Bacteria-free solution derived from Lactobacillus plantarum inhibits multiple NF-kappaB pathways and inhibits proteasome function.
BACKGROUND
Bacteria play a role in inflammatory bowel disease and other forms of intestinal inflammation. Although much attention has focused on the search for a pathogen or inciting inflammatory bacteria, another possibility is a lack of beneficial bacteria that normally confer anti-inflammatory properties in the gut. The purpose of this study was to determine whether normal commensal bacteria could inhibit inflammatory pathways important in intestinal inflammation.


METHODS
Conditioned media from Lactobacillus plantarum (Lp-CM) and other gut bacteria was used to treat intestinal epithelial cell (YAMC) and macrophage (RAW 264.7) or primary culture murine dendritic cells. NF-kappaB was activated through TNF-Receptor, MyD88-dependent and -independent pathways and effects of Lp-CM on the NF-kappaB pathway were assessed. NF-kappaB binding activity was measured using ELISA and EMSA. 1kappaB expression was assessed by Western blot analysis, and proteasome activity determined using fluorescence-based proteasome assays. MCP-1 release was determined by ELISA.


RESULTS
Lp-CM inhibited NF-kappaB binding activity, degradation of IkappaBalpha and the chymotrypsin-like activity of the proteasome. Moreover, Lp-CM directly inhibited the activity of purified mouse proteasomes. This effect was specific, since conditioned media from other bacteria had no inhibitory effect. Unlike other proteasome inhibitors, Lp-CM was not toxic in cell death assays. Lp-CM inhibited MCP-1 release in all cell types tested.


CONCLUSIONS
These studies confirm, and provide a mechanism for, the anti-inflammatory effects of the probiotic and commensal bacterium Lactobacillus plantarum. The use of bacteria-free Lp-CM provides a novel strategy for treatment of intestinal inflammation which would eliminate the risk of bacteremia reported with conventional probiotics.
Department of Medicine, GIDRU, Division of Infectious Diseases, Queen's University, Kingston, ON, Canada. eop@queensu.ca
Elaine O
EO
Petrof
Erika C
EC
Claud
Jun
J
Sun
Tatiana
T
Abramova
Yuee
Y
Guo
Tonya S
TS
Waypa
Shu-Mei
SM
He
Yasushi
Y
Nakagawa
Eugene B
EB
Chang
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D003713:Dendritic Cells / Q000378:metabolism / Q000382:microbiology; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007413:Intestinal Mucosa / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D007422:Intestines / Q000276:immunology / Q000382:microbiology; D048191:Lactiplantibacillus plantarum; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019936:Probiotics / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D051381:Rats; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D017077:Culture Media, Conditioned; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D061988:Proteasome Inhibitors; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex