PI3Kγ inhibition alleviates symptoms and increases axon number in experimental autoimmune encephalomyelitis mice

Abstract

Phosphoinositide 3-kinase γ (PI3Kγ) is a shared downstream component of chemokine-mediated signaling pathways and regulates migration, proliferation and activation of inflammatory cells. PI3Kγ has been shown to play a crucial role in regulating inflammatory responses during the progression of several diseases. We investigated the potential function of PI3Kγ in mediating inflammatory reactions and the development of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). We found that systemic treatment with selective PI3Kγ inhibitor AS-604850 significantly reduced the number of infiltrated leukocytes in the CNS and ameliorated the clinical symptoms of EAE mice. Treatment with this PI3Kγ inhibitor enhanced myelination and axon number in the spinal cord of EAE mice. Consistently, we demonstrated that PI3Kγ deletion in knockout mice mitigates the clinical sign of EAE compared to PI3Kγ+/+ controls. PI3Kγ deletion increased the number of axons in the lumbar spinal cord, including descending 5-HT-positive serotonergic fiber tracts. Our results indicate that PI3Kγ contributes to development of autoimmune CNS inflammation and that PI3Kγ blockade may provide a great potential for treating patients with MS.

Keywords: DMSO; EAE; GPCR; GTP-binding protein-coupled receptor; KO; LFB; Luxol fast blue; MBP; MOG; MS; NF; PBS; PI(3 4 5)P3; PI(4 5)P2; PI3 kinase γ; PI3Kγ; Phosphate-buffered saline; axon; dimethyl sulfoxide; experimental autoimmune encephalomyelitis; functional recovery; knockout; multiple sclerosis; myelin basic protein; myelin oligodendrocyte glycoprotein; myelination; neurofilament; phosphatidylinositol-3 4 5-trisphosphate; phosphatidylinositol-4 5-bisphosphate; phosphoinositide 3-kinase γ.

Fig. 1.

Systemic treatment with AS-604850 attenuated ED1+ macrophages and CD3+ lymphocytes in lumbar spinal cords of EAE mice. (A) The representative transverse spinal cord sections doubly stained for ED1 (green) and neurofilament (NF, red) display a number of ED1+ macrophages (arrows) in lateral white matter tracts in EAE mouse treated with vehicle DMSO. The similar sections reveal a reduced density of ED1+ macrophages in the same area of spinal cord from the EAE mice treated with AS-604850. Scale = 15 μm. (B) The schematic of molecular formula of AS-604850. (C) Quantification of ED1+ macrophages from each transverse section indicates significantly reduced number of ED1+ cells in the AS-604850-treated mice. (D) The representative transverse spinal cord sections doubly stained for CD3 (red) and NF (green) exhibit a number of CD3+ cells (arrowheads) in the dorsal column of lumbar spinal cord in EAE mouse treated with vehicle DMSO, but sections from AS-604850-treated EAE mice reveal a reduced density of CD3+ cells in the same area of spinal cord. Scale = 15 μm. (E) Quantification of CD3+ cells indicates significantly reduced number of CD3+ cells in AS-604850-treated mice. n = 5 mice in vehicle and AS-604850 groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

Treatment with selective PIK3γ inhibitor AS-604850 attenuates clinical symptoms of EAE mice. Graphs indicate clinical EAE scores as the function of time after EAE onset. Treatments with vehicle DMSO or AS-604850 (7.5 mg/kg/day) were initiated one day after EAE onset and sustained for 7 days (A) or for 14 days (B). AS-604850 treatment significantly reduced EAE scores at multiple time points. The bar in red indicates the time period for drug application in these EAE mice. n = 6 mice in each group in A; n = 9 and 8 mice in DMSO and AS-604850 groups in B. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

AS-604850 treatment enhances myelination and axon number in the lumbar spinal cord 25 days after EAE onset. (A) The representative images indicate transverse sections of the spinal cord immunostained for MBP (top images) and NF (bottom images) in normal, EAE + Veh and EAE + AS-604850 groups. In all the sections, dorsal is up. Scale = 150 (low power) or 30 (high power) μm. (B, C) The myelin signals stained by MBP and axonal signals labeled by NF were quantified from different white matter areas of transverse sections in a blind manner. n = 6 mice in each group.

Fig. 4.

AS-604850 treatment increases myelination in the spinal cord of EAE mice detected by LFB staining. Images indicate representative transverse sections of the lumbar spinal cord processed for LFB staining (A) or LFB + Cresyl Violet counterstaining (B) in normal, EAE + vehicle and EAE + AS-604850 groups. The images at higher power were collected from lateral (middle) or ventral (bottom) white matter areas. In all the sections, dorsal is up. Scale bars = 350 μm (low magnification) and 50 μm (high magnification).

Fig. 5.

PIK3γ deletion in knockout mice reduces clinical EAE symptoms. Graphs indicate clinical EAE scores as the function of time. PIK3γ−/− mice exhibit significantly reduced EAE scores at multiple time points compared to either PIK3γ+/+ or +/− control groups. n = 12 mice in each group in A; n =10 and 9 mice in PIK3γ+/+ and −/− groups in B.

Fig. 6.

PIK3γ deletion enhances serotonergic fibers in the cervical and lumbar spinal cord of EAE mice. (A, B) Transverse sections of the central and ventral spinal cord at C5 (A) and L4 (B) levels reveal high density of serotonergic fibers in normal control mice (left column). Transverse sections of the spinal cord in control EAE mice (PIK3γ+/+) displayed highly-reduced 5-HT fibers 5 weeks after EAE onset (center column), but sections from PIK3γ−/− mice (right column) exhibited increased serotonergic fibers in both central and ventral part of the spinal cord compared to those in vehicle-treated EAE mice. The dorsal is up in all these sections. Scale = 50 μm. (C, D) Serotonergic fiber length was measured from gray and white matter in dorso-central areas and from gray matter in the ventral horn of the spinal cord at C5 (C) or L4 (D). PIK3γ−/− group shows a greater number of 5-HT positive axons in the cervical and lumbar spinal cord. n = 10 and 9 mice in C and D.