Boston University OpenBU http://open.bu.edu Graduate Research Symposium Graduate Research Symposium 216 216-4-1 Identification of mirna differentially expressed in macrophages exposed to Porphyromonas gingivalis infection Al-Hashemi, Jacob https://hdl.handle.net/2144/16372 Boston University
Identification of mirna differentially expressed in macrophages exposed to Porphyromonas gingivalis infection Jacob Al-Hashemi, Olivier Huck, Eduard Drizik, Adam Gower, Jean-Luc Davideau, Henri Tenenbaum, Salomon Amar Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA ABSTRACT AIM OF THE STUDY We analyzed bacterial modulation of mirnas in bone-marrowderived macrophages (BMMs) induced by infection with either wild type Porphyromonas gingivalis (Pg) or mutant Pg ( FimA), through a microarray analysis. TNF-α and IL-1 concentrations in Pg infected BMMs transfected with selected mirnas were also assessed. The inhibition of mmu-mir-2137 increased the secretion of antiinflammatory IL-1, while mmu-mir-155-5p decreased TNF-α. In vivo: injecting these mirnas with Pg in mice reduced the size of the lesion significantly. Finding mirnas that could be a potential therapeutic target and analyzing their biological effects. Particularly, the ones that affect the immune host-response in relation to Pg infection. INTRODUCTION Periodontitis (PD) is a common chronic inflammatory disease inducing tooth-supporting tissue destruction (1) related to the dysbiosis of periodontal microbiota, associated with Pg. Macrophages are directly in contact with infectious agents or their byproducts (2). mirnas are involved in many physiological and pathological processes (3). The expression and role of mirnas in inflammatory and infectious diseases are not fully understood yet. METHODS Wild type and mutant Pg were utilized to infect BMMs. In the animal module only wild type tested. (7) In calvarial bone-resorption mousemodule, effect of two mirna was confirmed after Pg infection. ELISA technique were used to assess the level of cytokines in BMMs infected with Pg. (6) Murine bone marrow drive macrophages were collected for the study. L929 (M-CSF) stimulate hematopoietic stem cell into macrophages. (4) mirna expression monitored using microarray analysis. (5)
Identification of mirna differentially expressed in macrophages exposed to Porphyromonas gingivalis infection Jacob Al-Hashemi, Olivier Huck, Eduard Drizik, Adam Gower, Jean-Luc Davideau, Henri Tenenbaum, Salomon Amar Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA RESULTS Cont r ol ( 3h) I nf ect i on Pg ( 3h) I nf ect i on Fi m A ( 3h) TNF-α AA D G B E HH MM C F I N B 14 D E G L L 12 1 C p g/ml 8 Size of the les ion (mm2) 6 12 1 2 8 * * 6 4 2 Figure 2: TNF-α. Dosage of TNF-α in supernatants of transfected BMMs infected with Pg at 24h. * p<.5. All in vitro experiments were performed in triplicate. IL-1 IL-1 45 4 35 3 pg/ m l pg/ml i nf ect ed PG Fi ma cel l l i ne 3 i nf ect ed PG Fi ma cel l l i ne 2 i nf ect ed PG ATCC cel l l i ne 3 l i ne 2 i nf ect ed PG Fi ma cel l l i ne 1 i nf ect ed PG ATCC cel l c o n tro lt 3 C e lin e 3 cont r ol T3 Cel l l i ne 3 i nf ect ed PG ATCC cel l l i ne 1 cont r ol T3 Cel l l i ne 2 5 cont r ol T3 Cel l l i ne 1 I N 14 4 25 2 15 1 Figure 1: Heatmap of differentially expressed mirna of all of the conditions tested. Red and blue indicate z-scores of 2 or -2, respectively, and white indicates a z-score of (row-wise mean). p and q.1. F Figure 5: Histological Sections. Representative samples for skin & underlying calvarias bone at the middle of the lesion from each group. Sample from Pg (A-C); Pg + combination of mirna (D-F); Pg + mmumirna 2137 inhibitor (G-I); and Pg + mmumir 155 mimic (L-N). TRAP staining for bone (bottom raw 2x) and H&E staining for the skin (Top 1x and middle 2x raw) 5 Figure 3: IL-1. Dosage of IL-1 in supernatants of transfected BMMs infected with Pg at 24h. * p<.5. All in vitro experiments were performed in triplicate. Figure 4: Size of Lesions. Size of calvarial lesions 7 d after Pg (5x18 CFU) infection and the injection of the mmumir-155-5p mimic and mmu-mir-2137 inhibitor. * p<.5, n=4. CONCLUSIONS Our results indicate mirna-155 and mirna-2137-inhibitor could contribute to the pathogenicity of Pg in PD. This is supported by the decrease in TNF-α secretion when BMMs were transfected with this mirna in vitro, and the reduction of inflammation in vivo. Inhibition of mirna-2137 in vitro increased IL-1 secretion. More importantly, the inhibition of mirna 2137 lead to significantly reduced inflammation in vivo. Further research in this field may potentially lead to the identification of new therapeutics mirna aiming to reduce the manipulation of invasive bacteria on hostimmune, which in turn reduce bacterial survival. REFERENCES 1. Pihlstrom, B. L., B. S. Michalowicz, and N. W. Johnson. 25. Periodontal diseases. Lancet 366: 189 182 2. Hosin, A. A., A. Prasad, L. E. Viiri, A. H. Davies, and J. Shalhoub. 214. MicroRNAs in atherosclerosis. J. Vasc. Res. 51: 338 349. 3. Marques-Rocha, J. L., M. Samblas, F. I. Milagro, J. Bressan, J. A. Martínez, and A. Marti. 215. Noncoding RNAs, cytokines, and inflammation-related diseases. FASEB J. 4. http://www.nature.com/nrm/journal/v4/n5/fig_tab/nrm114_f2.html 5. http://www.arnoschrauwers.nl/weblog/tag/geheugen/ 6. http://www.elisa-antibody.com/uploads/images/sandwich_45.jpg 7. http://en.citizendium.org/images/8/8f/p.gingivalis_2.jpg
Control (3h) Infection Pg (3h) Infection FimA (3h) Figure 1: Heatmap of differentially expressed mirna of all of the conditions tested. Red and blue indicate z-scores of 2 or -2, respectively, and white indicates a z-score of (row-wise mean). p and q.1. control T3 Cell line 1 control T3 Cell line 2 T3 Celine 3 control T3 Cell line 3 infected PG ATCC cell line 1 infected PG ATCC cell line 2 infected PG ATCC cell line 3 infected PG FimA cell line 1 infected PG FimA cell line 2 infected PG FimA cell line 3
14 TNF-α 12 IL-1 IL-1 pg/ml 1 8 6 4 2 pg/ml pg/ml 5 45 4 35 3 25 2 15 1 5 Figure 2: TNF-α. Dosage of TNF-α in supernatants of transfected BMMs infected with Pg at 24h. * p<.5. All in vitro experiments were performed in triplicate. Figure 3: IL-1. Dosage of IL-1 in supernatants of BMMs infected with Pg at 24h. * p<.5. All in vitro experiments were performed in transfected triplicate.
Size of Size the of the Lesion lesion (mm2) (mm²) 14 12 1 8 6 4 2 * * Figure 4: Size of Lesions. Size of calvarial lesions 7 d after Pg (5x1 8 CFU) infection and the injection of the mmu-mir-155-5p mimic and mmu-mir-2137 inhibitor. * p<.5, n=4.
Pg only Pg + Combination group Pg + anti-mir-2137 Pg + mir-155 A D G L B E H M C F F I N Figure 5: Histological Sections. Representative samples for skin & underlying calvarial bone at the middle of the lesion from each of the following groups: Pg (A-C); Pg + combination group (D-F); Pg + anti-mirna-2137 (G-I); and Pg + mir 155 (L-N). TRAP staining for bone (bottom row, 2x) and H&E staining for the skin (Top row, 1x; middle row, 2x). Arrows indicate TRAP stained multi-nucleated osteoclasts attached to the bone and bone resorption lacunae.