaning in mice. One-day-old mice had colonic microbiota that resembled maternal vaginal communities, but at days 3 and 9 of age there was a substantial loss of intestinal bacterial diversity and dominance of Lactobacillus. By weaning (21 days), diverse intestinal bacteria had established, including strict anaerobes. Our results are consistent with vertical transmission of maternal microbiota and demonstrate a nonlinear ecological succession involving an early drop in bacterial diversity and shift in dominance from Streptococcus to Lactobacillus, followed by an increase in diversity of anaerobes, after the introduction of solid food. Mammalian newborns are born highly susceptible to colonization, and lactation may control microbiome assembly during early development.     

小鼠小肠四种消化酶活力的胎后发育模式

为明确晚成型小鼠胎后发育肠道消化酶活力的建立过程和发育模式,探讨其与适应性调节假说的关系,测定了从出生后至27日龄小鼠小肠前、中、后段的乳糖酶、蔗糖酶、麦芽糖酶和氨基肽酶的酶活力。结果发现单位组织酶活力方面,乳糖酶活力先增后降,小肠前段在9日龄而中后段在12日龄达到最高,至27日龄时仅中段有微弱的酶活力;蔗糖酶活力12日龄始出现,前段和后段自15日龄迅速升高,至18日龄达最高,但随后显著降低,而中段在15日龄后持续升高至21日龄达到最高,此后维持在较高水平;麦芽糖酶出生时已具有活力,但在15日龄前维持较低水平,此后迅速升高,前后段在18日龄,中段在21日龄达到峰值,此后下降;小肠前段的氨基肽酶活力出生后至27日龄持续下降,而后段和中段从出生到断乳前则持续升高,断乳后略有下降。除乳糖酶总酶活力先增后降,在15日龄达峰值外,其余3种酶的总酶活力均持续增加。在小肠不同位置4种酶活力的分布具有显著差异,且日龄对不同位置酶活力的影响趋势不同。总之,小鼠小肠4种消化酶的酶活力随时间的变化能够与其食物转变的消化需求相匹配,部分地支持适应性调节假说。     

Colitis development during the suckling-weaning transition in mucin Muc2-deficient mice

The mucin Muc2 is the structural component of the colonic mucus layer. Adult Muc2 knockout (Muc2(-/-)) mice suffer from severe colitis. We hypothesized that Muc2 deficiency induces inflammation before weaning of mother's milk [postnatal day (P) 14] with aggravation of colitis after weaning (P28). Muc2(-/-) and wild-type mice were killed at embryonic day 18.5 and P1.5, P7.5, P14, P21, and P28. Colonic morphology, influx of T cells, and goblet cell-specific protein expression was investigated by (immuno)histochemistry. Cytokine and Toll-like receptor (TLR) profiles in the colon were analyzed by quantitative RT-PCR. Muc2(-/-) mice showed an increased and persistent influx of Cd3ε-positive T cells in the colonic mucosa as of P1.5. This was accompanied by mucosal damage at P28 in the distal colon but not in the proximal colon. At P14, the proinflammatory immune response [i.e., increased interleukin (IL)-12 p35, IL-12 p40, and tumor necrosis factor-α, expression] in the distal colon of Muc2(-/-) mice presented with an immune suppressive response [i.e., increased Foxp3, transforming growth factor (TGF)-β1, IL-10, and Ebi3 expression]. In contrast, at P28, a proinflammatory response remained in the distal colon, whereas the immune suppressive response (i.e., Foxp3 and TGF-β1 expression) declined. The proximal colon of Muc2(-/-) mice did not show morphological damage and was dominated by an immune suppressive response at P14 and P28. Interestingly, changes in expression of TLRs and TLR-related molecules were observed in the distal colon at P14 and P28 and in the proximal colon only at P28. Colitis in Muc2(-/-) mice is limited before weaning by immune suppressive responses and exacerbates in the distal colon after weaning because of the decline in the immune suppressive response.     

Mucosal IgA increase in rats by continuous CLA feeding during suckling and early infancy

The aim of this work was to establish the effect of the cis9,trans11 conjugated linoleic acid (CLA) isomer on mucosal immunity during early life in rats, a period when mucosal immunoglobulin production is poorly developed, as is also the case in humans. CLA supplementation was performed during three life periods: gestation, suckling, and early infancy. The immune status of supplemented animals was evaluated at two time points: at the end of the suckling period (21-day-old rats) and 1 week after weaning (28-day-old rats). Secretory IgA was quantified in intestinal washes from 28-day-old rats by ELISA technique. IgA, TGFbeta, and PPARgamma mRNA expression was measured in small intestine and colon by real time PCR, using Taqman specific probes and primers. IgA mucosal production was enhanced in animals supplemented with CLA during suckling and early infancy: in 28-day-old rats, IgA mRNA expression was increased in small intestine and colon by approximately 6- and 4-fold, respectively, and intestinal IgA protein by approximately 2-fold. TGFbeta gene expression was independent of age and type of tissue considered, and was not modified by dietary CLA. Gene expression of PPARgamma, a possible mediator of CLA's effects was also upregulated in animals receiving CLA during early life. In conclusion, dietary supplementation with CLA during suckling and extended to early infancy enhances development of the intestinal immune response in rats.     

Segmented Filamentous Bacteria Are Indigenous Intestinal Bacteria That Activate Intraepithelial Lymphocytes and Induce MHC Class II Molecules and Fucosyl Asialo GM1 Glycolipids on the Small Intestinal Epithelial Cells in the Ex-Germ-Free Mouse

In ex-germ-free mice conventionalized by association with fecal microorganisms, the induction of major histocompatibility complex class II molecules and fucosylation of asialo GM1 glycolipid occur in the small intestinal epithelial cells (IEC). The intestinal intraepithelial lymphocytes (IEL), especially αβ T-cell receptor-bearing ones, also remarkably expand and show cytolytic activity. In this study, we investigated the immunological and physiological characteristics of the small intestine induced by a kind of indigenous bacteria of the small intestine, segmented filamentous bacteria (SFB), among chloroform-resistant intestinal bacteria. Monoassociation of SFB with germ-free mice was confirmed by the determination of the base sequences of polymerase chain reaction products of 16S rRNA genes of the fecal bacteria of these mice and in situ hybridization using fluorescein-labeled probes based on them. SFB increased the number of αβTCR-bearing IEL and induced Thy-1 expression and cytolytic activity of IEL. The induction of MHC class II molecules and fucosyl asialo GM1 glycolipids and the increases in the mitotic activity and the ratio of the number of columnar cells to those of goblet cells also occurred in the small intestinal epithelial cells on monoassociation of these bacteria. SFB are important indigenous bacteria for the development of the mucosal architecture and immune system in the small intestine, at least in mice.     

A strain of Lactobacillus plantarum affects segmented filamentous bacteria in the intestine of immunosuppressed mice

Segmented filamentous bacteria (SFB) are present in the gastrointestinal tract of mice from weaning until the maturation of the immune system. Probiotic bacteria also have an effect on host immunity. To study the relationships established between these bacteria, samples from a mouse model fed with Lactobacillus plantarum under different immunological conditions were analysed. SFB populations were measured by a newly designed group-specific quantitative PCR assay. The results confirmed the presence of the probiotic in the intestine and an expansion of SFB in the ileum of immunocompromised mice, which was abolished upon administration of L. plantarum, an effect not described to date.     

Different antiamebic antibody isotype patterns in the large and small intestine after local and systemic immunization of mice with glutaraldehyde fixed Entamoeba histolytica trophozoites

We have determined the major immunoglobulin isotypes (IgG, IgA, IgM) of antiamebic antibodies induced in the serum and in the large and small intestine after local (oral and rectal) or systemic (intraperitoneal and intramuscular) immunization of mice with glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). IgA predominated in the small intestine after immunization through all routes, whereas in the large intestine similar antibody levels of the major isotypes were induced by rectal, intraperitoneal and intramuscular immunization. The intramuscular route elicited intestinal responses lower than those induced by the rectal and intraperitoneal routes, but higher than the slight IgA antibody increase observed after oral immunization. The differences in antiamebic antibody response patterns at the large and small intestine suggest that there are different mucosal effector compartments. They also indicate that isotype analysis of mucosal antibodies from the sites where an infectious agent resides is needed to evaluate whether a vaccine candidate induces responses of higher protective value in the appropriate site, and that the study of antibody responses must not be limited to sampling the serum or mucosal sites distant to the relevant one.