The human monocyticcell line THP-1 (TIB-202) and Mycobacterium intracellulare were obtained from the American Type Culture Collection (Manassas, VA). Cell culture medium (RPMI 1640) was obtained from Cambrex (East Rutherford, NJ), reagents for Middlebrook 7H10 solid agar medium from Difco (Detroit, MI), and phorbolmyristate acetate (PMA) from Sigma (St. Louis, MO). TRIzol reagent from Invitrogen Inc, ISCRIP cDNA Synthesis Kit and the PCR Kit were purchased form Bio-Rad Inc (Bio-Rad, Hercules CA, USA). ELISA kit for detecting IL-8 was purchased from R & D System Inc (Minneapolis, MN), The TransAM NF-kB p65 Transcription Factor Assay Kit was obtained from Active Motif (Carlsbad, CA, USA).

THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 2 mM glutamine. Prior to experimentation, THP-1 cells were stimulated with 15 ng/mL PMA (phorbolmyristate acetate) for 24 h to allow differentiation into macrophages. The PMA-containing medium was removed and replaced with fresh medium just prior to exposing the cells to infection. Differentiated macrophages were plated with M. intracellulare (MAI) at a multiplicity of infection (MOI) of 10 bacilli to 1 macrophage as previously described [10] . For culture of cell-associated MAI, briefly, after one-hour infection, the cells were washed with a 1:1 solution of RPMI:1 × PBS. Adherent cells were lysed with 250 µl of 0.25% SDS solution per well, followed by addition of 250 uL of 7H9 plating broth. Serial dilution of cell lysates was prepared and then 5 µl of each dilution was plated on Middlebrook 7H10 agar. For 48 h and 96 h, the cells were washed after the initial 1 h of infection and replaced with fresh culture medium or the supernatant of S. boulardii-B508 as previously described [11] .

THP-1 cells were plated into each well of a six-well tissue culture plates (1 × 10⁶ cells/well), differentiated with PMA overnight for experiments. Total RNA was extracted using the TRIzol reagent, and cDNA was prepared using reverse transcriptase, following the manufacturer’s instructions (Invitrogen Life Technologies) and as previously described [12] . Briefly single-stranded cDNA products were used as templates in a 30 µL PCR reaction according to the instruction of the ISCRIP cDNA Synthesis Kit and the PCR Kit (Bio-Rad, Hercules CA, USA). The following primers were used: IL-8 sense primer: 5’-CAT GAC TTC CAA GCT GGC CGTG-3’ and IL-8 antisense primer: 5’-TCA CTG ATT CTT GGA TAC CAC AGAG-3’; GAPDH sense primer: 5’-TCC ATG ACA ACT TTG GTA TCG TG -3’ and GAPDH antisense primer: 5’-TGT CGC TGT TGA AGT CAG AGGA-3’. PCR amplification parameters were as follows: initial denaturation at 94C for 4 min, followed by 30 cycles of amplification (94˚C for 35 s, 55˚C for 50 s, 72˚C for 1 min), and finally terminated at 72˚C for 4 min and kept 4˚C until use. PCR products were analyzed by electrophoresis through 1.5% agarose gels containing ethidium bromide. The DNA bands corresponding to IL-8 (489 bp) and GAPDH (501 bp) were visualized using an ultraviolet dock. Images were captured and then quantified using Adobe Photoshop.

S.boulardii-B508 was cultured in RPMI 1640 cell culture medium for 48 h in 28˚C. The supernatant was then centrifuged at 6500 rpm for 20 min, the supernatant was prepared to use.

The THP-1 cells were plated in each well of a four-chamber culture slide, followed by differentiation with PMA of THP-1 cells and infection with MAI. Before of infection, the cells were washed and added with or without supernatant of S.boulardii-B508. For each condition, triplicate slides were prepared. To assay for apoptosis at single cell based on labeling of DNA strand breaks was used the in situ cell death detection kit from Roche Diagnostics Gmbh and followed according to manufacturer’s instructions and enumeration of apoptotic cells as previously described [10] .

Activation of the p65 subunit of NF-kB was quantified using the TransAMNF-kB p65 Transcription Factor Assay Kit according to manufacturer’s instructions. For each condition, 10 µg of nuclear protein extracts from treated cells were incubated in 96-well plates coated with an oligonucleotide containing the NF-kB consensus binding site (5’ GGGACTTTCC-3’ oligonucleotide) or a mutated oligonucleotide for one hour at room temperature. After washing three times, NF-kB p65 antibody was added for one hour followed by HRP-conjugated secondary antibody. Binding of activated p65 NF-kB was determined colorimetrically.

Replicated experiments were independent, and where appropriate, summary results are presented as means ± SEM. Differences were considered significant for P < 0.05, and all reported p-values used a two-sided test. For most experiments, group means were compared by ANOVA using Fisher’s least significance difference procedure.

To study the effects of S. boulardii-B508 on the growth of M. intracellulare in human macrophages, we cultured differentiated THP-1 cells with and without the supernatant of S. boulardii-B508, and then infected the cells with M. intracellularefor one hour, 48 or 96 hours. After the indicated times of infection, the cells were washed, lysed and M. intracellulare quantified. The number or M. intracellulare was significantly decreased after two days or four days of infection in cells cultured with S. boulardii-B508 supernatant compared with the control THP-1 cells (Figure 1). We conclude from these

findings that the secreted products of S. boulardii-B508 directly inhibit the proliferation of M. intracellulare enhances macrophage killing of M. intracellulare or both.

It is well established that IL-8 production is regulated at the level of gene transcription [13] . To examine whether the supernatant of S. boulardii-B508 affects IL-8 mRNA expression, we stimulated THP-1-derived differentiated macrophages seeded in 6 well plates with or without M. intracellulare infection in the presence or absence of the supernatant treatment of S. boulardii-B508 for 18 hours. Total RNA was isolated and RT-PCR was performed with sense and anti-sense primers for IL-8. M. intracellulare infection induced the production of IL-8 mRNA, with the stronger induction occurring after infection. Our data indicated that the total supernatant or 50% supernatant of S. boulardii-B508 inhibits significantly IL-8 mRNA expression (Figure 2).

Our previous studies suggested that induced apoptosis is known to inhibits the growth of intracellular mycobacteria [11] [12] . In order to determine whether the supernatant of S.boulardii-B508 induces the apoptosis of the macrophages THP-1 cells infected with M. intracellulare. TUNEL staining were performed, there was a dose-dependent increase in apoptosis in infected cell incubated with supernatant of S. boulardii-B508 (Figure 3). M. intracellulare infection of THP-1 cells also induced a modest increase in

apoptosis, but in the presence of supernatant of S.boulardii-B508, there was a significant increased in the percentage of TUNEL-positive cells, although the supernatant of S,boulardii-B508 were 1:1 dilution (Figure 3).

Since M. avium organisms have been showed to activate IκΒα kinase- NF-kB and the mitogen-activated protein kinase (MAPK) signaling pathway [14] [15] . Given that NF-kB is the prime regulator of IL-8 gene transcription in both epithelial cells and monocytes [13] [16] , we sought to determine the effects of S. boulardii on NF-kB activation by M. intracellulare infection. In our previous studies showed that NF-kB activation inhibits both apoptosis and autophagy in M. tuberculosis-infected human Macrophages, it was impairing their control of the infection [11] . THP-1-derived macrophages were infected with M. intracellulare for 30 minutes and three hours with or without adding the supernatant of S. boulardii-B508. p65NF-kB binding to its consensus oligonucleotide was then quantified. As shown in Figure 4(a), MAI induced NF-kB p65 binding at both time points and the supernatant of S. boulardii-B508 significantly inhibited that activation.