Neurons upregulate PD-L1 via IFN/STAT1/IRF1 to alleviate damage by CD8+ T cells in cerebral malaria

Abstract

Background: Cerebral malaria (CM) is the most lethal complication of malaria, and survivors usually endure neurological sequelae. Notably, the cytotoxic effect of infiltrating Plasmodium-activated CD8⁺ T cells on cerebral microvasculature endothelial cells is a prominent feature of the experimental CM (ECM) model with blood-brain barrier disruption. However, the damage effect of CD8⁺ T cells infiltrating the brain parenchyma on neurons remains unclear. Based on the immunosuppressive effect of the PD-1/PD-L1 pathway on T cells, our previous study demonstrated that the systemic upregulation of PD-L1 to inhibit CD8⁺ T cell function could effectively alleviate the symptoms of ECM mice. However, it has not been reported whether neurons can suppress the pathogenic effect of CD8⁺ T cells through the PD-1/PD-L1 negative immunomodulatory pathway. As the important inflammatory factor of CM, interferons can induce the expression of PD-L1 via different molecular mechanisms according to the neuro-immune microenvironment. Therefore, this study aimed to investigate the direct interaction between CD8⁺ T cells and neurons, as well as the mechanism of neurons to alleviate the pathogenic effect of CD8⁺ T cells through up-regulating PD-L1 induced by IFNs.

Methods: Using the ECM model of C57BL/6J mice infected with Plasmodium berghei ANKA (PbA), morphological observations were conducted in vivo by electron microscope and IF staining. The interaction between the ECM CD8⁺ T cells (immune magnetic bead sorting from spleen of ECM mice) and primary cultured cortical neurons in vitro was observed by IF staining and time-lapse photography. RNA-seq was performed to analyze the signaling pathway of PD-L1 upregulation in neurons induced by IFNβ or IFNγ, and verified through q-PCR, WB, IF staining, and flow cytometry both in vitro and in vivo using IFNAR or IFNGR gene knockout mice. The protective effect of adenovirus-mediated PD-L1 IgGFc fusion protein expression was verified in ECM mice with brain stereotaxic injection in vivo and in primary cultured neurons via viral infection in vitro.

Results: In vivo, ECM mice showed infiltration of activated CD8⁺ T cells and neuronal injury in the brain parenchyma. In vitro, ECM CD8⁺ T cells were in direct contact with neurons and induced axonal damage, as an active behavior. The PD-L1 protein level was elevated in neurons of ECM mice and in primary cultured neurons induced by IFNβ, IFNγ, or ECM CD8⁺ T cells in vitro. Furthermore, the IFNβ or IFNγ induced neuronal expression of PD-L1 was mediated by increasing STAT1/IRF1 pathway via IFN receptors. The increase of PD-L1 expression in neurons during PbA infection was weakened after deleting the IFNAR or IFNGR. Increased PD-L1 expression by adenovirus partially protected neurons from CD8⁺ T cell-mediated damage both in vitro and in vivo.

Conclusion: Our study demonstrates that both type I and type II IFNs can induce neurons to upregulate PD-L1 via the STAT1/IRF1 pathway mediated by IFN receptors to protect against activated CD8⁺ T cell-mediated damage, providing a targeted pathway to alleviate neuroinflammation during ECM.

Keywords: CD8+ T cell; Experimental cerebral malaria; Interferon; Neuron; PD-L1.

Fig. 1

Neurological damage and activated CD8⁺ T cell infiltration in the brains of ECM mice. A Electron microscope observation of neuron in the brainstem of ECM mice (yellow asterisk: change in nucleus shape; red arrow: demyelination; yellow arrow: mitochondria damage; blue arrow: autophagosome). B IF staining of TUNEL⁺ neurons (red arrow) in the brainstem and olfactory bulb of ECM mice. C IF staining of infiltrating CD8⁺ T cells (yellow arrow) which are close to neurons (NeuN⁺) in the brainstem and olfactory bulb of ECM mice. D IF staining of Ki67⁺CD8⁺ T cells (yellow arrow) in the brainstem of the ECM mice. E IF staining of IFNγ⁺CD8⁺ T cells (yellow arrow) in the brainstem of ECM mice

Fig. 2

Direct interaction between neurons and ECM CD8⁺ T cells in the co-culture system. Primary cortical neurons were co-cultured with CD8⁺ T cells isolated from the spleen of ECM mice for 24 h. A LDH activity in supernatants of neurons co-cultured with ECM CD8⁺ T cells or naïve CD8⁺ T cells. Data are expressed as mean ± SD; unpaired t-test, n = 2 per group. B Flow cytometry of PI and SyTox green in neurons co-cultured with ECM CD8⁺ T cells. C IF staining of ECM CD8⁺ T cells (yellow arrow) adhesion to neurons (MAP2⁺). D IF staining of MAP2 in neurons co-cultured with different effect-target ratios of ECM CD8⁺ T cells (yellow arrow: axon damage). E WB analysis of APP levels in neurons co-cultured with ECM CD8⁺ T cells or naïve CD8⁺ T cells

Fig. 3

IFNβ or IFNγ induces nerve cells to upregulate PD-L1 during ECM. A The violin plot of Pdcd1 expression in different types of cells from the brainstem of ECM mice (n = 3) through scRNA-seq analysis (P_adj < 0.05). B The violin plot of Cd274 expression in neurons from the brainstem of control mice (n = 2) and ECM mice (n = 3) through scRNA-seq analysis (P_adj < 0.05). C IHC staining of PD-L1 levels in the brainstem of control and ECM mice (red arrow: nerve cell). Data are expressed as mean ± SD; unpaired t-test; n = 8 fields per group. D WB analysis of PD-L1 levels in the olfactory bulb, cerebrum, cerebellum, and brainstem of control and ECM mice. Data are expressed as mean ± SD; unpaired t-test; n = 4. E IF staining of the IFNβ secretion in neurons (yellow arrow) and MAP2⁻ cells (red arrow) in the brainstem of ECM mice. F) Flow cytometry of PD-L1 levels on neurons stimulated with IFNβ (100 U/mL) or IFNγ (20 ng/mL)

Fig. 4

IFNβ or IFNγ induces PD-L1 expression in neurons through the STAT1/IRF1 pathway. A Violin plots of Cd274, Stat1, and Irf1 expression in neurons stimulated with IFNβ through RNA-seq analysis (n = 3 per group) (P_adj < 0.05). B q-PCR detection of Stat1 and Irf1 expression in neurons with IFNβ (100 U/mL, the same below) or IFNγ (20 ng/mL, the same below) stimulation. Data are expressed as mean ± SD; unpaired t-test, n = 3 per group, ****P < 0.0001. C WB analysis of p-STAT1, IRF1, and PD-L1 levels in neurons with IFNβ or IFNγ stimulation. D Flow cytometry of PD-L1 levels on neurons treated with fludarabine (20 µM), IFNβ, or IFNγ. E Violin plots of Stat1 and Irf1 expression in neurons in the brainstem of control mice (n = 2) and ECM mice (n = 3) through scRNA-seq analysis (P_adj < 0.05). F WB analysis of p-STAT1, IRF1, and PD-L1 levels in the brainstem of mice with PbA infection on days 3, 5, 7, and 9. G IHC staining of p-STAT1 in the brainstem of control and ECM mice (red arrow: neuron). H IHC staining of IRF1 in the brainstem of control and ECM mice (red arrow: neuron). G, H Data are expressed as mean ± SD; unpaired t-test; n = 8 fields per group

Fig. 5

The STAT1/IRF1 pathway in Ifnar1^−/− or Ifngr1^−/− mice after PbA infection. A Flow cytometry of PD-L1 levels on Ifnar1^−/− neurons stimulated with IFNβ or IFNγ. B Flow cytometry of PD-L1 levels on Ifngr1^−/− neurons stimulated with IFNβ or IFNγ. C WB analysis of p-STAT1, IRF1, and PD-L1 levels in Ifnar1^+/+ or Ifnar1^−/− neurons, and Ifngr1^+/+ or Ifngr1^−/− neurons stimulated with IFNβ or IFNγ. D WB analysis of p-STAT1, IRF1, and PD-L1 in the brainstem of Ifnar1^+/+ or Ifnar1^−/− mice, and Ifngr1^+/− or Ifngr1^−/− mice with PbA infection, uninfected littermates as control. E IF staining of p-STAT1, IRF1, and PD-L1 in neurons (NeuN⁺) in the brainstem of Ifnar1^+/− and Ifnar1^−/− mice with PbA infection. The mean fluorescence intensity is expressed as mean ± SD; unpaired t-test; n ≥ 10 per group. F IF staining of the infiltrating IFNγ⁺CD8⁺ T cells (yellow arrow) in the olfactory bulb, cerebrum, cerebellum, and brainstem of Ifnar1^−/− mice with PbA infection

Fig. 5

The STAT1/IRF1 pathway in Ifnar1^−/− or Ifngr1^−/− mice after PbA infection. A Flow cytometry of PD-L1 levels on Ifnar1^−/− neurons stimulated with IFNβ or IFNγ. B Flow cytometry of PD-L1 levels on Ifngr1^−/− neurons stimulated with IFNβ or IFNγ. C WB analysis of p-STAT1, IRF1, and PD-L1 levels in Ifnar1^+/+ or Ifnar1^−/− neurons, and Ifngr1^+/+ or Ifngr1^−/− neurons stimulated with IFNβ or IFNγ. D WB analysis of p-STAT1, IRF1, and PD-L1 in the brainstem of Ifnar1^+/+ or Ifnar1^−/− mice, and Ifngr1^+/− or Ifngr1^−/− mice with PbA infection, uninfected littermates as control. E IF staining of p-STAT1, IRF1, and PD-L1 in neurons (NeuN⁺) in the brainstem of Ifnar1^+/− and Ifnar1^−/− mice with PbA infection. The mean fluorescence intensity is expressed as mean ± SD; unpaired t-test; n ≥ 10 per group. F IF staining of the infiltrating IFNγ⁺CD8⁺ T cells (yellow arrow) in the olfactory bulb, cerebrum, cerebellum, and brainstem of Ifnar1^−/− mice with PbA infection

Fig. 6

The protective effects of PD-L1 expression vector in neurons and ECM brains. A IF staining of PD-L1 in neurons co-cultured with ECM CD8⁺ T cells, after treatment with recombinant adenovirus expressing PD-L1 IgGFc fusion protein (Ad: PD-L1) or recombinant adenovirus expressing IgGFc (Ad: IgGFc). B LDH activity in supernatants of Ad: PD-L1 or Ad: IgGFc treated neurons co-cultured with ECM CD8⁺ T cells. Data are expressed as mean ± SD; unpaired t-test, n = 2 per group. C IF staining of MAP2 in Ad: PD-L1 or Ad: IgGFc treated neurons co-cultured with ECM CD8⁺ T cells. D Flow cytometry for JC-1 (FL-1: monomer, FL-2: aggregates) in Ad: PD-L1 or Ad: IgGFc treated neurons co-cultured with ECM CD8⁺ T cells. E IF staining of NeuN in neurons on Ad: PD-L1 injected side or non-injected side of ECM brainstem. A–E Fluorescence statistical data are expressed as mean ± SD; unpaired t-test; n ≥ 3 per group. F) Nissl staining of neurons (red arrow) on Ad: PD-L1 injected side or non-injected side of ECM brainstem. Data are expressed as mean ± SD; unpaired t-test; n = 15 fields per group. G IF staining of Ki67 and CD8 on Ad: PD-L1 injected side or non-injected side of ECM brainstem (yellow arrow: Ki67⁺CD8⁺ T cell; green arrow: CD8⁺ T cell)

Fig. 6

The protective effects of PD-L1 expression vector in neurons and ECM brains. A IF staining of PD-L1 in neurons co-cultured with ECM CD8⁺ T cells, after treatment with recombinant adenovirus expressing PD-L1 IgGFc fusion protein (Ad: PD-L1) or recombinant adenovirus expressing IgGFc (Ad: IgGFc). B LDH activity in supernatants of Ad: PD-L1 or Ad: IgGFc treated neurons co-cultured with ECM CD8⁺ T cells. Data are expressed as mean ± SD; unpaired t-test, n = 2 per group. C IF staining of MAP2 in Ad: PD-L1 or Ad: IgGFc treated neurons co-cultured with ECM CD8⁺ T cells. D Flow cytometry for JC-1 (FL-1: monomer, FL-2: aggregates) in Ad: PD-L1 or Ad: IgGFc treated neurons co-cultured with ECM CD8⁺ T cells. E IF staining of NeuN in neurons on Ad: PD-L1 injected side or non-injected side of ECM brainstem. A–E Fluorescence statistical data are expressed as mean ± SD; unpaired t-test; n ≥ 3 per group. F) Nissl staining of neurons (red arrow) on Ad: PD-L1 injected side or non-injected side of ECM brainstem. Data are expressed as mean ± SD; unpaired t-test; n = 15 fields per group. G IF staining of Ki67 and CD8 on Ad: PD-L1 injected side or non-injected side of ECM brainstem (yellow arrow: Ki67⁺CD8⁺ T cell; green arrow: CD8⁺ T cell)