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Role of Fas and Treg cells in fracture healing as characterized in the fas-deficient (lpr) mouse model of lupus.

Al-Sebaei MO, Daukss DM, Belkina AC, Kakar S, Wigner NA, Cusher D, Graves D, Einhorn T, Morgan E, Gerstenfeld LC - J. Bone Miner. Res. (2014)

Bottom Line: The reduced bone fraction was related to elevated rates of coupled bone turnover in the B6.MRL/Fas(lpr) /J calluses, as evidenced by higher osteoclast numbers and increased osteogenesis.B6.MRL/Fas(lpr) /J mice had elevated Treg cells in both spleens and bones of B6.MRL/Fas(lpr) /J but decreased percentage of activated T cells in bone tissues.These results show that an active autoimmune state is inhibited during the period of cartilage resorption and suggest that iTreg cells play a functional role in this process.

View Article: PubMed Central - PubMed

Affiliation: Orthopaedic Research Laboratory, Boston University School of Medicine, Boston, MA, USA; King Abdul Aziz University, Faculty of Dentistry, Department of Oral and Maxillofacial Surgery, Jeddah, Saudi Arabia.

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Assessments of inflammatory state and T-cell activity. (A) Graphical representation of the expression of the temporal patterns and relative levels of mRNA expression of select pro- inflammatory and T-cell regulatory cytokines as determined by qRT-PCR. Days after fracture are denoted, and strain of mice is the same as denoted in Fig. 4. The nature of each mRNA that is assessed is indicated in the figure. Data presented are mean values from n = 6 replicates determined from two pools of mRNAs per time point. The error bars are + SD of the 6 replicates from the two pools. *Significance between the two strains for that time point p < 0.05 based on the comparisons of the replicates. (B) Flow cytometric analysis of Tregs and activated T cells in femoral bones at baseline before fracture and in both the contralateral (Cntl) unfractured and fracture (Fx) bone at 10 days after fracture. Tregs were identified as the percentage of CD25 + Foxp3+ of all live CD3 + CD4+ cells, and activated T cells were percentage CD69+ of all CD3 + CD4+ cells. Intragroup: *difference (p < 0.05) at day 0 and after-fracture day 10 in either WT or LPR strains. Intergroup: #difference in mice between WT and LPR strains. (C) Flow cytometric analysis of Tregs and activated T cells in spleens at baseline before fracture and at 10 days after fracture in both WT and LPR strains. Gating strategy and labeling are the same as in (B). A complete summary of the findings for the ANOVA statistical analysis of these data is presented in Supplemental Tables S3–S5.
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fig06: Assessments of inflammatory state and T-cell activity. (A) Graphical representation of the expression of the temporal patterns and relative levels of mRNA expression of select pro- inflammatory and T-cell regulatory cytokines as determined by qRT-PCR. Days after fracture are denoted, and strain of mice is the same as denoted in Fig. 4. The nature of each mRNA that is assessed is indicated in the figure. Data presented are mean values from n = 6 replicates determined from two pools of mRNAs per time point. The error bars are + SD of the 6 replicates from the two pools. *Significance between the two strains for that time point p < 0.05 based on the comparisons of the replicates. (B) Flow cytometric analysis of Tregs and activated T cells in femoral bones at baseline before fracture and in both the contralateral (Cntl) unfractured and fracture (Fx) bone at 10 days after fracture. Tregs were identified as the percentage of CD25 + Foxp3+ of all live CD3 + CD4+ cells, and activated T cells were percentage CD69+ of all CD3 + CD4+ cells. Intragroup: *difference (p < 0.05) at day 0 and after-fracture day 10 in either WT or LPR strains. Intergroup: #difference in mice between WT and LPR strains. (C) Flow cytometric analysis of Tregs and activated T cells in spleens at baseline before fracture and at 10 days after fracture in both WT and LPR strains. Gating strategy and labeling are the same as in (B). A complete summary of the findings for the ANOVA statistical analysis of these data is presented in Supplemental Tables S3–S5.

Mentions: To assess if the autoimmune phenotype of the B6.MRL-Faslpr/J strain affected endochondral bone formation or bone turnover during fracture healing, several different pro- and anti-inflammatory cytokines that have been shown to have altered expression during an active autoimmune state were assessed (Fig. 6A). As expected, these data showed that the expression of TNF-α, IL-1β, and IL-17F all were constitutively higher in the B6.MRL-Faslpr/J. In contrast, cytokines that would prevent the T cells from progressing to a Th1 phenotype (IL-10, IL-17A) were elevated in the mutant mice, whereas those that have been shown to inhibit Th17 development toward an iTreg (IL-6 IL-23) also showed diminished initial induction after fracture in the B6.MRL-Faslpr/J mice.(29) Using CD4 as a general assessment of T cell numbers also showed lower levels of CD4 mRNA initially after fracture in the wild-type mice. A comparison of these cytokine patterns across the time course of fracture healing further showed that there was reciprocal downregulation and upregulation of the cytokines that drive Th1 and iTreg differentiation, respectively, during the period of cartilage formation in both B6.MRL-Faslpr/J and wild-type callus tissues.


Role of Fas and Treg cells in fracture healing as characterized in the fas-deficient (lpr) mouse model of lupus.

Al-Sebaei MO, Daukss DM, Belkina AC, Kakar S, Wigner NA, Cusher D, Graves D, Einhorn T, Morgan E, Gerstenfeld LC - J. Bone Miner. Res. (2014)

Assessments of inflammatory state and T-cell activity. (A) Graphical representation of the expression of the temporal patterns and relative levels of mRNA expression of select pro- inflammatory and T-cell regulatory cytokines as determined by qRT-PCR. Days after fracture are denoted, and strain of mice is the same as denoted in Fig. 4. The nature of each mRNA that is assessed is indicated in the figure. Data presented are mean values from n = 6 replicates determined from two pools of mRNAs per time point. The error bars are + SD of the 6 replicates from the two pools. *Significance between the two strains for that time point p < 0.05 based on the comparisons of the replicates. (B) Flow cytometric analysis of Tregs and activated T cells in femoral bones at baseline before fracture and in both the contralateral (Cntl) unfractured and fracture (Fx) bone at 10 days after fracture. Tregs were identified as the percentage of CD25 + Foxp3+ of all live CD3 + CD4+ cells, and activated T cells were percentage CD69+ of all CD3 + CD4+ cells. Intragroup: *difference (p < 0.05) at day 0 and after-fracture day 10 in either WT or LPR strains. Intergroup: #difference in mice between WT and LPR strains. (C) Flow cytometric analysis of Tregs and activated T cells in spleens at baseline before fracture and at 10 days after fracture in both WT and LPR strains. Gating strategy and labeling are the same as in (B). A complete summary of the findings for the ANOVA statistical analysis of these data is presented in Supplemental Tables S3–S5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4305200&req=5

fig06: Assessments of inflammatory state and T-cell activity. (A) Graphical representation of the expression of the temporal patterns and relative levels of mRNA expression of select pro- inflammatory and T-cell regulatory cytokines as determined by qRT-PCR. Days after fracture are denoted, and strain of mice is the same as denoted in Fig. 4. The nature of each mRNA that is assessed is indicated in the figure. Data presented are mean values from n = 6 replicates determined from two pools of mRNAs per time point. The error bars are + SD of the 6 replicates from the two pools. *Significance between the two strains for that time point p < 0.05 based on the comparisons of the replicates. (B) Flow cytometric analysis of Tregs and activated T cells in femoral bones at baseline before fracture and in both the contralateral (Cntl) unfractured and fracture (Fx) bone at 10 days after fracture. Tregs were identified as the percentage of CD25 + Foxp3+ of all live CD3 + CD4+ cells, and activated T cells were percentage CD69+ of all CD3 + CD4+ cells. Intragroup: *difference (p < 0.05) at day 0 and after-fracture day 10 in either WT or LPR strains. Intergroup: #difference in mice between WT and LPR strains. (C) Flow cytometric analysis of Tregs and activated T cells in spleens at baseline before fracture and at 10 days after fracture in both WT and LPR strains. Gating strategy and labeling are the same as in (B). A complete summary of the findings for the ANOVA statistical analysis of these data is presented in Supplemental Tables S3–S5.
Mentions: To assess if the autoimmune phenotype of the B6.MRL-Faslpr/J strain affected endochondral bone formation or bone turnover during fracture healing, several different pro- and anti-inflammatory cytokines that have been shown to have altered expression during an active autoimmune state were assessed (Fig. 6A). As expected, these data showed that the expression of TNF-α, IL-1β, and IL-17F all were constitutively higher in the B6.MRL-Faslpr/J. In contrast, cytokines that would prevent the T cells from progressing to a Th1 phenotype (IL-10, IL-17A) were elevated in the mutant mice, whereas those that have been shown to inhibit Th17 development toward an iTreg (IL-6 IL-23) also showed diminished initial induction after fracture in the B6.MRL-Faslpr/J mice.(29) Using CD4 as a general assessment of T cell numbers also showed lower levels of CD4 mRNA initially after fracture in the wild-type mice. A comparison of these cytokine patterns across the time course of fracture healing further showed that there was reciprocal downregulation and upregulation of the cytokines that drive Th1 and iTreg differentiation, respectively, during the period of cartilage formation in both B6.MRL-Faslpr/J and wild-type callus tissues.

Bottom Line: The reduced bone fraction was related to elevated rates of coupled bone turnover in the B6.MRL/Fas(lpr) /J calluses, as evidenced by higher osteoclast numbers and increased osteogenesis.B6.MRL/Fas(lpr) /J mice had elevated Treg cells in both spleens and bones of B6.MRL/Fas(lpr) /J but decreased percentage of activated T cells in bone tissues.These results show that an active autoimmune state is inhibited during the period of cartilage resorption and suggest that iTreg cells play a functional role in this process.

View Article: PubMed Central - PubMed

Affiliation: Orthopaedic Research Laboratory, Boston University School of Medicine, Boston, MA, USA; King Abdul Aziz University, Faculty of Dentistry, Department of Oral and Maxillofacial Surgery, Jeddah, Saudi Arabia.

Show MeSH
Related in: MedlinePlus