Tuftsin-driven experimental autoimmune encephalomyelitis recovery requires neuropilin-1

Abstract

Experimental autoimmune encephalomyelitis (EAE) is an animal model of demyelinating autoimmune disease, such as multiple sclerosis (MS), which is characterized by central nervous system white matter lesions, microglial activation, and peripheral T-cell infiltration secondary to blood-brain barrier disruption. We have previously shown that treatment with tuftsin, a tetrapeptide generated from IgG proteolysis, dramatically improves disease symptoms in EAE. Here, we report that microglial expression of Neuropilin-1 (Nrp1) is required for tuftsin-driven amelioration of EAE symptoms. Nrp1 ablation in microglia blocks microglial signaling and polarization to the anti-inflammatory M2 phenotype, and ablation in either the microglia or immunosuppressive regulatory T cells (Tregs) reduces extended functional contacts between them and Treg activation, implicating a role for microglia in the activation process, and more generally, how immune surveillance is conducted in the CNS. Taken together, our findings delineate the mechanistic action of tuftsin as a candidate therapeutic against immune-mediated demyelinating lesions.

Keywords: EAE; Treg; anti-inflammatory; mice; microglia.

Figure 1. Tuftsin infusion has no effect on EAE disease symptoms in Nrp1-MgKO mice

EAE was induced by injection of MOG_35-55 in CFA and pertussis toxin. Osmotic pumps filled with 500μM tuftsin in PBS were implanted subcutaneously on day 0 after MOG immunization. Comparisons between WT control (untreated) and tuftsin-infused mice (A) and Nrp1-MgKO control (untreated) and tuftsin-infused mice (B). Data are mean ± SEM. n= 13-17, *, p<0.05; **, p<0.01, ***, p<0.001. (C) Frozen spinal cord sections were isolated from control and tuftsin-infused WT and KO mice at 0, 14, 21, and 28 days post induction of EAE. Demyelination was quantified by fluoromyelin staining and demyelinated areas were measured using ImageJ and quantified. (D) Quantification of Iba1 signal intensity staining was utilized to detect microglia in control and tuftsin-infused WT and Nrp-MgKO mice at 0, 14, 21, and 28 days post induction of EAE.

Figure 2. Tuftsin promotion of the M2 phenotype is abolished in Nrp1-MgKO mice

Pro-inflammatory (M1) and anti-inflammatory (M2) microglia were identified by co-localization of iNOS/Iba1 and Arg1/Iba1, respectively, in the spinal cords of control (C) and tuftsin-infused (T) WT EAE mice in (A) and control and tuftsin-infused EAE Nrp1-MgKO mice in (B). iNOS⁺/Iba1⁺ (C) and Arg1⁺/Iba1⁺ (D) cells were enumerated. Percentages of each population after treatment are indicated in (E). C: Control, T: Tuftsin. Nuclei are stained with DAPI (blue). Data are represented as mean ± SEM. n=3, **, p<0.01, ***, p<0.001. Scale bar: 50μm.

Figure 3. Nrp1 expression on microglia is necessary for increased phagocytic activity of microglia in the presence of tuftsin

Microglia isolated from Nrp1-MgKO or WT pups were untreated, treated with 100ng/ml LPS for 4 hours, or treated with 100ug/ml tuftsin in the presence or absence of neuronal conditioned medium (NCM) for 10 hours, and then incubated with 0.1ul/ml suspension of red fluorescent beads (0.8 um diameter, Sigma) (A). The number of beads in 25-31 cells per condition was quantified (B). ***, p<0.001; ns, not significant.

Figure 4. Loss of Nrp1 expression on microglia and/or Tregs reduces long contacts and TGFβ release

T cells isolated from the spleens of Nrp1^fl/fl mice were left as naïve T cells, polarized to Tregs, or polarized to Tregs in the presence of Cre retrovirus (A); microglia isolated from Nrp1-MgKO or WT pups were examined for Nrp1 expression in the presence or absence of LPS by PCR (B). Microglia were plated in chamber slides and allowed to return to a resting state. The next day, WT or Nrp1-TregKO were added and visualized using time-lapse video microscopy. Images were taken every 10 seconds for 20min and the duration of contacts were determined by the number of frames that Treg and microglia remained in steady contact. The interaction time for each individual contact is shown in (C). The percentage of interactions longer than 400sec (D), and average contact length within each biological replicate (E) were quantified. Significance is relative to WT microglia/WT Treg. After 4 days of co-culture, media were isolated and an ELISA assay used to quantify TGFβ cytokine levels (F), Data are represented as mean ± SEM. n=3-9, **, p<0.01; ***, p<0.001.