Mesenchymal stem cell-derived extracellular vesicles alleviate autism by regulating microglial glucose metabolism reprogramming and neuroinflammation through PD-1/PD-L1 interaction

Abstract

Neuroinflammation triggered by microglia activation is hallmark of autism spectrum disorder (ASD), and this process includes crucial metabolic reprogramming from oxidative phosphorylation to glycolysis, which may cause neuron loss and functional impairment. The inhibitory immune checkpoint programmed cell death protein 1 (PD-1) on immune cells is an important target for tumor immunotherapy. However, the immunomodulatory effects of PD-1 in ASD remains to be elusive. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) exhibit immunomodulatory capabilities in a range of neurological diseases. Our findings indicated the expression of PD-L1 on MSC-EVs, potentially facilitating signaling to PD-1-expressing microglia. Here, we showed how MSC-EVs activated of PD-L1/PD-1 axis and ameliorated glycolysis, neuroinflammation and autism-like behaviors. After first detecting elevated glycolysis and neuroinflammation in prefrontal cortex (PFC) tissue from the maternal immune activation (MIA) mice, we also demonstrated that PD-1 expression level was upregulated in microglia. Following given MSC-EVs carried PD-L1 into adult MIA offspring mice via intranasal administration, which bound with PD-1 on microglia and then the autism-like behaviors were alleviated as well. Further experiments verified that MSC-EVs could decreased the level of glycolysis and neuroinflammation by PD-1/ERK/HIF-1α pathway in the primary microglia in PFC of MIA offspring mice. Pharmacological blockade and genetic inhibition of PD-1 could weaken the effect of MSC-EVs and aggravate microglial dysfunction, glycolysis and autism-like behaviors in MIA offspring mice. Futhermore, PD-L1 deficient weakened the effect of MSC-EVs on neuroinflammation, glycolysis and autism-like behaviors in MIA offspring mice. Our research indicated the significant immunomodulatory capabilities of MSC-EVs, which play an important role in reprogramming microglial glucose metabolism and suppressing neuroinflammation in ASD. By activating the PD-L1/PD-1 axis and inhibiting the downstream ERK/HIF-1α pathway, MSC-EVs were found to alleviate autism-like behaviors, which revealing a novel pathological mechanism and offering promising therapeutic insights into ASD.

Keywords: Autism spectrum disorder; Glycolysis; MSC-EVs; Neuroinflammation; PD-L1/PD-1.

Fig. 1

MIA offspring mice showed typical autism-like phenotypes. (A) Schematic diagram illustrating the experimental procedure of MIA offspring mice. (B-E) The inflammatory factors detection in placenta, spleen, serum and lymph node by qPCR and ELISA between PBS and MIA maternal groups. n=6. Two-way ANOVA. (F) Trajectory route and statistical chart of sociability and social preference in the three-chamber test of PBS and MIA offspring mice. n=8. Two-way ANOVA. (G) Trajectory route and statistical chart of entries times in center zone and time in center zone in the OFT of PBS and MIA offspring mice. n=8. Student’s t-test. (H) Trajectory route and statistical chart of entries in open arms and time in open arms in EPM test of PBS and MIA offspring mice. n=8. Student’s t-test. (I) Statistical chart of number in marbles buried of PBS and MIA offspring mice. n=8. Student’s t-test. (J) Statistical diagram of number of grooming times and duration grooming time of PBS and MIA offspring mice. n=8. Student’s t-test. (K) Statistical diagram of exploration time for each object in the NOR test of MIA offspring mice. n=8. Two-way ANOVA. ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 2

The MIA offspring mice showed the activation of neuroinflammation and microglia. (A-B) IL-6 and TNF-α level detection of PFC and serum by qPCR and ELISA respectively between PBS and MIA offspring mice. n=6. Two-way ANOVA. (C) Immunostaining analysis of Iba-1⁺ of PFC in PBS and MIA offspring mice. Scale bar = 50 μm. Student’s t-test. (D) The results of GSEA enrichment in ASD and controls frontal cortex (GSE28521). (E) The IL-6 and TNF-α level assessment in microglia between PBS and MIA offspring mice. n=3. Two-way ANOVA. (F-I) Analysis of scRNA-seq dataset (PRJNA434002) in ASD patients and controls PFC: (F) tSNE visualization revealed 12 cell clusters; (G) Raincloud plot showed the inflammation score in microglia of ASD patients and controls; (H-I) GSEA enrichment analysis of 4 pathways according to the expressed profile in microglia cluster. ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 3

High expression of PD-L1 in MSC-EVs, and high expression of PD-1 in MIA offspring mice. (A) Schematic diagram illustrating the extraction–separation process of MSC-EVs. (B-D) Characterization of MSC-EVs: (B) Size distribution of MSC-EVs measured using a nanoparticle size meter; (C) TEM of MSCs-EVs isolated from MSCs. Scale bar = 200 nm; (D) Western blot analysis of CD9, CD63, TSG101 and calnexin in MSC-EVs. (E-F) Western blot analysis of the expression of PD-L1 in MSCs and MSC-EVs. n=3. Student’s t-test. (G) Western blot analysis of PD-1 and PD-L1 of PFC in PBS and MIA offspring mice. n=6. Two-way ANOVA. (H) Immunostaining analysis of co-localization of PD-1⁺ and Iba-1⁺ of PFC in MIA offspring mice. Scale bar = 50 μm. Student’s t-test. (I) Western blot analysis of PD-1 in microglia in PBS and MIA offspring mice. n=3. Student’s t-test. (J) Immunostaining analysis of co-localization of PD-1⁺ and Iba-1⁺ in microglia in PFC MIA offspring mice. Scale bar = 20 μm. Student’s t-test. (K) KEGG analysis of the differentially expressed genes of PFC in MIA offspring mice and controls (GSE77972). (L) KEGG analysis of the differentially expressed genes of FC in ASD patients and controls (GSE28521).ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 4

The activation level of the ERK/HIF-1α pathway and glycolysis were elevated in the MIA offspring mice. (A-B) The expression of glycolysis key genes of PFC by qPCR (n=8) and western blot (n=6) between the PBS and MIA offspring mice. Two-way ANOVA. (C) Lactate level in the PFC and serum between the PBS and MIA offspring mice. n=6. Student’s t-test. (D) Schematic diagram illustrating the experimental procedure of 2-DG administration. (E) Western blot analysis of HK2 and LDHA in PFC in MIA offspring mice after 2-DG administration. n=6. Two-way ANOVA. (F) Lactate level of the PFC and serum in MIA offspring mice after 2-DG administration. n=6. Student’s t-test. (G) IL-6 and TNF-α level of PFC and serum in MIA offspring mice after 2-DG administration. n=6. Two-way ANOVA. (H) Immunostaining analysis of Iba-1⁺ of PFC in MIA offspring mice after 2-DG administration. Scale bar = 50 μm. Student’s t-test. (I) Western blot analysis of HK2 and LDHA in microglia between the PBS and MIA offspring mice. n=3. Two-way ANOVA. (J) Lactate level of microglia supernatant between the PBS and MIA offspring mice. n=3. Student’s t-test. (K) Schematic diagram of 2-DG intervention for primary microglia from MIA offspring mice. (L-M) Western blot analysis of HK2 and LDHA in primary microglia with 2-DG intervention. n=3. Two-way ANOVA. (N) Lactate level of primary microglia supernatant with 2-DG intervention. n=3. Student’s t-test. (O) IL-6 and TNF-α level of primary microglia with 2-DG intervention. n=3. Two-way ANOVA. (P-S) Western blot analysis of p-ERK/ERK and HIF-1α of PFC (n=6) and primary microglia (n=3) between PBS and MIA offspring mice. Student’s t-test. ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 5

MSC-EVs alleviated neuroinflammation and autism-like behaviors of MIA offspring mice. (A) Schematic diagram illustrating the experimental procedure of MSC-EVs administration. (B-G) Autism-behaviors analysis between PBS and MIA offspring mice after MSC-EVs administration. (B) Statistical chart of sociability and social preference in the three-chamber test. Two-way ANOVA; (C) Statistical chart of entries times in center zone and time in center zone in the OFT. One-way ANOVA; (D) Statistical chart of entries in open arms and time in open arms in EPM test. One-way ANOVA; (E) Statistical chart of number in marbles buried. One-way ANOVA; (F) Statistical chart of grooming times and duration of grooming. One-way ANOVA; (G) Statistical chart of exploration time for each object in the NOR test. Two-way ANOVA. n=8. (H) Co-localization of Dil-labeled MSC-EVs of PFC in MIA offspring mice. Scale bar = 20 μm. (I) Western blot analysis of PD-1 and PD-L1 of PFC in MIA offspring mice after MSC-EVs administration. n=6. Two-way ANOVA. (J) IL-6 and TNF-α level of PFC and serum between PBS and MIA offspring mice after MSC-EVs administration. n=6. Two-way ANOVA. (K) Immunostaining analysis of Iba-1⁺ of PFC between PBS and MIA offspring mice after MSC-EVs administration. Scale bar = 50 μm. One-way ANOVA. (L) Co-localization of primary microglia with Dil-labeled MSC-EVs. Scale bar = 20 μm. (M) Western blot analysis of PD-1 and PD-L1 of primary microglia in MIA offspring mice with MSC-EVs intervention. n=3. Two-way ANOVA. (N) IL-6 and TNF-α level of primary microglia in MIA offspring mice with MSC-EVs intervention. n=3. Two-way ANOVA. ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 6

MSC-EV repressed the glycolysis process in MIA offspring mice. (A-C) Western blot analysis of p-ERK/ERK, HIF-1α, HK2 and LDHA of PFC between PBS and MIA offspring mice after MSC-EVs administration. n=6. One-way ANOVA, Two-way ANOVA. (D) Lactate level of PFC and serum between PBS and MIA offspring mice after MSC-EVs administration. n=6. One-way ANOVA. (E-G) Western blot analysis of p-ERK/ERK, HIF-1α, HK2 and LDHA in primary microglia with MSC-EVs intervention. n=3.One-way ANOVA, Two-way ANOVA. (H) Lactate level in primary microglia supernatant with MSC-EVs intervention. n=3. One-way ANOVA. ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 7

Pharmacological blockade of PD-1 weakened the effect of MSC-EVs on improving autism-like behaviors in MIA offspring mice. (A) Schematic diagram illustrating the experiment procedure of MIA offspring mice after PD-1 Ab alone or PD-1 Ab + MSC-EVs administration. (B-G) Autism-behaviors analysis of MIA offspring mice after PD-1 Ab alone or PD-1 Ab + MSC-EVs administration. (B) Statistical chart of sociability and social preference in the three-chamber test. Two-way ANOVA; (C) Statistical chart of entries times in center zone and time in center zone in the OFT. One-way ANOVA; (D) Statistical chart of entries in open arms and time in open arms in EPM test. One-way ANOVA; (E) Statistical chart of number in marbles buried. One-way ANOVA; (F) Statistical chart of grooming times and duration grooming. One-way ANOVA; (G) Statistical diagram of exploration time for each object in the NOR test. Two-way ANOVA. n=8. ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 8

Pharmacological blockade of PD-1 weakened the effect of MSC-EVs on neuroinflammation and glycolysis in MIA offspring mice. (A) IL-6 and TNF-α level of PFC and serum in MIA offspring mice after PD-1 Ab alone or PD-1 Ab + MSC-EVs administration. n=6. Two-way ANOVA. (B) Immunostaining analysis of Iba-1⁺ of PFC in MIA offspring mice after PD-1 Ab alone or PD-1 Ab + MSC-EVs administration. Scale bar = 50 μm. One-way ANOVA. (C-F) Western blot analysis of PD-1, PD-L1, p-ERK/ERK, HIF-1α, HK2 and LDHA of PFC in MIA offspring mice after PD-1 Ab alone or PD-1 Ab + MSC-EVs administration. n=6. One-way ANOVA, Two-way ANOVA. (G) Lactate level of PFC and serum in MIA offspring mice after PD-1 Ab alone or PD-1 Ab + MSC-EVs administration. n=6. One-way ANOVA. (H) Schematic diagram illustrating the experiment procedure of with PD-1 Ab alone or PD-1 Ab + MSC-EVs intervention in primary microglia. (I) IL-6 and TNF-α level of primary microglia with PD-1 Ab alone or PD-1 Ab + MSC-EVs intervention. n=3. Two-way ANOVA. (J-M) Western blot analysis of PD-1 and PD-L1. p-ERK/ERK, HIF-1α, HK2 and LDHA in primary microglia after PD-1 Ab alone or PD-1 Ab + MSC-EVs intervention. n=3. One-way ANOVA, Two-way ANOVA. (N) Lactate level in primary microglia supernatant after PD-1 Ab alone or PD-1 Ab + MSC-EVs intervention. n=3. One-way ANOVA. ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 9

Microglia-specific PD-1 knockdown repressed the effect of MSC-EVs on inhibiting neuroinflammation, glycolysis and autism-like behaviors in MIA offspring mice. (A) Schematic diagram illustrating the experimental procedure of MIA offspring mice after Ctrl + MSC-EVs or AAV-Iba-1-shPdcd1 + MSC-EVs administration. (B-G) Autism-behaviors analysis of MIA offspring mice after Ctrl + MSC-EVs or AAV-Iba-1-shPdcd1 + MSC-EVs administration. (B) Statistical chart of sociability and social preference in the three-chamber test. Two-way ANOVA; (C) Statistical chart of entries times in center zone and time in center zone in the OFT. One-way ANOVA; (D) Statistical chart of entries in open arms and time in open arms in EPM test. One-way ANOVA; (E) Statistical chart of number in marbles buried. One-way ANOVA; (F) Statistical chart of grooming times and duration grooming. One-way ANOVA; (G) Statistical diagram of exploration time for each object in the NOR test. Two-way ANOVA. (H) Immunostaining analysis of GFP⁺ cells stained with Iba-1⁺. Scale bar: 20 μm. (I) Immunostaining analysis of Iba-1⁺ of PFC in MIA offspring mice after Ctrl + MSC-EVs or AAV-Iba-1-shPdcd1 + MSC-EVs administration. Scale bar = 50 μm. One-way ANOVA. (J) IL-6 and TNF-α level of PFC and serum in MIA offspring mice after Ctrl + MSC-EVs or AAV-Iba-1-shPdcd1 + MSC-EVs administration. n=6. Two-way ANOVA. (K-N) Western blot analysis of PD-1, PD-L1, p-ERK/ERK, HIF-1α, HK2 and LDHA of PFC in MIA offspring mice after Ctrl + MSC-EVs or AAV-Iba-1-shPdcd1 + MSC-EVs administration. n=6. One-way ANOVA, Two-way ANOVA. (O) Lactate level of PFC and serum in MIA offspring mice after Ctrl + MSC-EVs or AAV-Iba-1-shPdcd1 + MSC-EVs administration. n=6. One-way ANOVA. ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 10

Genetic suppression of PD-1 weakened the effect of MSC-EVs on neuroinflammation and glycolysis in MIA offspring mice. (A-D) Western blot analysis of PD-1, p-ERK/ERK, HIF-1α, HK2 and LDHA in primary microglia with MSC-EVs + si-Pdcd1 intervention. n=3. One-way ANOVA, Two-way ANOVA. (E) Lactate level in primary microglia supernatant with MSC-EVs + si-Pdcd1 intervention. n=3. One-way ANOVA. (F-G) IL-6 and TNF-α level of primary microglia with MSC-EVs + si-Pdcd1 intervention. n=3. Two-way ANOVA.ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 11

MSC-EVs with PD-L1 knockdown weakened the effect on autism-like behaviors in MIA offspring mice. (A) Schematic diagram illustrating the experiment procedure of MIA offspring mice after MSC-EVs-Ctrl or MSC-EVs-shCd274 administration. (B-G) Autism-behaviors analysis of MIA offspring mice after MSC-EVs-Ctrl or MSC-EVs-shCd274 administration. (B) Statistical chart of sociability and social preference in the three-chamber test. Two-way ANOVA; (C) Statistical chart of entries times in center zone and time in center zone in the OFT. One-way ANOVA; (D) Statistical chart of entries in open arms and time in open arms in EPM test. One-way ANOVA; (E) Statistical chart of number in marbles buried. One-way ANOVA; (F) Statistical chart of grooming times and duration grooming. One-way ANOVA; (G) Statistical diagram of exploration time for each object in the NOR test. Two-way ANOVA. n=8. ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 12

MSC-EVs with PD-L1 knockdown weakened the effect on neuroinflammation and glycolysis in MIA offspring mice. (A) IL-6 and TNF-α level of PFC and serum in MIA offspring mice after MSC-EVs-Ctrl or MSC-EVs-shCd274 administration. n=6. Two-way ANOVA. (B) Immunostaining analysis of Iba-1⁺ of PFC in MIA offspring mice after MSC-EVs-Ctrl or MSC-EVs-shCd274 administration. n=6. Scale bar = 50 μm. One-way ANOVA. (C-F) Western blot analysis of PD-L1/PD-1, p-ERK/ERK, HIF-1α, HK2 and LDHA of PFC in MIA offspring mice after MSC-EVs-Ctrl or MSC-EVs-shCd274 administration. n=6. One-way ANOVA, Two-way ANOVA. (G) Lactate level of PFC and serum in MIA offspring mice after MSC-EVs-Ctrl or MSC-EVs-shCd274 administration. n=6. One-way ANOVA. (H) Schematic diagram illustrating the experiment procedure of primary microglia with MSC-EVs-Ctrl or MSC-EVs-shCd274 intervention. (I) IL-6 and TNF-α level of primary microglia with MSC-EVs-Ctrl or MSC-EVs-shCd274 intervention. n=3. Two-way ANOVA. (J-M) Western blot analysis of PD-L1/PD-1, p-ERK/ERK, HIF-1α, HK2 and LDHA in primary microglia with MSC-EVs-Ctrl or MSC-EVs-shCd274 intervention. n=3. One-way ANOVA, Two-way ANOVA. (N) Lactate level of primary microglia supernatant with MSC-EVs-Ctrl or MSC-EVs-shCd274 intervention. n=3. One-way ANOVA. ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001

Fig. 13

MSC-EVs inhibited microglial activation and glycolysis via the PD-1/ERK/HIF-1α pathway. (A) Schematic diagram illustrating the experiment procedure of combining MSC-EVs with ERK activator honokiol intervention in primary microglia. (B-D) Western blot analysis of p-ERK, ERK, HIF-1α, HK2 and LDHA in primary microglia with combining MSC-EVs and ERK activator honokiol intervention. n=3. One-way ANOVA, Two-way ANOVA. (E) Lactate level in primary microglia supernatant with combining MSC-EVs with ERK activator honokiol intervention. n=3. One-way ANOVA. (F) IL-6 and TNF-α level of primary microglia with combining MSC-EVs with ERK activator honokiol intervention. n=3. Two-way ANOVA. (G) Schematic diagram illustrating the experiment procedure of MSC-EVs combining PD-1 Ab and ERK inhibitor SL327 intervention in primary microglia. (H-J) Western blot analysis of p-ERK, ERK, HIF-1α, HK2 and LDHA in primary microglia of MSC-EVs combining PD-1 Ab and ERK inhibitor SL327 intervention. n=3. One-way ANOVA, Two-way ANOVA. (K) Lactate level of primary microglia supernatant of MSC-EVs combining PD-1 Ab and ERK inhibitor SL327 intervention. n=3. One-way ANOVA. (L) IL-6 and TNF-α level of primary microglia of MSC-EVs combining PD-1 Ab and ERK inhibitor SL327 intervention. n=3. Two-way ANOVA. ns P≥0.05, *P<0.05, **P<0.01, ***P<0.001