Selective promotion of sensory innervation-mediated immunoregulation for tissue repair

Abstract

Sensory innervation triggers the regenerative response after injury. However, dysfunction and impairment of sensory nerves, accompanied by excessive inflammation impede tissue regeneration. Consequently, specific induction of sensory innervation to mediate immunoregulation becomes a promising therapeutic approach. Herein, we developed a cell/drug-free strategy to selectively boost endogenous sensory innervation to harness immune responses for promoting tissue rehabilitation. Specifically, a dual-functional phage was constructed with a sensory nerve-homing peptide and a β-subunit of nerve growth factor (β-NGF)-binding peptide. These double-displayed phages captured endogenic β-NGF and localized to sensory nerves to promote sensory innervation. Furthermore, regarding bone regeneration, phage-loaded hydrogels achieved rapid sensory nerve ingrowth in bone defect areas. Mechanistically, sensory neurotization facilitated M2 polarization of macrophages through the Sema3A/XIAP/PAX6 pathway, thus decreasing the M1/M2 ratio to induce the dissipation of local inflammation. Collectively, these findings highlight the essential role of sensory innervation in manipulating inflammation and provide a conceptual framework based on neuroimmune interactions for promoting tissue regeneration.

Fig. 1.. Schematic illustration.

The promoted bone regenerative processes induced by dual-functional phage-loaded composite hydrogels, progressed with selective promotion of sensory innervation and regulation of immune responses via neuroimmune interactions. The phages released from the hydrogel capture the autogenous β-NGF in the impaired microenvironment and home to local sensory nerve fibers to facilitate sensory innervation. The sensory neurons subsequently mediate macrophage polarization toward M2 phenotype through the Sema3A/XIAP/PAX6 pathway, which further induces the dissipation of local inflammation and improved bone healing outcome. β-NGF, β subunit of nerve growth factor; M1 and M2, M1 and M2 polarized macrophage.

Fig. 2.. Sensory denervation impairs bone healing after injury with aggravation of the inflammatory response.

(A and B) Schematic representation of the rat mandibular defect and IAN innervation models. (C) Time durations of the animal experiments. w, weeks. (D) Photographs of mandibular samples and (E) the corresponding 3D reconstruction micro-CT images after 2 and 8 weeks of surgery. The black dotted circles in mandible photographs and the blue circles in reconstructed images represent the initial defect areas (diameter = 4 mm). Scale bars, 1 cm. (F and G) Quantitative analysis of coverage percentage and new bone volume at the defect site. (H) Venn diagram of the numbers of DEGs in the denervation and sham groups. (I) Volcano plot of differential gene expression between the two groups. (J) Heatmap of differential gene expression in the denervation and sham groups. (K) Enriched KEGG pathways in the denervation and sham groups. (L) GSEA of up-regulated and down-regulated macrophage-related genes. (M) Panoramic and partially enlarged images of H&E, Masson, and immunohistochemical stained tissues (for CGRP, dopamine, iNOS, and CD206) at 1 and 2 weeks after surgery. Scale bars, 1 mm and 60 μm, respectively. (N to P) Corresponding semiquantitative results of immunohistochemistry analysis at 1 week and (Q to S) 2 weeks after surgery. ***P < 0.001; NS, not significant.

Fig. 3.. Discovery of sensory neuron targeting peptides and functional verification of an NGF-binding peptide.

(A) Schematic diagram of the screening of sensory neuron targeting peptides from a library of random M13 phage–displayed peptides. (B) Occurrence frequencies of selected sensory neuron–binding peptides after biopanning. (C) The phage ELISA demonstrated the binding between sensory neurons and the phages displayed peptides with a high occurrence frequency in biopanning. (D) Fluorescence staining to demonstrate the binding abilities of AV-displaying phages in vitro. The WT, AV-displaying, and AV^R (the reversed sequence of AV)–displaying M13 phages were labeled with FITC dyes and added to the medium containing sensory neurons (marked with TUBB3). Scale bar, 30 μm. (E) MD simulation of the binding between the β-NGF protein (gray) and the identified NK peptide (rainbow). CABS-docking modeling of NK and NK^R (reversed sequence of NK) peptides bound to β-NGF. (F) Density of the peptide clusters bound to the β-NGF protein. (G) MD simulation snapshots of β-NGF interacting with NK and NK^R visualized using Visual MD with a 10-ns interval and a total duration of 50 ns. (H) ELISA results of the interaction between β-NGF and the phage-displayed NK and NK^R peptides. ***P < 0.001.

Fig. 4.. Construction and functional verification of the dual-function phages.

(A) Schematic representation of the doubled-displaying phages. The genetically engineered M13-AV-NK phages displayed sensory neuron–targeting (AV, fused to pIII at the tip) and NFG-binding peptides (NK, fused to pVIII on the side wall). (B) DNA sequencing of the dual-displaying phages to verify the successful insertion of the sequences encoding the AV and NK peptides. (C) Nucleic acid electrophoresis images of M13-WT and M13-AV-NK phages. (D and E) ELISAs showed the binding abilities of the M13-AV-NK phages for sensory neurons and β-NGF. (F) Fluorescence staining to determine the binding abilities of M13-AV-NK phages for sensory neurons (marked with TUBB3). Scale bar, 30 μm. (G) Schematic diagram of the sensory neuron culture model with periodic administration of exogenous β-NGF in vitro to simulate the in vivo microenvironment. β-NGF was added to the basic culture medium containing sensory neurons, which were pretreated with M13-AV-NK phages, and left for a short period of time before being removed by washing. The same process was conducted once a day for 3 days. (H) Immunofluorescence staining and (I) the corresponding quantification of TUBB3 and CGRP expression in sensory neurons. Scale bar, 50 μm. (J) Relative mRNA expression levels of Cgrp and Tac1 in different groups at 72 hours. (K) Schematic representation of DRG explant culture in the same model as illustrated in (G) and evaluations, including Sholl analysis and axon analysis. (L) Immunofluorescence staining of TUBB3 to evaluate the axon growth of DRG explants in different groups after 72 hours of culturing. Scale bar, 500 μm. (M) Sholl plots and (N) axon tracing analysis results of M13-AV-NK–promoted neurite growth in in vitro DRG explants cultures with added β-NGF. h, hours. *P < 0.05; ***P < 0.001; NS, not significant.

Fig. 5.. Fabrication and characterization of the bone repair composite system.

(A) Schematic diagram and (B) the specific construction processes of the M13-AV-NK/β-TCP/Col I composite hydrogel. (C) TEM images of M13-AV-NK, β-TCP, and M13-AV-NK–loaded β-TCP. The red arrows represent the M13 phages attached to β-TCP particles. Scale bars, 2 μm in the bottom left image and 200 nm for the other images. (D) Schematic diagram of the determination of phage release from the M13-AV-NK/β-TCP/Col I composite hydrogel. (E) Representative images of the phage locus coeruleus count and (F) the corresponding titer of the phages released from the M13-AV-NK–loaded β-TCP complex at different time points. (G) Representative images and (H) quantification of agar plate cultivation of E. coli treated with the released phages from various time points. (I) ATR-FTIR spectra of the four different groups. The dotted lines (a to c) represent specific absorption peaks related to genipin-mediated cross-linking. (J) Mechanical properties of the different hydrogels. (K) SEM images of the different groups. The red arrow in the lower-right image indicates DRGCs growing on the surface of the M13-AV-NK/β-TCP/Col I hydrogel. ***P < 0.001; NS, not significant. h, hours.

Fig. 6.. Bone repair system promotes sensory innervation in vivo to inhibit the inflammatory response and improve bone healing.

(A and B) Schematic representation of the rat mandibular critical-sized defect model and injection of the M13-AV-NK/β-TCP/Col I hydrogel. (C) Timelines of the animal experiments. (D) Panoramic and enlarged images and (E) the corresponding quantitative analysis of H&E, Masson, and immunohistochemical staining (CGRP, Dopamine, iNOS, and CD206) results at 1 week after implantation. The black asterisks represent residual hydrogels. Scale bars, 1 mm and 60 μm, respectively. (F and G) Micro-CT photographs of mandibular samples and the corresponding 3D reconstruction images at 2 and 8 weeks after implantation. The black dotted circles in mandible photographs and the blue circles in reconstructed images represent the initial defect areas (diameter = 5 mm). Scale bars, 1 cm. (H and I) Quantitative analysis of coverage percentage and new bone volume at the defect site for both time points. (J) Panoramic and enlarged images and (K) the corresponding quantification of H&E, Masson, and immunohistochemistry staining (ALP and OCN staining) at 8 weeks after implantation. β-TCP/g-Col I, WT/β-TCP/g-Col I, and M13-AV-NK/β-TCP/g-Col I (abbreviated as Blank, WT, and M13-AV-NK, respectively). The black asterisks represent residual hydrogels. “FT” and “NB” in H&E images indicate fiber tissue and new bone tissue, respectively. Scale bars, 1 mm and 60 μm, respectively. **P < 0.01; ***P < 0.001; NS, not significant.

Fig. 7.. IAN denervation leads to therapeutic failure of the bone repair system.

(A) Schematic diagram of the rat mandibular critical-sized defect model with IAN denervation. Before M13-AV-NK/β-TCP/Col I hydrogel administration, the IAN was transected from rats in the denervation group, and a sham operation was conducted for the control group. (B) Timelines of the animal experiments. (C) Panoramic and partially enlarged images and (D) the corresponding quantitative results of H&E, Masson, and immunohistochemistry staining (CGRP, Dopamine, iNOS, and CD206) after 1 week of implantation. The black asterisks represent residual hydrogels. Scale bars, 1 mm and 60 μm, respectively. (E) Photographs of mandibular samples and the corresponding 3D reconstruction micro-CT images at 2 and 8 weeks after implantation. The black dotted circles in mandible photographs and the blue circles in reconstructed images represent the initial defect areas (diameter = 5 mm). Scale bars, 1 cm. (F) Quantitative analysis of 3D reconstruction results, including coverage percentage and the new bone volume of the defect region, of the two groups at different time points. (G) Panoramic and partially enlarged images and (H) the corresponding quantification results of H&E, Masson, and immunohistochemistry staining (ALP and OCN) after 8 weeks of implantation. The black asterisks represent residual hydrogels. “FT” and “NB” in the H&E images indicate fiber tissue and new bone tissue, respectively. Scale bars, 1 mm and 60 μm. (I) Schematic diagram of early-stage and late-stage inflammatory processes during bone healing in models administered the phage-loaded bone repair system with or without IAN transection. *P < 0.05; ***P < 0.001; NS, not significant.

Fig. 8.. Sema3A mediates XIAP expression to promote the M2 polarization of macrophages.

(A) Schematic representation of macrophage conditioned culture. The supernatant of DRGC culture medium was collected and mixed with the conventional culture medium of macrophages in equal proportion to obtain the CM. (B) Immunofluorescence staining and (C) quantification of CD206 (M2 marker) and iNOS (M1 marker) expression in macrophages. Scale bar, 30 μm. (D) ELISAs of anti-inflammatory cytokine IL-10 and pro-inflammatory cytokines TNF-α and IL-1β. (E) Immunofluorescence staining and (F) the corresponding quantification of CD206 and iNOS expression in macrophages treated with CM from Sema3A-specific knockdown DRGCs and rSema3A. Scale bars, 30 μm. (G) ELISAs of IL-10, TNF-α, and IL-1β expression after culturing with different CMs. (H) Volcano plot of the differential gene expression in macrophages after Sema3A transfection. (I) Heatmap of the mRNA transcription profiles of the top 15 up-/down-regulated genes. (J) Venn diagram indicating the number of genes overlapping with those collected from the GSEA database (inflammation-related gene sets containing 303 different genes) and the top 15 up-/down-regulated genes screened using RNA-seq. Two genes (Xiap and Mmp13) were overlapping. (K) Representative Western blotting images of XIAP and MMP13 in macrophages after culturing with Sema3A. (L and M) Immunofluorescence images and the corresponding fluorescence intensity of CD206 and iNOS expression in macrophages after shXIAP transfection. Scale bar, 30 μm. (N) ELISAs of IL-10, TNF-α, and IL-1β after shXIAP transfection. (O and P) Immunofluorescence images and the corresponding quantitative analysis of CD206 and iNOS in XIAP-overexpressing macrophages transfected with pcDNA3.1-XIAP. Scale bar, 30 μm. (Q) ELISAs of IL-10, TNF-α, and IL-1β in macrophages overexpressing XIAP. IAN, inferior alveolar nerve; rSema3A, recombinant Sema3A. **P < 0.01; ***P < 0.001; NS, not significant.

Fig. 9.. XIAP-mediated PAX6 ubiquitination regulates macrophage polarization.

(A) Heatmap of the top 5 proteins with up-regulated and down-regulated expression identified by proteomic analysis between macrophages with or without XIAP knockdown. (B) Overlap between selected proteomic results and the UbiBrowser database. PAX6 was the only overlapping protein. (C) Representative Western blotting images of PAX6 expression after XIAP knockdown. (D) Co-IP assay to analyze the interaction between XIAP and PAX6 after cotransfection of Myc-XIAP and Flag-PAX6 in HEK293T cells. (E) The ubiquitin level of PAX6 was detected in HEK293T cells using an IP assay after cotransfection by Myc-tagged XIAP (Myc-XIAP), Flag-tagged PAX6 (Flag-PAX6), and HA-tagged ubiquitin (HA-UB) plasmids. (F) HEK293T cells were transfected with plasmids of Myc-XIAP (WT, H467A, or ΔRing), Flag-PAX6, and HA-UB and then subjected to ubiquitin detection. (G) Immunofluorescence images of macrophages (expressing CD206 and iNOS) after knockdown of XIAP or PAX6 using shXIAP or shPAX6, respectively. (H) ELISAs of IL-10, TNF-α, and IL-1β. (I) Schematic diagram of the possible mechanism of macrophage polarization mediated by Sema3A via the Sema3A/XIAP/Pax6 pathway. **P < 0.01; ***P < 0.001.