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The alternatively spliced fibronectin CS1 isoform regulates IL-17A levels and mechanical allodynia after peripheral nerve injury.

Liu H, Dolkas J, Hoang K, Angert M, Chernov AV, Remacle AG, Shiryaev SA, Strongin AY, Nishihara T, Shubayev VI - J Neuroinflammation (2015)

Bottom Line: The CS1 peptide effects were analyzed in cultured primary Schwann cells, the major source of FN-CS1 in CCI nerves.Acute CS1 therapy attenuated mechanical allodynia (pain from innocuous stimulation) but not thermal hyperalgesia and reduced the levels of IL-17A expression in the injured nerve.CS1 peptide therapy can be developed for pharmacological control of neuropathic pain.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, University of California, 9500 Gilman Dr., Mail Code 0629, La Jolla, San Diego, CA, 92093-0629, USA. hliu@salk.edu.

ABSTRACT

Background: Mechanical pain hypersensitivity associated with physical trauma to peripheral nerve depends on T-helper (Th) cells expressing the algesic cytokine, interleukin (IL)-17A. Fibronectin (FN) isoform alternatively spliced within the IIICS region encoding the 25-residue-long connecting segment 1 (CS1) regulates T cell recruitment to the sites of inflammation. Herein, we analyzed the role of CS1-containing FN (FN-CS1) in IL-17A expression and pain after peripheral nerve damage.

Methods: Mass spectrometry, immunoblotting, and FN-CS1-specific immunofluorescence analyses were employed to examine FN expression after chronic constriction injury (CCI) in rat sciatic nerves. The acute intra-sciatic nerve injection of the synthetic CS1 peptide (a competitive inhibitor of the FN-CS1/α4 integrin binding) was used to elucidate the functional significance of FN-CS1 in mechanical and thermal pain hypersensitivity and IL-17A expression (by quantitative Taqman RT-PCR) after CCI. The CS1 peptide effects were analyzed in cultured primary Schwann cells, the major source of FN-CS1 in CCI nerves.

Results: Following CCI, FN expression in sciatic nerve increased with the dominant FN-CS1 deposition in endothelial cells, Schwann cells, and macrophages. Acute CS1 therapy attenuated mechanical allodynia (pain from innocuous stimulation) but not thermal hyperalgesia and reduced the levels of IL-17A expression in the injured nerve. CS1 peptide inhibited the LPS- or starvation-stimulated activation of the stress ERK/MAPK pathway in cultured Schwann cells.

Conclusions: After physical trauma to the peripheral nerve, FN-CS1 contributes to mechanical pain hypersensitivity by increasing the number of IL-17A-expressing (presumably, Th17) cells. CS1 peptide therapy can be developed for pharmacological control of neuropathic pain.

No MeSH data available.


Related in: MedlinePlus

Acute CS1 therapy reduces IL-17A levels in CCI nerve. aTaqman qRT-PCR of IL-17A in sciatic nerve after acute intraneural CS1 or sCS1 treatment (50 μg/ml in 5 μl) performed at day 7 post-CCI after the completion of behavioral testing (Fig. 2). The mean relative mRNA ± SEM of n = 5/group normalized to GAPDH compared to naive nerve (*p < 0.05). b Methylene blue/azure II staining in 1-μm-thick sciatic nerve sections after acute intraneural CS1 or sCS1 treatment (50 μg/ml in 5 μl) performed at day 7 post-CCI, after the completion of behavioral testing (Fig. 2). Control nerve showed intact nerve morphology. CCI nerves displayed Wallerian degeneration (axonal degeneration, edema, myelin ovoids, and immune cell infiltration) after sCS1 treatment. In contrast, a greater number of uncompromised axons were observed in CCI nerves treated with CS1. Representative micrographs of n = 3/group. Scale bars, 20 μm
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Fig3: Acute CS1 therapy reduces IL-17A levels in CCI nerve. aTaqman qRT-PCR of IL-17A in sciatic nerve after acute intraneural CS1 or sCS1 treatment (50 μg/ml in 5 μl) performed at day 7 post-CCI after the completion of behavioral testing (Fig. 2). The mean relative mRNA ± SEM of n = 5/group normalized to GAPDH compared to naive nerve (*p < 0.05). b Methylene blue/azure II staining in 1-μm-thick sciatic nerve sections after acute intraneural CS1 or sCS1 treatment (50 μg/ml in 5 μl) performed at day 7 post-CCI, after the completion of behavioral testing (Fig. 2). Control nerve showed intact nerve morphology. CCI nerves displayed Wallerian degeneration (axonal degeneration, edema, myelin ovoids, and immune cell infiltration) after sCS1 treatment. In contrast, a greater number of uncompromised axons were observed in CCI nerves treated with CS1. Representative micrographs of n = 3/group. Scale bars, 20 μm

Mentions: IL-17, a Th17 cell cytokine in CCI nerves [10, 13], selectively controls mechanical (but not thermal) pain hypersensitivity [13]. Given these comparable effects of the CS1 therapy and IL-17 gene deletion [13], we tested a model in which CS1 therapy acts by reducing IL-17A gene expression in the CCI nerves. Following the behavioral testing, we employed Taqman qRT-PCR to measure the IL-17A mRNA levels (Fig. 3a) and ultrastructural analysis (Fig. 3b) of the nerves exposed to CS1 or sCS1 peptides. The IL-17A mRNA was undetectable in naïve nerve (Fig. 3a), consistent with the report by others [10]. Accordingly, control nerve displayed the uniform morphology of intact axons (Fig. 3b). The IL-17A levels were significantly elevated in the injured nerves at day 7 post-CCI in the rats that received sCS1 (Fig. 3a). These nerves exhibited the characteristic features of Wallerian degeneration, including endoneurial edema, axon degeneration, the presence of myelin ovoids, and immune cell clusters (Fig. 3b). Both the IL-17A expression (Fig. 3a) and the features of degeneration (Fig. 3b) were significantly reduced in the animals that received CS1. These data suggested that acute CS1 therapy decreased a number of IL-17A+ (presumably, Th17) cells in the rodent neuropathy model.Fig. 3


The alternatively spliced fibronectin CS1 isoform regulates IL-17A levels and mechanical allodynia after peripheral nerve injury.

Liu H, Dolkas J, Hoang K, Angert M, Chernov AV, Remacle AG, Shiryaev SA, Strongin AY, Nishihara T, Shubayev VI - J Neuroinflammation (2015)

Acute CS1 therapy reduces IL-17A levels in CCI nerve. aTaqman qRT-PCR of IL-17A in sciatic nerve after acute intraneural CS1 or sCS1 treatment (50 μg/ml in 5 μl) performed at day 7 post-CCI after the completion of behavioral testing (Fig. 2). The mean relative mRNA ± SEM of n = 5/group normalized to GAPDH compared to naive nerve (*p < 0.05). b Methylene blue/azure II staining in 1-μm-thick sciatic nerve sections after acute intraneural CS1 or sCS1 treatment (50 μg/ml in 5 μl) performed at day 7 post-CCI, after the completion of behavioral testing (Fig. 2). Control nerve showed intact nerve morphology. CCI nerves displayed Wallerian degeneration (axonal degeneration, edema, myelin ovoids, and immune cell infiltration) after sCS1 treatment. In contrast, a greater number of uncompromised axons were observed in CCI nerves treated with CS1. Representative micrographs of n = 3/group. Scale bars, 20 μm
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Fig3: Acute CS1 therapy reduces IL-17A levels in CCI nerve. aTaqman qRT-PCR of IL-17A in sciatic nerve after acute intraneural CS1 or sCS1 treatment (50 μg/ml in 5 μl) performed at day 7 post-CCI after the completion of behavioral testing (Fig. 2). The mean relative mRNA ± SEM of n = 5/group normalized to GAPDH compared to naive nerve (*p < 0.05). b Methylene blue/azure II staining in 1-μm-thick sciatic nerve sections after acute intraneural CS1 or sCS1 treatment (50 μg/ml in 5 μl) performed at day 7 post-CCI, after the completion of behavioral testing (Fig. 2). Control nerve showed intact nerve morphology. CCI nerves displayed Wallerian degeneration (axonal degeneration, edema, myelin ovoids, and immune cell infiltration) after sCS1 treatment. In contrast, a greater number of uncompromised axons were observed in CCI nerves treated with CS1. Representative micrographs of n = 3/group. Scale bars, 20 μm
Mentions: IL-17, a Th17 cell cytokine in CCI nerves [10, 13], selectively controls mechanical (but not thermal) pain hypersensitivity [13]. Given these comparable effects of the CS1 therapy and IL-17 gene deletion [13], we tested a model in which CS1 therapy acts by reducing IL-17A gene expression in the CCI nerves. Following the behavioral testing, we employed Taqman qRT-PCR to measure the IL-17A mRNA levels (Fig. 3a) and ultrastructural analysis (Fig. 3b) of the nerves exposed to CS1 or sCS1 peptides. The IL-17A mRNA was undetectable in naïve nerve (Fig. 3a), consistent with the report by others [10]. Accordingly, control nerve displayed the uniform morphology of intact axons (Fig. 3b). The IL-17A levels were significantly elevated in the injured nerves at day 7 post-CCI in the rats that received sCS1 (Fig. 3a). These nerves exhibited the characteristic features of Wallerian degeneration, including endoneurial edema, axon degeneration, the presence of myelin ovoids, and immune cell clusters (Fig. 3b). Both the IL-17A expression (Fig. 3a) and the features of degeneration (Fig. 3b) were significantly reduced in the animals that received CS1. These data suggested that acute CS1 therapy decreased a number of IL-17A+ (presumably, Th17) cells in the rodent neuropathy model.Fig. 3

Bottom Line: The CS1 peptide effects were analyzed in cultured primary Schwann cells, the major source of FN-CS1 in CCI nerves.Acute CS1 therapy attenuated mechanical allodynia (pain from innocuous stimulation) but not thermal hyperalgesia and reduced the levels of IL-17A expression in the injured nerve.CS1 peptide therapy can be developed for pharmacological control of neuropathic pain.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, University of California, 9500 Gilman Dr., Mail Code 0629, La Jolla, San Diego, CA, 92093-0629, USA. hliu@salk.edu.

ABSTRACT

Background: Mechanical pain hypersensitivity associated with physical trauma to peripheral nerve depends on T-helper (Th) cells expressing the algesic cytokine, interleukin (IL)-17A. Fibronectin (FN) isoform alternatively spliced within the IIICS region encoding the 25-residue-long connecting segment 1 (CS1) regulates T cell recruitment to the sites of inflammation. Herein, we analyzed the role of CS1-containing FN (FN-CS1) in IL-17A expression and pain after peripheral nerve damage.

Methods: Mass spectrometry, immunoblotting, and FN-CS1-specific immunofluorescence analyses were employed to examine FN expression after chronic constriction injury (CCI) in rat sciatic nerves. The acute intra-sciatic nerve injection of the synthetic CS1 peptide (a competitive inhibitor of the FN-CS1/α4 integrin binding) was used to elucidate the functional significance of FN-CS1 in mechanical and thermal pain hypersensitivity and IL-17A expression (by quantitative Taqman RT-PCR) after CCI. The CS1 peptide effects were analyzed in cultured primary Schwann cells, the major source of FN-CS1 in CCI nerves.

Results: Following CCI, FN expression in sciatic nerve increased with the dominant FN-CS1 deposition in endothelial cells, Schwann cells, and macrophages. Acute CS1 therapy attenuated mechanical allodynia (pain from innocuous stimulation) but not thermal hyperalgesia and reduced the levels of IL-17A expression in the injured nerve. CS1 peptide inhibited the LPS- or starvation-stimulated activation of the stress ERK/MAPK pathway in cultured Schwann cells.

Conclusions: After physical trauma to the peripheral nerve, FN-CS1 contributes to mechanical pain hypersensitivity by increasing the number of IL-17A-expressing (presumably, Th17) cells. CS1 peptide therapy can be developed for pharmacological control of neuropathic pain.

No MeSH data available.


Related in: MedlinePlus