HMGB1 protein does not mediate the inflammatory response in spontaneous spinal cord regeneration: a hint for CNS regeneration

Abstract

Uncontrolled, excessive inflammation contributes to the secondary tissue damage of traumatic spinal cord, and HMGB1 is highlighted for initiation of a vicious self-propagating inflammatory circle by release from necrotic cells or immune cells. Several regenerative-competent vertebrates have evolved to circumvent the second damages during the spontaneous spinal cord regeneration with an unknown HMGB1 regulatory mechanism. By genomic surveys, we have revealed that two paralogs of HMGB1 are broadly retained from fish in the phylogeny. However, their spatial-temporal expression and effects, as shown in lowest amniote gecko, were tightly controlled in order that limited inflammation was produced in spontaneous regeneration. Two paralogs from gecko HMGB1 (gHMGB1) yielded distinct injury and infectious responses, with gHMGB1b significantly up-regulated in the injured cord. The intracellular gHMGB1b induced less release of inflammatory cytokines than gHMGB1a in macrophages, and the effects could be shifted by exchanging one amino acid in the inflammatory domain. Both intracellular proteins were able to mediate neuronal programmed apoptosis, which has been indicated to produce negligible inflammatory responses. In vivo studies demonstrated that the extracellular proteins could not trigger a cascade of the inflammatory cytokines in the injured spinal cord. Signal transduction analysis found that gHMGB1 proteins could not bind with cell surface receptors TLR2 and TLR4 to activate inflammatory signaling pathway. However, they were able to interact with the receptor for advanced glycation end products to potentiate oligodendrocyte migration by activation of both NFκB and Rac1/Cdc42 signaling. Our results reveal that HMGB1 does not mediate the inflammatory response in spontaneous spinal cord regeneration, but it promotes CNS regeneration.

Keywords: CNS; Evolution; HMGB1; Inflammation; Receptor for Advanced Glycation End Products (RAGE); Regeneration; Toll-like receptors (TLR).

FIGURE 1.

Phylogenetic tree of gHMGB1 paralogs and those of other representative vertebrates constructed by the neighbor joining method within the package PHYLIP 3.5c. Bootstrap majority consensus values on 1000 replicates are indicated at each branch point in percent. Amphioxus HMGB was designated as outgroup. Sequences obtained from GenBank^TM are as follows: zebrafish HMGB1a (NM_199555); zebrafish HMGB1b (NM_001099251); Xenopus HMGB1 (U21933); Xenopus HMGB1-like (BC054148); chicken HMGB1 (NP_990233); rat HMGB1 (NP_037095); rat HMGB1-like (XP_003753270); mouse HMGB1 (NP_034569); mouse HMGB1-like (XP_889413); and human HMGB1 (NP_002119).

FIGURE 2.

Transcriptional expression of gHMGB1 paralogs in tissues and differential responses to injury and infectious challenges. A and B, Northern blotting analysis of gHMGB1a and gHMGB1b transcripts in different tissues. C, quantitative real time PCR analysis of gHMGB1a transcripts in different gecko tissues. Gecko EF-1α was used for normalization. D and E, quantitative RT-PCR amplification of gHMGB1a and gHMGB1b in the spinal cord from L13 to the 6th caudal vertebra following tail amputation at 0 day (con), 1 and 3 days (d) and 1 and 2 weeks (w). F and G, quantitative RT-PCR amplification of gHMGB1a and gHMGB1b in the different tissues after injection of LPS (7.5 mg/kg body weight) intraperitoneally. Quantities were normalized to endogenous EF-1α expression. Error bars represent the standard deviation (*, p < 0.01).

FIGURE 3.

Distribution of gHMGB1a and gHMGB1b in the spinal cord. A, preparation of gHMGB1a and gHMGB1b recombinant proteins by eukaryotic expression in 293T cells. B, gHMGB1a and gHMGB1b polyclonal antibodies react specifically with spinal cord tissue by Western blot analysis. C, colocalization of gHMGB1a and gHMGB1b with NF-positive cells (arrowhead). D, colocalization of gHMGB1a and gHMGB1b with CD11b-positive cells (arrowhead). Lane M indicates protein marker. Rectangle indicates region magnified below. Scale bars, 100 μm.

FIGURE 4.

Production of inflammatory cytokines in macrophage RAW 264.7 cells transfected with pEGFP-gHMGB1a, pEGFP-gHMGB1b, and mutation plasmids. A, alignment of first 20 amino acids from B-box domain of gHMGB1 paralogs, human and mouse HMGB1, showing an amino acid difference in gHMGB1b. B, overexpression of pEGFP-N1, pEGFP-gHMGB1a, pEGFP-gHMGB1b, mutation plasmids (Lys ↔ Glu) pEGFP-gHMGB1ma and pEGFP-gHMGB1mb in RAW 264.7 cells for 48 h. C, transfected cells were selected by flow cytometry. D and E, transcriptional analysis of TNF-α and IL-1α in macrophages following transfected with pEGFP-gHMGB1a and pEGFP-gHMGB1b. F and G, transcriptional analysis of TNF-α and IL-1α in macrophages following transfected with pEGFP-gHMGB1ma and pEGFP-gHMGB1mb. Control means cells transfected by pEGFP-N1. Quantities were normalized to endogenous GAPDH expression. Error bars in D–G represent the standard deviation (*, p < 0.01). Scale bars, 30 μm.

FIGURE 5.

Effects of intracellular gHMGB1 paralogs on the neurons. A, Western blot analysis of cleaved caspase3 in SH-SY5Y cells after transfection with pEGFP-gHMGB1a, pEGFP-gHMGB1b, pEGFP-gHMGB1ma, and pEGFP-gHMGB1mb for 72 h. Control means cells transfected with pEGFP-N1; B, statistics of A, and quantities were normalized to endogenous β-actin. Error bars in B represent the standard deviation (*, p < 0.01); C–Q, immunohistochemistry showing colocalization of transfected cells with annexin-V. Scale bars, 40 μm.

FIGURE 6.

Effects of gHMGB1 paralogs recombinant proteins on the release of inflammatory cytokines. A and B, determination of TNF-α and IL-1α transcripts in macrophage RAW 264.7 cells after treatment with 0, 1, 5, and 10 μg/ml recombinant proteins prepared from gHMGB1a, gHMGB1b, gHMGB1ma and gHMGB1mb, for 24 h, respectively. C and D, ELISA of TNF-α from supernants (C) and pellets (D) of macrophages cultured with 1 μg/ml gHMGB1a, gHMGB1b, gHMGB1ma. and gHMGB1mb recombinant proteins for 24 h. E and F, ELISA of IL-1α from supernants (E) and pellets (F) of macrophages cultured with 1 μg/ml gHMGB1a, gHMGB1b, gHMGB1ma, and gHMGB1mb recombinant proteins for 24 h. G, ELISA of TNF-α from spinal cord segments proximal to amputation following injection of 5 μl of 300 μg/ml gHMGB1 recombinant proteins for 24, 48, and 72 h, respectively. H, ELISA of IL-1α from spinal cord segments proximal to amputation following injection of 5 μl of 300 μg/ml gHMGB1 recombinant proteins for 24, 48, and 72 h, respectively. A total of 5 μl of 300 μg/ml mHMGB1 recombinant proteins, commercial hHMGB1, or 5 μl of 200 μg/ml LPS was performed as positive control. Negative control means ELISA of cytokines immediately after amputation or injection with a dose of 5 μl of saline. Error bars in C–H represent the standard deviation (*. p < 0.01).

FIGURE 7.

Binding assays of gHMGB1a and gHMGB1b with RAGE, TLR2, and TLR4 receptors. A, Western blot analysis of RAGE, TLR2, TLR4, and p65NFκB after macrophages and SH-SY5Y cells were cultured with 1 μg/ml gHMGB1a or gHMGB1b recombinant proteins for 24 h, respectively; B, statistical analysis of A; C–E, immunoprecipitation (IP) using anti-RAGE (C), -TLR2 (D), or -TLR4 antibody (E) and detection of the components of the RAGE- (C), TLR2- (D), or TLR4-associated complexes (D) with anti-His antibody.

FIGURE 8.

Effects of gHMGB1 paralog recombinant proteins on the neurons and oligodendrocytes. A, Western blot analysis of cleaved caspase3 in the SH-SY5Y cells cultured with 1 μg/ml exogenous recombinant gHMGB1a, gHMGB1b, or mutant proteins. B, immunofluorescence of SH-SY5Y cells detected by NF-200 antibody, and analysis of the neurite length. C–G, assays of oligodendrocytes Gsn3 migration by transwell experiments after cells cultured with PBS, pH 7.4 (C), recombinant gHMGB1a (D), or gHMGB1b proteins (E) for 36 h; anti-gHMGB1a antibody (F) and anti-gHMGB1b antibody (G) were able to accordingly block the effects. H, statistical analysis of migrated cell numbers in C–G. Error bars in H represent the standard deviation (*, p < 0.01). Scale bars, 25 μm in B and 50 μm in C–G.

FIGURE 9.

Examination of HMGB1/RAGE signal pathway associated with oligodendrocyte migration. A, Western blot analysis of RAGE, p65NFκB, CREB, p-CREB, JNK, p-JNK, p38, p-p38, ERK1/2, and p-ERK1/2 implicated in the pathway activation. B, determination of Rac1/Cdc42/RhoA in signaling activation. Error bars in A and B represent the standard deviation (*, p < 0.01). Con, control.

FIGURE 10.

Illustration of gHMGB1 paralog functions on different cells of traumatic spinal cord. Red ellipsoids indicate gHMGB1a, and green ones indicate gHMGB1b.