[Bifidobacterium DNA upregulates Th1 type response of umbilical cord blood mononuclear cell].
OBJECTIVE
To study the effect of bifidobacterium genomic DNA on umbilical cord blood mononuclear cell (CBMC), and investigate the immunoregulation of bifidobacterium DNA and explore possible mechanisms by which bifidobacterium acts against allergic reaction.


METHODS
Bifidobacterium genomic DNA (bDNA) and human DNA (hDNA) were extracted with phenol/chloroform/isoamyl alcohol and stored at -20 degrees C for later use. Parts of bDNA were completely digested with DNaseI (d-bDNA) at 37 degrees C. CBMCs were isolated with Ficoll from umbilical cord blood and incubated at 37 degrees C in a 5% CO2 humidified incubator. These cells were divided into four groups, control group: without any stimulant; bDNA group: stimulated with 25 microg/ml bDNA; d-bDNA group: stimulated with 25 microg/ml d-bDNA; hDNA group: stimulated with 25 microg/ml hDNA. The cells were stimulated with different stimulants in vitro, at the end of incubation culture supernatant and cells were collected. IL-12 and IL-10 levels in the culture supernatant were measured by enzyme linked immuno sorbent assay (ELISA); cells secreting IL-4 and IFN-gamma were counted by enzyme linked immunospot (ELISPOT) assay; and total RNA was isolated from the cells to assay T-bet and GATA3 mRNA expression levels by reverse transcription polymerase chain reaction (RT-PCR).


RESULTS
Six hours after stimulation there was no significant difference in IL-12 level in supernatant among the four groups; 12 hours after stimulation, IL-12 level in supernatant of bDNA treated group was significantly higher than that of each of the other groups, so were the results obtained at 24 hours and 48 hours after stimulation (P < 0.05). No significant difference could be detected in IL-12 level in supernatant among the other 3 groups. On the other hand, 6 hours after stimulation there was no significant difference in IL-10 level in supernatant among the four groups. But 12 and 24 hours after stimulation IL-10 level in supernatant of bDNA treated group was lower than that of each of the other groups, but the difference was not statistically significant. The count of IFN-gamma secreting cells of bDNA treated group was higher than that of the other groups, while IL-4 secteting cells of bDNA treated group were lower than that of the other groups. After bDNA stimulation, nuclear factor T-box expressed in T cells (T-bet) mRNA expression was conspicuously enhanced as compared to the other three groups (P < 0.05). GATA3 mRNA transcription in CBMC had no significant change after bDNA stimulation.


CONCLUSIONS
bDNA could promote secretion of Th1 type cytokine IL-12, while Th2 type cytokine IL-10 level of cell supernatant was decreased. bDNA could stimulate secretion of IFN-gamma by CBMC and inhibit secretion of IL-4. T-bet mRNA expression was highly enhanced after bDNA stimulation. bDNA could upregulate Th1 type response, which may be one of important mechanisms by which bifidobacterium inhibit allergic response.
Zhao
Hui
H
Department of Immunology, Children's Hospital, Fudan University, Shanghai 200032, China.
Wang
Xiao-chuan
XC
Wang
Jing-yi
JY
Yu
Ye-heng
YH
Wang
Chuan-qing
CQ
Yang
Yi
Y
D001644:Bifidobacterium / Q000166:cytology / Q000235:genetics*; D018929:Cell Culture Techniques; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005312:Fetal Blood / Q000166:cytology* / Q000276:immunology; D050990:GATA3 Transcription Factor / Q000235:genetics; D006801:Humans; D007231:Infant, Newborn; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D016753:Interleukin-10 / Q000276:immunology / Q000378:metabolism; D018664:Interleukin-12 / Q000276:immunology / Q000378:metabolism; D015847:Interleukin-4 / Q000276:immunology / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020825:T-Box Domain Proteins / Q000235:genetics; D018417:Th1 Cells / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D000098815:T-bet Transcription Factor
D004269:DNA, Bacterial; D050990:GATA3 Transcription Factor; D012333:RNA, Messenger; D020825:T-Box Domain Proteins; D000098815:T-bet Transcription Factor; D016753:Interleukin-10; D018664:Interleukin-12; D015847:Interleukin-4; D007371:Interferon-gamma