CX3CR1+ monocytes modulate learning and learning-dependent dendritic spine remodeling via TNF-α

Abstract

Impaired learning and cognitive function often occurs during systemic infection or inflammation. Although activation of the innate immune system has been linked to the behavioral and cognitive effects that are associated with infection, the underlying mechanisms remain poorly understood. Here we mimicked viral immune activation with poly(I:C), a synthetic analog of double-stranded RNA, and longitudinally imaged postsynaptic dendritic spines of layer V pyramidal neurons in the mouse primary motor cortex using two-photon microscopy. We found that peripheral immune activation caused dendritic spine loss, impairments in learning-dependent dendritic spine formation and deficits in multiple learning tasks in mice. These observed synaptic alterations in the cortex were mediated by peripheral-monocyte-derived cells and did not require microglial function in the central nervous system. Furthermore, activation of CX3CR1^highLy6C^low monocytes impaired motor learning and learning-related dendritic spine plasticity through tumor necrosis factor (TNF)-α-dependent mechanisms. Taken together, our results highlight CX3CR1^high monocytes and TNF-α as potential therapeutic targets for preventing infection-induced cognitive dysfunction.

Figure 1. Systemic immune challenge increases dendritic spine turnover in the cortex

(a) Left, cartoon depicting the experimental approach for longitudinal imaging of synaptic structural plasticity in Thy1-YFP transgenic mice, which express yellow fluorescent protein (YFP) in L5 pyramidal neurons. Right, representative images of transcranial two-photon imaging of dendritic spines on apical dendritic segments of L5 pyramidal neurons in the primary motor cortex of vehicle- or poly(I:C)-treated mice. Empty and filled arrowheads indicate individual spines that were eliminated or newly formed, respectively, on the same dendritic segment after 2 d. Asterisks indicate dendritic filopodia. Scale bar, 2 μm. (b) Summary quantification of dendritic spine elimination and formation from P30 to P32 in mice that were injected with a vehicle solution (n = 5 mice), with 0.5 mg/kg (n = 4 mice), 5 mg/kg (n = 6 mice) or 50 mg/kg (n = 4 mice) poly(I:C) i.p. at P30 or with 5 mg/kg poly(I:C) at P26 (n = 5 mice). (c) Spine elimination and formation 24 h after vehicle (n = 4 mice) or poly(I:C) (n = 6 mice) injection. (d) Net change in spine number after vehicle or poly(I:C) injection at P30 for the mice in c. (e) Quantification of dendritic spine elimination and formation after 2 d in mice that were injected with vehicle (n = 6 mice) or 5 mg/kg poly(I:C) (n = 5 mice) at P60. (f) Net change in spine number after 2 d in the vehicle- or poly(I:C)-treated mice in e. Throughout, individual circles represent data from a single mouse. Summary data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; by the Kruskal–Wallis (b) or Mann–Whitney (c–f) test. See also Supplementary Table 1.

Figure 2. Systemic immune challenge impairs learning-dependent spine remodeling and performance improvement

(a) Schematic showing the timeline for in vivo imaging, vehicle or poly(I:C) (5 mg/kg i.p.) administration, and rotarod training and testing. (b,c) Effect of poly(I:C) treatment on dendritic spine elimination and formation after a 2-d rotarod training period in P30 (n = 4 mice per group) (b) or P60 (n = 7 mice per group) (c) mice. (d,e) Net change in spine number after a 2-d rotarod training period in the P30 (d) and P60 (e) mice in b,c, respectively. (f,g) Effect of poly(I:C) treatment on baseline rotarod performance in P30 (n = 9 mice per group) (f) and P60 (vehicle-treated, n = 10 mice; poly(I:C)-treated, n = 7 mice) (g) mice. Rotarod performance is expressed as the average speed reached during the training day. (h,i) Rotarod performance improvement assessed after 2 d for the mice that were treated at P30 (h) or P60 (i) in f,g, respectively. (j) Number of persistent new spines in mice that were injected with vehicle (n = 4) or poly(I:C) (n = 4). Persistent new spines are expressed as the number of new spines formed during the 2-d training period (P60 to P62) that persist on P67, as a percentage of the total number of spines quantified at P60. (k) Rotarod performance improvement, as assessed at P67, for the mice in g. (l) Correlation between the number of persistent new spines and the mouse’s rotarod performance on P67 (r = 0.7857; P = 0.0279; by Spearman correlation). Throughout, individual circles represent data from a single mouse. Summary data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; by Mann–Whitney test. See also Supplementary Table 1.

Figure 3. CX₃CR1⁺ cells are required for altered dendritic spine plasticity after systemic immune challenge

(a) Schematic representation of the strategy to deplete CX₃CR1⁺ cells, which include peripheral monocytes and CNS-resident microglia. (b) Representative flow cytometry analysis showing the percentages of CX₃CR1–EYFP⁺CD11b⁺ cells in the cortex (left), liver (middle) and blood (right) of Cx3cr1^CreER/+ (top) or Cx3cr1^CreER/+;R26^iDTR/+ (bottom) mice after administration of the last dose of DT. Gate: singlets, DAPI⁻LIN⁻CD45^low (cortex) or CD45^high (periphery). (c) Quantification of the data shown in b (n = 6 mice for each group). (d) Representative coronal sections (n = 8 sections per group) of the motor cortex from control (top) and CX₃CR1⁺-cell-depleted (bottom) mice that were stained with the microglia marker Iba1 1 d after administration of the last dose of DT. Scale bar, 50 μm. (e) Quantification of dendritic spine elimination and formation over 2 d in Cx3cr1^CreER/+ control mice that were injected with vehicle or poly(I:C) (n = 4 mice per group). (f) Dendritic spine elimination and formation after 2 d in Cx3cr1^CreER/+;R26^iDTR/+ mice (which are depleted of CX₃CR1⁺ cells) that were injected with vehicle (n = 6 mice) or poly(I:C) (n = 4 mice). (g) Dendritic spine elimination and formation after 2 d in Rag1^−/− mice that were injected with vehicle or poly(I:C) (n = 4 mice per group). Throughout, individual circles represent data from a single mouse. Summary data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant; by Mann–Whitney test. See also Supplementary Table 1.

Figure 4. CX₃CR1⁺ monocytes, but not microglia, mediate synaptic and learning deficits after systemic immune challenge

(a) Schematic of the strategy to selectively deplete CNS-resident microglia. (b) Quantification of CX₃CR1⁺CD11b⁺ cells in the cortex, liver and blood of Cx3cr1^CreER/+ (control) or Cx3cr1^CreER/+;R26^iDTR/+ mice 1 d after administration of the last dose of DT (n = 4 mice per group). (c) Effect of poly(I:C) treatment on dendritic spine elimination and formation after 2 d in Cx3cr1^CreER/+;R26^iDTR/+ mice that were depleted of microglia (n = 4 mice per group). (d) Schematic of the strategy to selectively deplete CX₃CR1⁺ monocytes using Cx3cr1^CreER/+;R26^iDTR/+→WT chimeras. Cx3cr1^CreER/+→WT chimeras were used as controls. Irradiation was performed with or without head protection (HP). (e) Percentages of CD11b⁺CD45^low cells (microglia) in the cortex and CX₃CR1⁺CD11b⁺ cells in blood and liver after administration of the last DT injection (n = 4 mice per group). (f,g) Effect of poly(I:C) treatment on baseline spine elimination and formation after 2 d in Cx3cr1^CreER/+→WT (f) or Cx3cr1^CreER/+;R26^iDTR/+→WT (g) chimeras (n = 4 mice per group). (h,i) Effect of poly(I:C) treatment on dendritic spine elimination and formation after a 2-d motor-training period in Cx3cr1^CreER/+→WT (n = 6 mice per group) (h) or in Cx3cr1^CreER/+;R26^iDTR/+→WT chimeras (vehicle-treated, n = 8; poly(I:C)-treated, n = 9 mice) (i). (j,k) Net change in spine number after a 2-d motor-training period for the mice in h (j) and i (k). (l–o) Effect of poly(I:C) treatment on rotarod performance in Cx3cr1^CreER/+→WT (vehicle-treated, n = 5 mice; poly(I:C)-treated, n = 6 mice) (l,m) or in Cx3cr1^CreER/+;R26^iDTR/+→WT chimeras (vehicle-treated, n = 9 mice; poly(I:C)-treated, n = 11 mice) (n,o) that were injected with vehicle or poly(I:C) on P60. Throughout, individual circles represent data from a single mouse. Summary data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01; n.s., not significant; by Mann–Whitney test. See also Supplementary Table 1.

Figure 5. Cx3cr1^−/− chimeric mice do not show synaptic and learning deficits during systemic immune challenge

(a) Schematic showing the timeline of BM chimera generation, vehicle or poly(I:C) (5 mg/kg i.p.) administration, rotarod training and in vivo two-photon imaging. (b,c) Effect of poly(I:C) treatment on baseline spine elimination and formation after 2 d in WT→WT (b) or Cx3cr1^−/−→WT (c) chimeras (n = 4 mice per group). (d,e) Effect of poly(I:C) treatment on dendritic spine elimination and formation after a 2-d motor-training period in WT→WT (n = 6 mice per group) (d) or Cx3cr1^−/−→WT chimeras (n = 4 mice per group) (e). (f,g) Net change in total spine numbers after a 2-d motor-training period in WT→WT (f) or Cx3cr1^−/−→WT (g) chimeras. (h–k) Effect of poly(I:C) administration on rotarod performance in WT→WT (n = 6 mice per group) (h,i) or Cx3cr1^−/−→WT (vehicle-treated, n = 9 mice; poly(I:C)-treated, n = 7 mice) (j,k) chimeras. Throughout, individual circles represent data from a single mouse. Summary data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01; n.s., not significant; by Mann–Whitney test. See also Supplementary Table 1.

Figure 6. TNF-α mediates synaptic and learning deficits after systemic immune challenge

(a,b) Levels of TNF-α in the plasma (F_3,14 = 23.37, P < 0.0001) (a) or cortex (F_3,26 = 5.717, P = 0.0038) (b) of WT mice over time after injection with 5 mg/kg poly(I:C). (c) Summary quantification of dendritic spine remodeling after 2 d (imaged at P60 and P62) in Tnf^−/− mice that were injected with vehicle (n = 3 mice), 5 mg/kg poly(I:C) (n = 5 mice) or 30 μg/kg exogenous TNF-α (n = 4 mice). (d) Summary quantification of dendritic spine elimination and formation after 2 d (imaged at P30 and P32) in WT mice that were injected with vehicle (n = 5 mice), 30 mg/kg DN-TNF (n = 4 mice), 5 mg/kg poly(I:C) (n = 6 mice), or poly(I:C) + DN-TNF (n = 3 mice). (e) Schematic of the timeline of bone marrow (BM) chimera generation, vehicle or poly(I:C) (5 mg/kg i.p.) injection, rotarod training and in vivo two-photon imaging. (f) Baseline spine elimination and formation after 2 d in Tnf^−/−→WT chimeras that were injected with vehicle (n = 4 mice) or poly(I:C) (n = 5 mice). (g, h) Effect of poly(I:C) treatment on dendritic spine remodeling (g) or total spine number (h) after a 2-d motor-training period in Tnf^−/−→WT chimeras (n = 8 mice per group). (i,j) Rotarod performance in Tnf^−/−→WT chimeras that were injected with vehicle (n = 14 mice) or poly(I:C) (n = 12 mice) at the time of injection (i) or after a 2-d motor-training period (j). Throughout, individual circles represent data from a single mouse. Summary data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant; by one-way analysis of variance (ANOVA) and post hoc Bonferroni (a,b), Kruskal–Wallis (c,d) or Mann–Whitney (f–j) test. See also Supplementary Table 1.