The spinal TREM2 increases along with the development of PDN
To induce the experimental type 1 diabetes (T1DM), we injected the mice i.p. with a single dose of STZ (150 mg/kg, Sigma). Then, we determined the behavioral changes in mice receiving STZ (T1DM) or saline injection (Saline). The results showed that compared with the Control group, the PMWTs of the STZ-injected mice began to decrease 3 days after STZ injection (D3) and reached the bottom on D14 (Fig. 1B, F1, 10 = 144.4, P < 0.0001, n = 6, two-way ANOVA; mean ± SD: T1DM vs. Saline on D14, 2.70 ± 0.33 g vs. 5.12 ± 0.33 g, n = 6, P < 0.0001, Bonferroni post hoc analysis), indicating the presence of mechanical allodynia in the STZ-treated mice. We also tested the thermal nociception behaviors after STZ injection using a thermal paw stimulation system. The results suggested that, compared with the Control, the PTWL of the STZ-injected mice dropped after STZ injection, with the statistically significant difference on D7 and D14 (Fig. 1C, F1, 10 = 43.93, P < 0.0001, n = 6, two-way ANOVA; mean ± SD: T1DM vs. Saline on D14, 1.95 ± 0.22 s vs. 3.63 ± 0.16 s, n = 6, P < 0.0001, Bonferroni post hoc analysis), suggesting evident thermal hyperalgesia in the STZ-injected mice.
Next, we determined the pattern of TREM2 expression in the lumbar cord of the diabetic mice (T1DM) on baseline (BL), D7, and D14. The Western blot results showed that the protein expression of TREM2 and its downstream DAP-12 in the lumbar spinal cord markedly increased on D7 and D14 (Fig. 1D, P = 0.0492 on D7 and P = 0.0007 on D14, n = 5). These results indicated that the spinal TREM2 was activated along with the development of PDN.
The immunofluorescence double-staining results showed that on D14, TREM2 was mostly expressed in microglia in the spinal dorsal horn of STZ-treated mice (Fig. 1E). The statistical analysis indicated that the number of Iba-1 + microglia and TREM2 + Iba-1 + cells was minimal in the lumbar cord of the Control mice but substantially increased in the STZ-treated diabetic mice (Fig. 1F; mean ± SD: T1DM vs. Saline on D14, Iba-1 + microglia, 49.64 ± 5.24/mm2 vs. 10.26 ± 2.03/mm2, P = 0.0926, n = 5; Trem2 + Iba-1 + microglia, 19.62 ± 4.96 /mm2 vs. 1.85 ± 0.27 /mm2, P < 0.0001, n = 5).
Both M1 and M2 microglia increase in parallel to the development of PDN
The switch between M1 and M2 microglial phenotypes regulates the inflammatory response in the CNS[41]. Numerous studies have demonstrated that microglial activation, but not astrocytes, contributes to PDN[12, 13]. Thus, we examined the protein expression of the M1 (CD86) and M2 (CD206 and Arg-1) microglia phenotype markers in the lumbar spinal cord. The results revealed that the protein levels of both M1 (CD86) and M2 (CD206 and Arg-1) microglia phenotype markers were markedly elevated on D7 and D14 after STZ injection (Fig. 2A-D, P < 0.05, P < 0.01, or P < 0.0001, n = 5). This observation was further verified by the immunofluorescence test, which indicated that the number of CD86 + Iba-1 + and CD206 + Iba-1 + cells was significantly augmented in the lumbar spinal dorsal horn of the STZ-treated mice on D14 (Fig. 2E-G; mean ± SD: T1DM vs. Saline on D14, CD86 + Iba-1 + microglia, 17.72 ± 2.32/mm2 vs. 2.73 ± 1.90/mm2, P < 0.0001, n = 5; CD206 + Iba-1 + microglia, 7.56 ± 2.19/mm2 vs. 1.20 ± 1.13/mm2, P = 0.0004, n = 5). We also determined the expression of IL-1β, TGF-β, and IL-10 in the spinal cord, as IL-1β is derived explicitly from M1 phenotype microglia, while the primary source of TGF-β and IL-10 is M2 phenotype. We found that the spinal protein levels of IL-1β, TGF-β, and IL-10 were increased on D7 and D14 after STZ injection (Fig. 2H-K, P < 0.05, P < 0.01, or P < 0.0001, n = 5).
Overexpression of TREM2 in microglia aggravates PDN in mice
Previous studies have demonstrated that TREM2 inhibits neuroinflammatory response in neurodegenerative diseases such as Alzheimer's disease[23, 24], and this effect may be related to its promotion of M2 phenotype microglia. Given the anti-inflammatory properties of M2 phenotype microglia and the above results, we speculated that overexpression of the TREM2 gene in microglia might alleviate PDN by suppressing spinal inflammation. In this regard, we injected intrathecally (i.t.) the mice with microglia-specific TREM2 overexpression LV (LV-TREM2, 5×107 TU) or the control LV (LV-control, 5×107 TU, 7 days before STZ injection) 7 days before STZ injection.
We found that the injection of LV-control did not alter the PMWTs and PTWLs compared with T1DM mice that only received an i.t. injection of saline (Fig. 3A and B; T1DM vs. T1DM + LV-control: PMWT, F1, 14 = 2.543, P > 0.05, n = 8, two-way ANOVA; PTWL, F1, 14 = 2.372, P > 0.05, n = 8, two-way ANOVA). In contrast to our expectation, the injection of LV-TREM2 decreased the PMWTs and extended the PTWLs of the T1DM + LV-TREM2 mice (Fig. 3A and B; T1DM vs. T1DM + LV-TREM2: PMWT, F1, 14 =35.21, P < 0.0001, n = 8, two-way ANOVA; PTWL, F1, 14 = 14.27, P = 0.0020, n = 8, two-way ANOVA), while the protein expression of TREM2 in the lumbar spinal cord was elevated (Fig. 3D, P < 0.0001, n = 4).
This unexpected result was confirmed by the immunofluorescence test, which showed that the injection of LV-TREM2 amplified the number of Iba-1 microglia and CD86 + Iba-1 + cells (M1 phenotype), but decreased the CD206 + Iba-1 + cells (M2 phenotype) in the spinal dorsal horn (Fig. 3E-H; mean ± SD, T1DM vs. T1DM + LV-TREM2 on D14: Iba-1 + microglia, 36.83 ± 7.25/mm2 vs. 65.17 ± 10.29/mm2, P = 0.0002, n = 4; CD86 + Iba-1 + microglia, 12.98 ± 1.25/mm2 vs. 17.20 ± 2.89/mm2, P = 0.0261, n = 4; CD206 + Iba-1 + microglia, 9.44 ± 3.01/mm2 vs. 2.79 ± 1.14/mm2, P = 0.0032, n = 4). Furthermore, the protein expression of pro-inflammatory cytokine IL-1β was increased by the injection of LV-TREM2, whereas anti-inflammatory cytokines TGF-β and IL-10 were reduced (Fig. 3I, P < 0.05 or P < 0.001 compared with T1DM, n = 5). Together, these results indicated that the overexpression of TREM2 in spinal microglia was detrimental by intensifying microglial M1 response and suppressing microglial M2 polarization.
Intrathecal treatment with anti-TREM2 neutralizing antibody ameliorates PDN in mice
Based on the above data, it was reasonable to argue that inhibition of TREM2 signalling might relieve PDN. Next, we investigate whether inhibition of the spinal TREM2 with a TREM2 neutralizing antibody can attenuate the pain-like behavior in T1DM mice. The behavioral test showed that the i.t. treatment with TREM2 neutralizing antibody (1 µg/d, from D3 to D5 after STZ injection, i.t.) elevated the PMWTs and TWLs in the T1DM + anti-TREM2 mice as compared with the T1DM mice receiving i.t. injection of saline (Fig. 4B and C; T1DM vs. T1DM + anti-TREM2: PMWT, F1, 16 =23.17, P = 0.0002, n = 9, two-way ANOVA; PTWL, F1, 16 = 19.65, P = 0.0004, n = 9, two-way ANOVA), while the IgG-treated T1DM mice did not present with any behavioral change (Fig. 4B and C; T1DM vs. T1DM + IgG: PMWT, F1, 16 =0.8287, P = 0.3762, n = 9, two-way ANOVA; PTWL, F1, 16 = 0.1968, P = 0.6632, n = 9, two-way ANOVA).
The immunofluorescence results showed that the injection of TREM2 neutralizing antibody reduced the spinal number of Iba-1 microglia and CD86 + Iba-1 + cells (M1 phenotype) and increased the CD206 + Iba-1 + cells (M2 phenotype) in the spinal dorsal horn of T1DM + anti-TREM2 mice (Fig. 4D-G; mean ± SD, T1DM vs. T1DM + anti-TREM2 on D14: CD86 + Iba-1 + microglia, 14.51 ± 2.01/mm2 vs. 8.21 ± 2.87/mm2, P = 0.0024, n = 4; CD206 + Iba-1 + microglia, 7.34 ± 0.80/mm2 vs. 10.08 ± 1.48/mm2, P = 0.0052, n = 4). Furthermore, the protein level of pro-inflammatory cytokine IL-1β was decreased by the injection of TREM2 neutralizing antibody, whereas anti-inflammatory cytokines TGF-β and IL-10 were increased (Fig. 4H-K, P < 0.01 or P < 0.001 compared with T1DM, n = 5).
GLT-3 is the potential ligand of the microglial TREM2 receptor in facilitating the progression of PDN
Furthermore, we asked what ligand triggered the TREM2 signalling in the development of PDN. Recent studies suggested that ApoE[42, 43] and GLT-3[44] were the potential ligands of TREM2 in mediating a series of downstream actions. Therefore, we detected the protein expression of ApoE and GLT-3 in the lumbar spinal cord of the T1DM mice. We found that the spinal protein expression of GLT-3, but not ApoE, was significantly increased in the STZ-treated diabetic mice on D7 and D14 after STZ injection (Fig. 5A-C; ApoE, P = 0.9248 on D7, P = 0.6238 on D14, n = 5; GLT-3, P = 0.0288 on D7, P < 0.0001 on D14, n = 5).
To corroborate these proteins' attachment with the TREM2 receptor, we performed immunoprecipitation by antibodies against TREM2 and tested its efficiency using western blot analysis. We found that the immunoprecipitated GLT-3 with TREM2 antibodies from the lumbar spinal cord of diabetic mice on D14 after STZ injection was more than that of saline-treated mice (Fig. 5D), while the difference in the blotted ApoE was not found (Fig. 5D).
We also identified the colocalization of ApoE or GLT-3 with the immunofluorescence double-staining procedure. We observed that in the spinal dorsal horn, ApoE expression was primarily colocalized with neurons (NeuN+) and astrocyte (GFAP+) (Fig. 5E), while GLT-3 expression was largely overlapped with Iba-1 + microglia (Fig. 5F).
Next, we examine whether neutralizing GLT-3 in the spinal cord can affect the behavioral manifestation of PDN and the microglial M1/M2 balance. The behavioral data showed that the injection of GLT-3 neutralizing antibody (1 µg/d, from D3 to D5 after STZ injection, i.t.) increased the PMWTs and PTWLs in the T1DM + anti-GLT-3 mice (Fig. 6B and C; T1DM vs. T1DM + anti-GLT-3: PMWT, F1, 16 =16.09, P = 0.001, n = 9, two-way ANOVA; PTWL, F1, 16 = 40.63, P < 0.0001, n = 9, two-way ANOVA). The immunofluorescence assays suggested that the i.t. injection of GLT-3 neutralizing antibody reduced the spinal number of Iba-1 microglia and CD86 + Iba-1 + cells (M1 phenotype) and increased the CD206 + Iba-1 + cells (M2 phenotype) in the spinal dorsal horn of T1DM + anti-GLT-3 mice (Fig. 6D-G; mean ± SD, T1DM vs. T1DM + anti-TREM2 on D14: CD86 + Iba-1 + microglia, 15.34 ± 1.40/mm2 vs. 6.60 ± 2.42/mm2, P < 0.0001, n = 4; CD206 + Iba-1 + microglia, 7.34 ± 1.47/mm2 vs. 11.66 ± 3.46/mm2, P = 0.0301, n = 4). Furthermore, the protein level of pro-inflammatory cytokine IL-1β was decreased by the injection of GLT-3 neutralizing antibody, whereas anti-inflammatory cytokines TGF-β and IL-10 were increased (Fig. 6H-K, P < 0.05, P < 0.01, or P < 0.001, n = 5).