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Mycoplasma synoviae induces upregulation of apoptotic genes, secretion of nitric oxide and appearance of an apoptotic phenotype in infected chicken chondrocytes.

Dusanic D, Bencina D, Oven I, Cizelj I, Bencina M, Narat M - Vet. Res. (2012)

Bottom Line: We found that M. synoviae significantly reduces chondrocyte respiration.Increased production of nitric oxide was also confirmed in cell supernates.The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Ljubljana, Department of Animal Science, Chair for Genetics, Animal Biotechnology and Immunology, Groblje 3, 1230 Domzale, Slovenia. Daliborka.Dusanic@bf.uni-lj.si.

ABSTRACT
The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans.

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Expression of selected genes after exposure of CCH to 5-fluorouracil or M. synoviae WVU 1853. Exposure agent and time of exposure (in hours) are indicated below columns. Control (CTRL) represents in all graphs non-exposed CCH. The results are given as mean values ± standard error for three independent cell treatment experiments with three RT-qPCR replicates for each experiment. Means marked with stars are significantly different from controls (p < 0.05 to p < 0.001) by Student t-test.
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Figure 4: Expression of selected genes after exposure of CCH to 5-fluorouracil or M. synoviae WVU 1853. Exposure agent and time of exposure (in hours) are indicated below columns. Control (CTRL) represents in all graphs non-exposed CCH. The results are given as mean values ± standard error for three independent cell treatment experiments with three RT-qPCR replicates for each experiment. Means marked with stars are significantly different from controls (p < 0.05 to p < 0.001) by Student t-test.

Mentions: Expression of 15 genes (Table 2) was assayed in CCH exposed to 5-FU or infected with M. synoviae. The level of expression was normalized to untreated control cells and calibrated with reference to GAPDH expression. Upregulation of several genes was already observed in the cells sampled 24 h after infection. The gene encoding inducible nitric oxide synthase (NOS2) was upregulated 46-fold (p < 0.001), Casp3 was upregulated 2.6-fold (p < 0.001), Mapk11 3.2-fold (p < 0.01) and CASP8 2.9-fold (p < 0.01) (Figure 4). In CCH infected with M. synoviae for 48 h, Casp3, NOS2 and Mapk11 remained upregulated (2.8-fold, 20.1-fold and 3.1-fold, respectively, p < 0.001), while another gene, htrA3, became slightly upregulated (1.6-fold, p < 0.01) (Figure 4). Seventy-two hours after infection, Casp3 and NOS2 remained upregulated (4.4-fold and 7.7-fold, respectively, p < 0.001), while Mapk11 was no longer upregulated. A strong increase in transcription was noticed in genes AIFM1 (6.7-fold, p < 0.001), NFκB1 (2.5-fold, p < 0.001) and htrA3 (2.7-fold, p < 0.001). BCL2 was also upregulated (2-fold, p < 0.001) (Figure 4). No change in gene expression at all tested timepoints was noticed for Bak1, endog, tp53, CD44, XIAP, Fas and FASLG (Additional file 1, Figure S1).


Mycoplasma synoviae induces upregulation of apoptotic genes, secretion of nitric oxide and appearance of an apoptotic phenotype in infected chicken chondrocytes.

Dusanic D, Bencina D, Oven I, Cizelj I, Bencina M, Narat M - Vet. Res. (2012)

Expression of selected genes after exposure of CCH to 5-fluorouracil or M. synoviae WVU 1853. Exposure agent and time of exposure (in hours) are indicated below columns. Control (CTRL) represents in all graphs non-exposed CCH. The results are given as mean values ± standard error for three independent cell treatment experiments with three RT-qPCR replicates for each experiment. Means marked with stars are significantly different from controls (p < 0.05 to p < 0.001) by Student t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293721&req=5

Figure 4: Expression of selected genes after exposure of CCH to 5-fluorouracil or M. synoviae WVU 1853. Exposure agent and time of exposure (in hours) are indicated below columns. Control (CTRL) represents in all graphs non-exposed CCH. The results are given as mean values ± standard error for three independent cell treatment experiments with three RT-qPCR replicates for each experiment. Means marked with stars are significantly different from controls (p < 0.05 to p < 0.001) by Student t-test.
Mentions: Expression of 15 genes (Table 2) was assayed in CCH exposed to 5-FU or infected with M. synoviae. The level of expression was normalized to untreated control cells and calibrated with reference to GAPDH expression. Upregulation of several genes was already observed in the cells sampled 24 h after infection. The gene encoding inducible nitric oxide synthase (NOS2) was upregulated 46-fold (p < 0.001), Casp3 was upregulated 2.6-fold (p < 0.001), Mapk11 3.2-fold (p < 0.01) and CASP8 2.9-fold (p < 0.01) (Figure 4). In CCH infected with M. synoviae for 48 h, Casp3, NOS2 and Mapk11 remained upregulated (2.8-fold, 20.1-fold and 3.1-fold, respectively, p < 0.001), while another gene, htrA3, became slightly upregulated (1.6-fold, p < 0.01) (Figure 4). Seventy-two hours after infection, Casp3 and NOS2 remained upregulated (4.4-fold and 7.7-fold, respectively, p < 0.001), while Mapk11 was no longer upregulated. A strong increase in transcription was noticed in genes AIFM1 (6.7-fold, p < 0.001), NFκB1 (2.5-fold, p < 0.001) and htrA3 (2.7-fold, p < 0.001). BCL2 was also upregulated (2-fold, p < 0.001) (Figure 4). No change in gene expression at all tested timepoints was noticed for Bak1, endog, tp53, CD44, XIAP, Fas and FASLG (Additional file 1, Figure S1).

Bottom Line: We found that M. synoviae significantly reduces chondrocyte respiration.Increased production of nitric oxide was also confirmed in cell supernates.The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Ljubljana, Department of Animal Science, Chair for Genetics, Animal Biotechnology and Immunology, Groblje 3, 1230 Domzale, Slovenia. Daliborka.Dusanic@bf.uni-lj.si.

ABSTRACT
The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans.

Show MeSH
Related in: MedlinePlus