Lactobacillus and Lactobacillus cell-free culture supernatants modulate chicken macrophage activities.

Lactobacilli are commensal microbes that reside in the intestines of several species, including chickens. Structural constituents of lactobacilli are able to stimulate the host immune system. Macrophages are crucial players in both innate and adaptive immune systems. Here, we investigated the effects of Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius and their cell-free culture supernatants on the pro-inflammatory gene expression profile, nitric oxide (NO) production and phagocytosis by chicken macrophages. Substantial differences were found among Lactobacillus strains in their capacity to induce pro-inflammatory cytokines. L. acidophilus only up-regulated interferon (IFN)-γ, while L. reuteri and L. salivarius up-regulated interleukin (IL)-1ß, IL-6, IL-8 and IL-12 expression. Supernatant of L. salivarius up-regulated IL-1ß, IL-8 and IFN-γ expression, while the other cell-free supernatants did not induce significant changes. Moreover, L. reuteri and L. salivarius increased macrophage phagocytosis, but all cell-free supernatants increased macrophage NO production and did not change phagocytosis activity.

Vitamin D3 modulates the function of chicken macrophages.

Vitamin D3 is known to modulate both innate and adaptive immune responses in mammals, but there is little information on its effects on avian immune system cells. Here, we studied the effects of vitamin D3 on chicken macrophages. Chicken macrophages expressed vitamin D receptor (VDR) and lipopolysaccharide (LPS) stimulation increased their VDR expression. Macrophages were treated with 1,25(OH)2D3 in the presence or absence of Toll-like receptor ligands, such as LPS and Pam3CSK4. Subsequently, macrophage activation was assessed by measuring nitric oxide (NO) and expression of CXCL8 and interleukin (IL)-1ß. In addition, changes in major histocompatibility complex (MHC)-II and CD86 were examined. Treatment of cells with 1,25(OH)2D3 increased the ability of macrophages to respond to stimuli and produce NO, but vitamin D3 alone did not activate macrophages and resulted in the down-regulation of CD86, MHC-II, CXCL8 and IL-1ß. These findings suggest that vitamin D3 has an immunomodulatory role in chicken macrophages.

Differential effects of lactobacilli on activation and maturation of mouse dendritic cells.

Lactic acid bacteria (LAB) are of interest because of their potential to modulate immune responses. The effects of LAB range from regulation to stimulation of the immune system. A series of studies were performed in vitro to study the effects of six lactic acid bacteria (LAB), Lactobacillus helveticus LH-2, Lactobacillus acidophilus La-5, La-115, La-116 and La-14, and Lactobacillus salivarius, on maturation and activation of mouse dendritic cells. Production of tumour necrosis factor (TNF)-?, interleukin (IL)-6 and IL-10 by dendritic cells (DCs) was determined after treating cells with live LAB. The expression of DC maturation markers, CD80 and CD40, was also measured using flow cytometry after stimulation with LAB. In addition, the expression of Toll-like receptors (TLRs) 2, 4 and 9 by DCs stimulated with LAB was measured. Our results revealed that LAB act differentially on pro-inflammatory and anti-inflammatory cytokine production and induction of co-stimulatory molecules by DCs. Specifically, L. salivarius was found to be the most effective LAB to induce pro-inflammatory cytokine production and expression of co-stimulatory molecules. Moreover, La-14, La-116 and La-5 induced moderate maturation and activation of DCs. On the other hand, LH-2 and La-115 were the least effective lactobacilli to induce DC responses. The present study also revealed that L. salivarius was able to induce the expression of TLR2, 4 and 9 by DCs. In conclusion, various strains and species of LAB can differentially regulate DC activation and maturation, providing further evidence that these bacteria may have the ability to influence and steer immune responses in vivo.

Differential cytokine expression in T-cell subsets of chicken caecal tonsils co-cultured with three species of Lactobacillus.

Members of the intestinal microbiota play an important role in the development of T-cells. Little is known about responses of intestinal T-cell subsets of chickens to commensal bacteria. Therefore, we set out to characterise cytokine responses in T-cells after exposure to lactobacilli. Caecal tonsil mononuclear cells were isolated and co-cultured with Lactobacillus acidophilus, Lactobacillus reuteri and Lactobacillus salivarius for 12 hours. Subsequently the CD4+ and CD8+ cells were fractionated by flow cytometry and the expression of pro- and anti-inflammatory cytokines as well as Toll-like receptor 21 (TLR21) was determined. The results demonstrated that chicken CD4+ and CD8+ T-cells express TLR21 and that the various isolates of lactobacilli differentially induces the expression of interleukin 10, interferon-gamma and transforming growth factor beta. Our results demonstrate that different Lactobacillus species have the capacity to regulate intestinal T-cell responses and that these responses may be important to intestinal homeostasis.

Influence of in-feed virginiamycin on the systemic and mucosal antibody response of chickens.

Subtherapeutic and prophylactic doses of virginiamycin are capable of altering the intestinal microbiota as well as increasing several growth parameters in chickens. In spite of the fact that the microbiota plays a role in shaping the host's immune system, little information is available on the effects of in-feed antibiotics on the chicken immune system. The objective of this study was to examine the effects of an antibiotic, virginiamycin, on the development of antibody responses. Chickens were fed diets containing no antibiotics, along with either subtherapeutic (11 ppm) or prophylactic (22 ppm) doses of virginiamycin. Chickens were then immunized with keyhole limpet hemocyanin (KLH) and sheep red blood cells systemically, and with BSA and KLH orally. Although antibodies were detected against BSA in the intestinal contents of birds that were orally immunized, there was no difference among different treatment groups. Systemic IgG, and to a lesser extent IgM, antibody responses to KLH were greater (P < 0.05) in birds fed a diet containing 11 or 22 ppm of virginiamycin compared with control birds fed no antibiotic. No treatment effect was found in the sheep red blood cell-immunized birds. Results of the present study implicate virginiamycin in enhancing antibody responses to some antigens in chickens. Further studies are required to determine to what extent these effects on antibody response are mediated through changes in the composition of the microbiota.

Appropriate chicken sample size for identifying the composition of broiler intestinal microbiota affected by dietary antibiotics, using the polymerase chain reaction-denaturing gradient gel electrophoresis technique.

The bacterial microbiota in the broiler gastrointestinal tract are crucial for chicken health and growth. Their composition can vary among individual birds. To evaluate the composition of chicken microbiota in response to environmental disruption accurately, 4 different pools made up of 2, 5, 10, and 15 individuals were used to determine how many individuals in each pool were required to assess the degree of variation when using the PCR-denaturing gradient gel electrophoresis (DGGE) profiling technique. The correlation coefficients among 3 replicates within each pool group indicated that the optimal sample size for comparing PCR-DGGE bacterial profiles and downstream applications (such as identifying treatment effects) was 5 birds per pool for cecal microbiota. Subsequently, digesta from 5 birds was pooled to investigate the effects on the microbiota composition of the 2 most commonly used dietary antibiotics (virginiamycin and bacitracin methylene disalicylate) at 2 different doses by using PCR-DGGE, DNA sequencing, and quantitative PCR techniques. Thirteen DGGE DNA bands were identified, representing bacterial groups that had been affected by the antibiotics. Nine of them were validated. The effect of dietary antibiotics on the microbiota composition appeared to be dose and age dependent. These findings provide a working model for elucidating the mechanisms of antibiotic effects on the chicken intestinal microbiota and for developing alternatives to dietary antibiotics.