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Low-dose TNF augments fracture healing in normal and osteoporotic bone by up-regulating the innate immune response.

Chan JK, Glass GE, Ersek A, Freidin A, Williams GA, Gowers K, Espirito Santo AI, Jeffery R, Otto WR, Poulsom R, Feldmann M, Rankin SM, Horwood NJ, Nanchahal J - EMBO Mol Med (2015)

Bottom Line: Fragility, or osteoporotic, fractures represent a major medical problem as they are associated with permanent disability and premature death.Using a murine model of fragility fractures, we found that local rhTNF treatment improved fracture healing during the early phase of repair.If translated clinically, this promotion of fracture healing would reduce the morbidity and mortality associated with delayed patient mobilization.

View Article: PubMed Central - PubMed

Affiliation: Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK.

No MeSH data available.


Related in: MedlinePlus

TNF promotes recruitment of innate immune cells and CCL2 expressionAnti-Ly6G treatment depletes local monocytes/macrophages at the fracture site. Counts of positively stained infiltrative F4/80+ cells in the adjacent muscle to the fracture site following treatment with control or anti-Ly6G at day 1 and day 7. Data are presented as mean ± SEM. **P = 0.005 at day 1 and **P = 0.002 at day 7, and *P = 0.013 for control at day 1 versus control at day 7, by unpaired two-sided t-test.Addition of 1 ng rhTNF to fracture supernatant in the air pouch model led to increased numbers of neutrophils (Ly6G+, CD11b+ cells) and monocytes/macrophages (Ly6G−, CD11b+, CD115+ cells) in the cellular infiltrate. These effects were abrogated by the addition of anti-CCL2. Neutrophils: FS versus FS + TNF *P = 0.015, FS + TNF versus FS + TNF + anti-CCL2 *P = 0.028, and FS + TNF + anti-CCL2 versus FS + TNF + IgG *P = 0.042 by one-way ANOVA followed by Bonferroni's multiple comparison test. Monocytes/macrophages: FS versus FS + TNF **P = 0.0063, FS + TNF versus FS + TNF + anti-CCL2 ****P < 0.0001, and FS + TNF + anti-CCL2 v FS + TNF + IgG *P = 0.015 by one-way ANOVA followed by Bonferroni's multiple comparisons test.Addition of 1 ng rhTNF to fracture supernatant increased CCL2 protein levels in the air pouch. “Neat” indicates level of CCL2 in fracture supernatant before injection into air pouch. Data are presented as mean ± SEM. FS versus FS + TNF **P = 0.0020 by one-way ANOVA with Bonferroni's multiple comparisons test.Addition of rhTNF to enriched bone marrow-derived murine neutrophils pre-exposed to fracture supernatant in vitro led to up-regulation of CCL2 production at 1 h. Data are presented as mean ± SEM. No TNF versus TNF 0.01 ng/ml *P = 0.012, TNF 0.1 ng/ml ***P < 0.0001, TNF 1.0 ng ***P = 0.0001, TNF 10 ng/ml ***P < 0.0001, and TNF 100 ng/ml *P = 0.023 by one-way ANOVA with Dunnett's multiple comparisons test.
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fig03: TNF promotes recruitment of innate immune cells and CCL2 expressionAnti-Ly6G treatment depletes local monocytes/macrophages at the fracture site. Counts of positively stained infiltrative F4/80+ cells in the adjacent muscle to the fracture site following treatment with control or anti-Ly6G at day 1 and day 7. Data are presented as mean ± SEM. **P = 0.005 at day 1 and **P = 0.002 at day 7, and *P = 0.013 for control at day 1 versus control at day 7, by unpaired two-sided t-test.Addition of 1 ng rhTNF to fracture supernatant in the air pouch model led to increased numbers of neutrophils (Ly6G+, CD11b+ cells) and monocytes/macrophages (Ly6G−, CD11b+, CD115+ cells) in the cellular infiltrate. These effects were abrogated by the addition of anti-CCL2. Neutrophils: FS versus FS + TNF *P = 0.015, FS + TNF versus FS + TNF + anti-CCL2 *P = 0.028, and FS + TNF + anti-CCL2 versus FS + TNF + IgG *P = 0.042 by one-way ANOVA followed by Bonferroni's multiple comparison test. Monocytes/macrophages: FS versus FS + TNF **P = 0.0063, FS + TNF versus FS + TNF + anti-CCL2 ****P < 0.0001, and FS + TNF + anti-CCL2 v FS + TNF + IgG *P = 0.015 by one-way ANOVA followed by Bonferroni's multiple comparisons test.Addition of 1 ng rhTNF to fracture supernatant increased CCL2 protein levels in the air pouch. “Neat” indicates level of CCL2 in fracture supernatant before injection into air pouch. Data are presented as mean ± SEM. FS versus FS + TNF **P = 0.0020 by one-way ANOVA with Bonferroni's multiple comparisons test.Addition of rhTNF to enriched bone marrow-derived murine neutrophils pre-exposed to fracture supernatant in vitro led to up-regulation of CCL2 production at 1 h. Data are presented as mean ± SEM. No TNF versus TNF 0.01 ng/ml *P = 0.012, TNF 0.1 ng/ml ***P < 0.0001, TNF 1.0 ng ***P = 0.0001, TNF 10 ng/ml ***P < 0.0001, and TNF 100 ng/ml *P = 0.023 by one-way ANOVA with Dunnett's multiple comparisons test.

Mentions: Since rhTNF was only effective when given in the first 24 h following injury and the major inflammatory cell infiltrate during this period consisted primarily of TNF-expressing neutrophils, we investigated whether exogenous rhTNF, which led to augmented fracture repair in vivo, acted via neutrophils. We hypothesized that additional rhTNF at the fracture site would enhance the innate immune response to promote the recruitment of neutrophils, which in turn attract monocytes that have been shown to be associated with fracture healing (Alexander et al, 2011) and orchestrate wound healing in other tissues (Scapini et al, 2000; Nathan, 2006; Soehnlein & Lindbom, 2010). We found that anti-Ly6G treatment also depleted the number of neutrophils and F4/80+ cells at the fracture site on histology on days 0 and 7 (Figs2B, C and 3A). The analysis of numerous sections at multiple time points and the sacrifice of many animals would be required to translate the static pictures provided by histology into an appreciation of the dynamics of cell migration and chemokine release in vivo. Therefore, we used the air pouch model of inflammation, a widely accepted and validated in vivo model for studying the regulation of the early events of local inflammation (Romano et al, 1997; Lawrence et al, 2001), to accurately quantify the neutrophils and monocytes attracted to the complex local fracture cytokine milieu. Media or murine fracture supernatants prepared as described above were injected into the air pouch either alone or in combination with 1 ng rhTNF, and the cellular infiltrates assessed at 4 h. Addition of rhTNF to fracture supernatant into the air pouch resulted in an increase in both the numbers of neutrophils (Ly6G+, CD11b+ cells) and monocytes/macrophages (Ly6G−, CD11b+, CD115+ cells) (Fig3B and Supplementary Fig S1D).


Low-dose TNF augments fracture healing in normal and osteoporotic bone by up-regulating the innate immune response.

Chan JK, Glass GE, Ersek A, Freidin A, Williams GA, Gowers K, Espirito Santo AI, Jeffery R, Otto WR, Poulsom R, Feldmann M, Rankin SM, Horwood NJ, Nanchahal J - EMBO Mol Med (2015)

TNF promotes recruitment of innate immune cells and CCL2 expressionAnti-Ly6G treatment depletes local monocytes/macrophages at the fracture site. Counts of positively stained infiltrative F4/80+ cells in the adjacent muscle to the fracture site following treatment with control or anti-Ly6G at day 1 and day 7. Data are presented as mean ± SEM. **P = 0.005 at day 1 and **P = 0.002 at day 7, and *P = 0.013 for control at day 1 versus control at day 7, by unpaired two-sided t-test.Addition of 1 ng rhTNF to fracture supernatant in the air pouch model led to increased numbers of neutrophils (Ly6G+, CD11b+ cells) and monocytes/macrophages (Ly6G−, CD11b+, CD115+ cells) in the cellular infiltrate. These effects were abrogated by the addition of anti-CCL2. Neutrophils: FS versus FS + TNF *P = 0.015, FS + TNF versus FS + TNF + anti-CCL2 *P = 0.028, and FS + TNF + anti-CCL2 versus FS + TNF + IgG *P = 0.042 by one-way ANOVA followed by Bonferroni's multiple comparison test. Monocytes/macrophages: FS versus FS + TNF **P = 0.0063, FS + TNF versus FS + TNF + anti-CCL2 ****P < 0.0001, and FS + TNF + anti-CCL2 v FS + TNF + IgG *P = 0.015 by one-way ANOVA followed by Bonferroni's multiple comparisons test.Addition of 1 ng rhTNF to fracture supernatant increased CCL2 protein levels in the air pouch. “Neat” indicates level of CCL2 in fracture supernatant before injection into air pouch. Data are presented as mean ± SEM. FS versus FS + TNF **P = 0.0020 by one-way ANOVA with Bonferroni's multiple comparisons test.Addition of rhTNF to enriched bone marrow-derived murine neutrophils pre-exposed to fracture supernatant in vitro led to up-regulation of CCL2 production at 1 h. Data are presented as mean ± SEM. No TNF versus TNF 0.01 ng/ml *P = 0.012, TNF 0.1 ng/ml ***P < 0.0001, TNF 1.0 ng ***P = 0.0001, TNF 10 ng/ml ***P < 0.0001, and TNF 100 ng/ml *P = 0.023 by one-way ANOVA with Dunnett's multiple comparisons test.
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fig03: TNF promotes recruitment of innate immune cells and CCL2 expressionAnti-Ly6G treatment depletes local monocytes/macrophages at the fracture site. Counts of positively stained infiltrative F4/80+ cells in the adjacent muscle to the fracture site following treatment with control or anti-Ly6G at day 1 and day 7. Data are presented as mean ± SEM. **P = 0.005 at day 1 and **P = 0.002 at day 7, and *P = 0.013 for control at day 1 versus control at day 7, by unpaired two-sided t-test.Addition of 1 ng rhTNF to fracture supernatant in the air pouch model led to increased numbers of neutrophils (Ly6G+, CD11b+ cells) and monocytes/macrophages (Ly6G−, CD11b+, CD115+ cells) in the cellular infiltrate. These effects were abrogated by the addition of anti-CCL2. Neutrophils: FS versus FS + TNF *P = 0.015, FS + TNF versus FS + TNF + anti-CCL2 *P = 0.028, and FS + TNF + anti-CCL2 versus FS + TNF + IgG *P = 0.042 by one-way ANOVA followed by Bonferroni's multiple comparison test. Monocytes/macrophages: FS versus FS + TNF **P = 0.0063, FS + TNF versus FS + TNF + anti-CCL2 ****P < 0.0001, and FS + TNF + anti-CCL2 v FS + TNF + IgG *P = 0.015 by one-way ANOVA followed by Bonferroni's multiple comparisons test.Addition of 1 ng rhTNF to fracture supernatant increased CCL2 protein levels in the air pouch. “Neat” indicates level of CCL2 in fracture supernatant before injection into air pouch. Data are presented as mean ± SEM. FS versus FS + TNF **P = 0.0020 by one-way ANOVA with Bonferroni's multiple comparisons test.Addition of rhTNF to enriched bone marrow-derived murine neutrophils pre-exposed to fracture supernatant in vitro led to up-regulation of CCL2 production at 1 h. Data are presented as mean ± SEM. No TNF versus TNF 0.01 ng/ml *P = 0.012, TNF 0.1 ng/ml ***P < 0.0001, TNF 1.0 ng ***P = 0.0001, TNF 10 ng/ml ***P < 0.0001, and TNF 100 ng/ml *P = 0.023 by one-way ANOVA with Dunnett's multiple comparisons test.
Mentions: Since rhTNF was only effective when given in the first 24 h following injury and the major inflammatory cell infiltrate during this period consisted primarily of TNF-expressing neutrophils, we investigated whether exogenous rhTNF, which led to augmented fracture repair in vivo, acted via neutrophils. We hypothesized that additional rhTNF at the fracture site would enhance the innate immune response to promote the recruitment of neutrophils, which in turn attract monocytes that have been shown to be associated with fracture healing (Alexander et al, 2011) and orchestrate wound healing in other tissues (Scapini et al, 2000; Nathan, 2006; Soehnlein & Lindbom, 2010). We found that anti-Ly6G treatment also depleted the number of neutrophils and F4/80+ cells at the fracture site on histology on days 0 and 7 (Figs2B, C and 3A). The analysis of numerous sections at multiple time points and the sacrifice of many animals would be required to translate the static pictures provided by histology into an appreciation of the dynamics of cell migration and chemokine release in vivo. Therefore, we used the air pouch model of inflammation, a widely accepted and validated in vivo model for studying the regulation of the early events of local inflammation (Romano et al, 1997; Lawrence et al, 2001), to accurately quantify the neutrophils and monocytes attracted to the complex local fracture cytokine milieu. Media or murine fracture supernatants prepared as described above were injected into the air pouch either alone or in combination with 1 ng rhTNF, and the cellular infiltrates assessed at 4 h. Addition of rhTNF to fracture supernatant into the air pouch resulted in an increase in both the numbers of neutrophils (Ly6G+, CD11b+ cells) and monocytes/macrophages (Ly6G−, CD11b+, CD115+ cells) (Fig3B and Supplementary Fig S1D).

Bottom Line: Fragility, or osteoporotic, fractures represent a major medical problem as they are associated with permanent disability and premature death.Using a murine model of fragility fractures, we found that local rhTNF treatment improved fracture healing during the early phase of repair.If translated clinically, this promotion of fracture healing would reduce the morbidity and mortality associated with delayed patient mobilization.

View Article: PubMed Central - PubMed

Affiliation: Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK.

No MeSH data available.


Related in: MedlinePlus