Bridging immune-neurovascular crosstalk via the immunomodulatory microspheres for promoting neural repair

Abstract

The crosstalk between immune cells and the neurovascular unit plays a pivotal role in neural regeneration following central nervous system (CNS) injury. Maintaining brain immune homeostasis is crucial for restoring neurovascular function. In this study, an interactive bridge was developed via an immunomodulatory hydrogel microsphere to link the interaction network between microglia and the neurovascular unit, thereby precisely regulating immune-neurovascular crosstalk and achieving neural function recovery. This immunomodulatory crosstalk microsphere (MP/RIL4) was composed of microglia-targeted RAP12 peptide-modified interleukin-4 (IL-4) nanoparticles and boronic ester-functionalized hydrogel using biotin-avidin reaction and air-microfluidic techniques. We confirmed that the immunomodulatory microspheres reduced the expression of pro-inflammatory factors including IL-1β, iNOS, and CD86, while upregulating levels of anti-inflammatory factors such as IL-10, Arg-1, and CD206 in microglia. In addition, injection of the MP/RIL4 significantly mitigated brain atrophy volume in a mouse model of ischemic stroke, promoted neurobehavioral recovery, and enhanced the crosstalk between immune cells and the neurovascular unit, thus increasing angiogenesis and neurogenesis of stroke mice. In summary, the immunomodulatory microspheres, capable of orchestrating the interaction between immune cells and neurovascular unit, hold considerable therapeutic potential for ischemic stroke and other CNS diseases.

Keywords: Angiogenesis; Crosstalk; Immune modulating microsphere; Ischemic stroke; Neurogenesis.

Graphical abstract

Fig. 1

Schematic diagram of immunomodulatory microsphere (MP/RIL4) for bridging immune neurovascular crosstalk. (A) Preparation of the immunomodulatory microsphere (MP/RIL4). RAP12 modified nanoparticle with IL-4 was prepared via biotin-avidin reaction, and the boronic ester-functionalized PVA microspheres were fabricated using air-microfluidic technique and crosslinking with TSPBA. MP/RIL4 was obtained by adding the RIL4 nanoparticles into the lyophilized MP microspheres through negative pressure suction. (B) MP/RIL4 promoted the polarization of microglia to anti-inflammatory phenotype in vitro and in vivo, and improved neurogenesis and angiogenesis of ischemic stroke mice. The schematic diagram was created with BioRender.com.

Fig. 2

Preparation and characterization of PVA microspheres loaded with RAP12-PLGA/IL-4 nanoparticles. (A) Representative TEM images of nanoparticles loaded with IL-4 (IL4-NP), and IL4-NP modified with RAP12 (RIL4). (B) Quantitative size distribution analysis of IL4-NP and RIL4. (C, D) Bright filed images and quantitative size distribution analysis of PVA microsphere (MP) precursor and PVA microspheres (MPs). (E, F) Representative SEM images of MP and MP/RIL4. (G) Drug release of RIL4 in PBS, MP/RIL4 in PBS, and H₂O₂. n = 3. All data are presented as mean ± SEM.

Fig. 3

Microspheres loaded with RAP12-PLGA/IL-4 did not affect the proliferation and survival of microglia. (A) Cell viability of PBS, MP, RIL4 and MP/RIL4 co-cultured with BV2 microglia after 24 h n = 4. (B) Representative images of Calcein AM/PI staining images of PBS, MP, RIL4 and MP/RIL4 co-cultured with BV2 microglia after 24 h. Scale bar = 100 μm. (C) Cell viability of PBS, MP, RIL4 and MP/RIL4 co-cultured with BV2 microglia after 48 h n = 4. (D) Representative Calcein AM/PI staining images of PBS, MP, RIL4 and MP/RIL4 co-cultured with BV2 microglia after 48 h. Scale bar = 100 μm. (E) Cell viability of PBS, MP, RIL4 and MP/RIL4 co-cultured with BV2 microglia after 72 h n = 4. (F) Representative Calcein AM/PI staining images of PBS, MP, RIL4 and MP/RIL4 co-cultured with BV2 microglia after 72 h. Scale bar = 100 μm. All data are presented as mean ± SEM.

Fig. 4

Immunoregulation effect of the “immunomodulator” in vitro. (A, B) Quantitative RT-PCR results of pro-inflammatory marker (IL-1β, iNOS) and anti-inflammatory marker (Arg-1, IL-10) expression of PBS, MP, RIL4 and MP/RIL4 co-cultured with LPS stimulated BV2 microglia after 24 h n = 3–4. (C, D) Western blot and quantitative analysis of Arg-1 expression after PBS, MP, RIL4 and MP/RIL4 co-cultured with LPS stimulated BV2 microglia after 24h. n = 3. (E) NO production of PBS, LPS, L-MP, L-RIL4 and L-MP/RIL4 co-cultured with BV2 microglia after 24 h measured by NO assay kit. n = 3. (F) IL-10 protein concentration of PBS, LPS, L-MP, L-RIL4 and L-MP/RIL4 co-cultured with BV2 microglia after 24 h determined by ELISA. n = 3. (G–J) Representative immunostaining images and relative total fluorescence intensity of iNOS, and Agr-1 with IBA-1 after PBS, MP, RIL4 and MP/RIL4 co-cultured with LPS stimulated BV2 microglia. Scale bar = 100 μm. n = 4. All data are presented as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 5

The “immunomodulator” promoted neurobehavioral function and reduced brain infarct volume of ischemic stroke mice. (A) Experimental diagram. (B) Analysis of body weight of each group. (C) mNSS analysis, (D) EBST, and (E) hanging wire test of ischemic mice. n = 7–15 mice. These mouse patterns were created with BioRender.com. (F, G) Brain atrophy volume was quantified by cresyl violet staining at 14 d after ischemic stroke. n = 3 mice. All data are presented as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 6

Immunoregulation effect of the “immunomodulator” at day 7 post-tMCAO in vivo. (A) Experimental diagram. (B) Diagram of the analyzed peri-infarct regions of 1, 2 and 3. The pattern was created with BioRender.com. (C, D) Representative co-immunostaining images of CD86 and IBA-1 and quantitative analysis of the ratio of CD86⁺/IBA-1⁺ cells in IBA-1⁺ cells. Scale bar = 50 μm. n = 3 mice. (E, F) Representative co-immunostaining images of CD206 and CD11b and quantitative analysis of the ratio of CD206⁺/CD11b⁺ cells in CD11b⁺ cells. Scale bar = 50 μm. n = 3 mice. All data are presented as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 7

Immunoregulation effect of the “immunomodulator” at day 14 post-tMCAO in vivo. (A) Experimental diagram. (B) Diagram of the analyzed peri-infarct regions of 1, 2 and 3. The pattern was created with BioRender.com. (C, E) Representative co-immunostaining images of CD86 and IBA-1 and quantitative analysis of the ratio of CD86⁺/IBA-1⁺ cells in IBA-1⁺ cells. Scale bar = 50 μm. n = 3 mice. (D, F) Representative co-immunostaining images of CD206 and CD11b and quantitative analysis of the ratio of CD206⁺/CD11b⁺ cells in CD11b⁺ cells. Scale bar = 50 μm. n = 3 mice. RT-PCR analysis of (G) pro-inflammatory marker (IL-1β) and (H) anti-inflammatory marker (Arg-1) expression of MP, RIL4 and MP/RIL4 injection after ischemic stroke. n = 3 mice. All data are presented as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 8

The “immunomodulator” regulated immune-neurovascular crosstalk in vitro. (A) Experimental diagram of BV2-HUVECs/NSCs cell interaction model. The schematic diagram was created with BioRender.com. (B–C) Representative images and analysis of tube formation of HUVECs which were co-cultured with BV2 microglia or MP/RIL4-treated BV2 microglia with or without LPS. Scale bar = 50 μm. n = 3. (D–E) Representative immunostaining images and differentiation of NSCs which were co-cultured with BV2 microglia or MP/RIL4-treated BV2 microglia with or without LPS. Scale bar = 50 μm. n = 3. All data are presented as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 9

The “immunomodulator” improved angiogenesis and neurogenesis of ischemic stroke mice. (A) Experimental diagram. (B) Diagram of the analyzed peri-infarct regions of C1, C2, C3, E and F in corresponding brain slices. The pattern was created with BioRender.com. Representative images of (C) CD31⁺ blood vessels and (D) CD31⁺/Ki67⁺ vessels. Scale bar = 50 μm. n = 3 mice. Representative images of DCX⁺ cells in (E) SVZ and (F) striatum. Scale bar = 50 μm. n = 3 mice. Quantitative analysis of (G) blood vessel density and (H) numbers of CD31⁺/Ki67⁺ vessels, (I) DCX area in SVZ and (J) DCX migration. n = 3 mice. All data are presented as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.