PIF* promotes brain re-myelination locally while regulating systemic inflammation- clinically relevant multiple sclerosis M.smegmatis model

Abstract

Neurologic disease diagnosis and treatment is challenging. Multiple Sclerosis (MS) is a demyelinating autoimmune disease with few clinical forms and uncertain etiology. Current studies suggest that it is likely caused by infection(s) triggering a systemic immune response resulting in antigen/non-antigen-related autoimmune response in central nervous system (CNS). New therapeutic approaches are needed. Secreted by viable embryos, PreImplantation Factor (PIF) possesses a local and systemic immunity regulatory role. Synthetic PIF (PIF) duplicates endogenous peptide's protective effect in pre-clinical autoimmune and transplantation models. PIF protects against brain hypoxia-ischemia by directly targeting microglia and neurons. In chronic experimental autoimmune encephalitis (EAE) model PIF reverses paralysis while promoting neural repair. Herein we report that PIF directly promotes brain re-myelination and reverses paralysis in relapsing remitting EAE MS model. PIF crosses the blood-brain barrier targeting microglia. Systemically, PIF decreases pro-inflammatory IL23/IL17 cytokines, while preserving CNS-specific T-cell repertoire. Global brain gene analysis revealed that PIF regulates critical Na+/K+/Ca++ ions, amino acid and glucose transport genes expression. Further, PIF modulates oxidative stress, DNA methylation, cell cycle regulation, and protein ubiquitination while regulating multiple genes. In cultured astrocytes, PIF promotes BDNF-myelin synthesis promoter and SLC2A1 (glucose transport) while reducing deleterious E2F5, and HSP90ab1 (oxidative stress) genes expression. In cultured microglia, PIF increases anti-inflammatory IL10 while reducing pro-inflammatory IFNγ expression. Collectively, PIF promotes brain re-myelination and neuroprotection in relapsing remitting EAE MS model. Coupled with ongoing, Fast-Track FDA approved clinical trial, NCT#02239562 (immune disorder), current data supports PIF's translation for neurodegenerative disorders therapy.

Keywords: M. smegmatis bacteria; RR-EAE clinically-relevant model; neuroprotection; neuroregeneration; preImplantation factor.

Figure 1. Continuous and intermittent PIF administration reduces clinical score (RR-EAE model)

SJL mice (4-7 per group) were infected sc with 4×106 CFU of live recombinant M Smegmatis expressing a recombinant chimeric protein MPT64-PLP139-151 (rMSp139), as previously published [43]. Starting on day 3 after infection, mice were treated daily with PIF (closed symbols and black bars) or vehicle only (PBS, open symbols and white bars). (A) Disease course and average total score of disease in mice treated continuously until day 50 after infection with 0.75 mg/Kg of PIF (n=6) or with vehicle only (n=4) (B) Disease course and average total score of disease in mice treated from day 3 until day 18 and then from day 51 until day 70 with 0.75 mg/Kg of PIF (n=6) or with vehicle only (n=4). (C) Disease course and average total score of disease in mice treated from day 3 until day 25 and then from day 51 until day 65 with 1.5 mg/Kg of PIF (n=7) or with vehicle only (n=6). Disease score was monitored by two independent examiners, blinded with respect to treatment. *p< 0.05; Mann-Whitney test. PIF: PreImplantation Factor, ATDS: average total disease score.

Figure 2. PIF promotes brain re-myelination

PIF effect on myelin expression was compared to vehicle treated control and naïve SJL mice. Briefly, at day 28-30 after RR-EAE induction (control n=3; Injury n=5; Injury+PIF n=8) mice fixed brains were embedded in paraffin and sectioned into 7μm slices. Slides were stained in Cresyl violet (Nissl body staining for neuronal structure and gross brain morphology) and Luxol Fast Blue revealing areas of myelination in the subcortical white matter. (A) Representative images of subcortical white matter comparing the three groups. Upper panel shows the brain morphology and myelination. PIF treated injured brains (Injury+PIF) are similar to naïve mice (control) and vehicle treated injured brains (Injury) show impaired myelination in the subcortical region. Lower panels: Representative images of the subcortical region (blue staining: luxol fast blue) display PIF`s induced myelination (compare Injury+PIF versus Injury and Injury versus Control). (B) Quantitative analysis of myelin positive cells in the groups. PIF treatment results in restored myelination in the brain. *p<0.05 and ** p<0.01. PIF: PreImplantation Factor; scale bar: 100μm

Figure 3. PIF targets microglia in the brain

SJL mice previously infected with rMSp139 and in late phase of chronic disease (> 60 days after infection) treated with PIF or PBS for 3 weeks were injected with a single FITC-PIF or PBS dose and sacrificed 3 hours later. We used FITC-scrambled PIF as negative control as well. Brains were prepared for histology. (A) In the PBS injected mice the Iba-1 positive cells (microglia) showed predominantly amoeboid state and negative PIF staining. Right panels show Iba-1 positive cell (upper), negative PIF immunofluorescence (middle) and merged images (lower). (B) In PIF treated animals microglia was predominantly in ramified state and PIF positive. Right panels show Iba-1 positive cell (upper), positive PIF immunofluorescence (middle) and merged images (lower). (C) In the PBS injected mice with FITC-scrambled PIF (negative control) the Iba-1 positive cells (microglia) showed predominantly amoeboid state and expectantly negative PIF staining. Right panels show Iba-1 positive cell (upper), negative PIF immunofluorescence (middle) and merged images (lower). Scale bar 50 μm.

Figure 4. PIF modulates cytokine expression in draining lymph nodes

SJL mice (5 each group) were infected with rMSp139 and treated daily with 0.75 mg/Kg of PIF (black bars) or vehicle only (white bars). Ten days later, cells from draining lymph nodes were obtained and cultured for 3 hours. Levels of mRNA specific for the indicated cytokines and transcription factors were measured by quantitative RT-PCR. PIF reduced the expression of IL-17 and IL23 significantly. *p<0.05 (Mann-Whitney Test); PIF: PreImplantation Factor.

Figure 5. PIF does not modulate splenic T cell repertoire

SJL mice (7 each group) were infected sc with rMSp139 and treated daily with 0.75 mg/Kg PIF or vehicle (PBS). Thirty days later, cells from spleen were obtained and cultured in the presence or absence of p139. mRNA was obtained and submitted to TCR BV-BJ spectratyping for the shared rearrangements characterizing the induced CD4+ T cells specific for p139 (Vb 4-Jb1.6; Vb10-Jb1.1), the T cells spontaneously responding to this epitope (Vb18-Jb1.2; Vb19-Jb1.2), and the induced CD8+ T cells specific for p139 (Vb17-Jb1.6; Vb20-Jb2.3). Each column reports data from one individual mouse, and a black square indicates the detection of T cells bearing the indicated TCR rearrangement. TCR: T cell repertoire; PIF: PreImplantation Factor.

Figure 6. PIF effect of global brain genome pathways (Ingenuity analysis)

Schematic pathways that are significantly affected by PIF and their interaction. Leading among them was the ubiquitin pathway that is responsible for protein degradation which was down-regulated by PIF. This was coupled with reduction in hypoxia signaling that creates vascular inflammation thereby the oxidative stress is reduced as well. On the other hand, the EIF2 signaling pathway increased which is responsible for the promotion of protein synthesis.

Figure 7. PIF effect on global genome (Ingenuity statistics)

Evaluation of the pathways involved describing the effect PIF whether it is up or down regulated as well the associated level of significance. Namely the reduction in ubiquitination and EIF2 signaling were the most pronounced. On the other hand, the most remarkable increase was present in asparagine biosynthesis which protects against vascular damage.

Figure 8. PIF promotes BDNF, SLC2A1, and reduces HSP90AB1, and E2F5 expression in astrocytes

Primary astrocytes were cultured with increasing PIF concentrations up to 48 hours. PIF promotes BDNF expression (myelin synthesis inducer), SLC2A1 expression (glucose transporter) while reducing HSP90AB1 expression (oxidative stress), and E2F5 expression (neuro-injury activated). mRNA levels were determined using RT-PCR of the specific genes compared to S12 expression. For each factor the control levels were set as 1 and the expression of the treated samples was compared to the control. *** p<0.001. PIF: PreImplantation Factor.

Figure 9. PIF promotes IL10 and reduces IFNγ expression in microglia cultures

Primary microglial cells were cultured with increasing PIF concentrations up to 48 hours. PIF increased anti-inflammatory IL10 while reducing pro-inflammatory IFNγ expression in a dose dependent manner. mRNA levels were determined using RT-PCR of the specific genes compared to S12 expression. For each factor the control levels were set as 1 and the expression of the treated samples was compared to the control *** p<0.001. PIF: PreImplantation Factor.