Mucopolysaccharidosis IIIB, a lysosomal storage disease, triggers a pathogenic CNS autoimmune response

MPS IIIB knock-out mice [2] were maintained on an inbred background (C57BL/6) by backcrosses of heterozygotes. This colony has been through 16 backcrosses, alternated by a cross with wt C57BL/6 to generate heterozygotes for next backcross. MPS IIIB mice were housed in the barrier area in the Vivarium at the Research Institute at Nationwide Children's Hospital (NCH-RI). Progeny mice were genotyped by PCR. The MPS IIIB mice and their age-matched wt littermates were used in the experiments. All procedures using MPS IIIB mice in this study have been approved by the Institutional Animal Care and Use Committee at NCH-RI. All care and procedures were in accordance with the Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23]. MPS IIIB mice and control wt littermate were used in all experiments in this study.

Mice were anesthetized with an intraperitoneal injection of Avertin (0.3 - 0.4 μg/g) and spleens were dissected for lymphocyte preparation, following established procedures. 1) Under sterile conditions, cell suspensions in RPMI media containing 2% FBS were filtered through a 70 μm nylon cell strainer. 2) The cells were washed and resuspended in Fac's buffer (PBS, 2% fetal calf serum), 3) then incubated with RBC lysis buffer (eBioscience) to remove red blood cells, and 4) washed and resuspended in Fac's buffer or RPMI media for further analysis. The number of splenocytes was determined using Sysmex KX-2IN Automated Hematology Analyzer after step 1) and again at the end of the process.

Splenocytes from 2-3-month-old MPS IIIB mice were isolated as described above. Single cell suspension of splenocytes isolated from multiple mice were pooled and stimulated in vitro for 48 hours with Concanavalin A (ConA, 1 μg/ml) in RPMI 1640 growth media (10% FBS, 2 mM L-glutamine, 100 U/ml penicillin 100 μg/ml streptomycin and 50 μM 2-ME). The non-adherent cells were collected and washed with PBS for adoptive transfer. The isolated lymphocytes (2 × 10⁷/mouse) were injected intravenously (IV) into 10-12-week-old wt male littermates through tail vein. The mice were then observed daily for their wellbeing and clinical disease signs over 2-6 weeks. Lymphocytes isolated from age-matched wt littermates were IV injected into age-matched wt littermates as controls.

Mice were examined daily to identify any abnormalities. After observing a mild ascending paralytic clinical disease in animals receiving MPS IIIB lymphocytes, we adopted a scoring system used in evaluation of experimental autoimmune encephalomyelitis (EAE)[20] to assess mice in this study. The scoring system was on a scale of 0-5: 0 = no clinical disease; 1 = tail weakness; 1.5 = tail paralysis; 2 = hind limb weakness; 3 = hind limb paralysis; 3.5 forelimb weakness; 4 = forelimb paralysis; 5 = moribund or death.

Grip tests were performed at 14 days after the adoptive transfer to evaluate the strength of limbs, using a digital grip strength meter (Columbus instruments). The grip strength was presented as peak tension (g).

Exploratory activity in a novel environment was assessed in a one-hour trial in a photobeam open field chamber [21](San Diego instruments). Counts were taken of the number of photobeams broken during the trial, with separate measures for locomotor, vertical, and rearing activity.

Subjects were tested on an accelerating rotarod (Med Associates Inc.) to assess motor coordination [21]. For the first test session, animals were given three trials, with 45 seconds between each trial. Two additional trials were given 48 hours later. Revolutions per minute (rpm) was set at an initial value of 3, increasing to a maximum of 30 rpm across five minutes (the trial length). Measures were taken for latency to fall from the rotating barrel (sec).

Tissue samples were collected at 14 days, and 5 weeks after adoptive transfer. The animals were anesthetized with an intrperitoneal injection of 2.5% Avertin, blood samples were collected. The mice were then perfused transcardially with 20 ml of cold PBS (0.1 M, pH7.4), and, for histological examinations, followed by 4% paraformalde, prior to tissue collection. Brain, spinal cord and spleen samples were collected. The half brain samples and spinal cords were stored at -80°C before being processed for total RNA extraction. Tissue samples from paraformaldehyde-perfused animals were further fixed in 4% paraformaldehyde overnight, then infiltrated in 3% sucrose, and then embedded in OCT for cryosectioning, and histology and immunohistochemistry staining. Each assay was performed using samples from at least 4 mice (n ≥ 4/group).

Flow cytometry was performed on donor splenocytes (4 × 10⁶ lymphocytes per sample), expanded with ConA, using antibodies against mouse CD3e, CD4, CD8, CD25, CD45R/B220, and F4/80 (BD Biosciences). The labeled cells were sorted using a BD-LSRII flow cytometer (BD Biosciences) and data were analyzed using BD FACSDiva software.

Freshly isolated splenocytes were assayed for T-cell proliferation following published procedures [22, 23] using freshly isolated cells with anti-mouse-CD3e antibody (eBioscience) as stimulator, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as cell proliferation marker. Cells in RPMI growth media (2 × 10⁵ in 200 μl/well, in triplicates or more) were plated onto 96-well tissue culture plates pre-coated with anti-CD3e antibody (50 μl, 10 μg/ml in PBS) or PBS, and incubated at 37°C for 72 hr. The cell clusters formed in each well were then counted under a microscope, before the addition of 20 μl MTT buffer (5 mg/ml in PBS), followed by 4-hour incubation. The MTT lysis buffer (20% SDS in 50% DMF) was then added to each well and incubated at 37°C overnight. T-cell proliferation was expressed as: Stimulation index = A₅₇₀ with anti-CD3/A₅₇₀ with PBS.

Splenocytes (10⁶ cells/ml) were incubated in RPMI 1640 growth media, as described above, in the presence or absence of ConA. Media were collected after 42 hour incubation, and assayed for cytokine production by Bio-plex cytokine assay (BioRad), following the procedures provided by the manufacturer. The Bio-plex panel used targets IL2, IL4, IL5, IL10, IL17, IFNγ and TNFα.

Total RNA was isolated from half brain of each individual mouse (n = 4/group), using SV Total RNA Isolation System (Promega), following the manufacturer's protocols. The isolated total RNA samples were then further purified through an RNeasy spin column (Qiagen). The mouse brain total RNA was assayed for the expression of immune-associated genes, using sequence specific primers. A pair of primers for murine GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene was used as internal control. Superscript™ First-strand Synthesis System for RT-PCR (Invitrogen) was used for cDNA synthesis. The qRT-PCR was performed using SYBR^® Green PCR Master Mix (Applied Biosystems). Comparative threshold (C_T) method was used for data analysis. Data were expressed as relative quantitation of gene expression (2^{^-ΔΔCt})[24], in the brain of mice given an adoptive transfer of lymphocytes from MPS IIIB mice vs. lymphocytes from wt mice.

Mouse brain tissues were processed for serial transverse cryostat sections (10-15 μm). IHC staining was performed following the procedures provided by manufacturers, using primary antibodies against mouse CD3 (AbD Serotec) and CD8 (Biolegend), and secondary antibodies conjugated with horse radish peroxidase or Alexa Fluo 568.

Data are reported as means ± SD. P-values between datasets were determined by two-tailed Student's t-test. Significance was defined as P < 0.05.

To test our hypothesis that MPS IIIB primes an autoimmune response which subsequently provokes the onset or exacerbates MPS IIIB neuropathology, we analyzed the phenotype and functional potential of MPS IIIB mouse lymphocytes. T-cell proliferation assay showed a significant increase in stimulation index of splenic lymphocytes from MPS IIIB mice, compared to wt cells, when stimulated with anti-CD3e ex vivo (data not shown), suggesting T-cell activation in MPS IIIB mice. To be used in the adoptive transfer experiments below, splenic lymphocytes isolated from wt (10-12 weeks old) or MPS IIIB mice (n = 8/group) were pooled within a group. Leukocytes from wt or MPS IIIB mice were comprised of a mixed population of T cells, B cells and macrophages (Table 1). Interestingly, the splenic T:B cell ratio was reduced 26% in MPS IIIB mice and was accompanied by an increase in F4/80+ macrophages, suggesting the activation of B-cells and macrophages. T cells from MPS IIIB mice spontaneously (i.e., without ex vivo stimulation) secreted more cytokine at higher concentrations than T cells from wt mice. Specifically, we found IL2, IL4, IL5, IL10, IL17 and IFNγ to be present in supernatants of T cells from MPS IIIB mice but not wt mice (data not shown). After stimulation with concanavilin A (ConA), MPS IIIB lymphocytes secreted greater amounts of IL2, IL5, IL10, IFNγ and TNFα as compared to ConA-activated wt lymphocytes (Fig. 1). These data suggest that T cells are activated in vivo during the progression of MPS IIIB and develop a predominantly Th1 cytokine profile (i.e., high levels of IL-2 and IFNγ).

Adoptive transfer of isolated lymphocytes was performed to assess the pathogenic potential of MPS IIIB lymphocytes. Because our previous studies showed that the CNS immune activation in MPS IIIB mice involves virtually every aspect of the immune system, total mouse splenic lymphocytes were used for adoptive transfer in this study.

Lymphocytes that were pooled from the spleen of age-matched donor MPS IIIB or wt mice were activated with ConA ex vivo for 48 hours then were injected intravenously into naïve wt recipient mice (n = 26-38 recipient mice/group, 8-10 weeks old). All recipient mice were monitored daily for signs of neurological impairment using a 0-5 "clinical scale" that is widely used to quantify the magnitude of ascending paralysis in experimental autoimmune encephalomyelitis (EAE)[20]. None of the mice treated with wt lymphocytes developed disease. In contrast, all mice receiving lymphocytes from MPS IIIB donor mice developed mild ascending paralysis characterized by weak tail and lower trunk (score of ~1) (Table 2, Fig. 2a, Additional files 1, 2, 3, 4). Tail weakness emerged 6-9 days after adoptive transfer and persisted for up to 15 days reaching peak disease at ~2 weeks post transfer (Table 2; Fig. 2a).

Given the subjective nature of the clinical scale, additional behavioral assays were completed to confirm and quantify the magnitude of neurological impairment. Specifically, we quantified grip strength, volitional open field activity and latency on rotarod at 2 weeks post transfer, i.e., the time of peak clinical disease. As shown in Fig. 2b, hind limb grip strength was significantly impaired in mice injected with MPS IIIB lymphocytes (P < 0.05 vs. mice injected with wild-type lymphocytes). Fore limb grip strength was unaffected (not shown). Hind limb deficits were confirmed in the open field and on the rotorod, where a significant decrease in rearing activity (P < 0.05) and shorter latencies to fall were noted in mice treated with MPS IIIB lymphocytes (Fig. 2c&2d).

To further assess the pathological impact of adoptively-transferred MPS IIIB lymphocytes, we monitored the body weight of mice born on the same day that received either wt or MPS IIIB lymphocytes (n = 6-8 recipient mice/group). All mice had equal access to food and water each day during the analysis period. Mice treated with wild-type lymphocytes increased their body weight ~16% over baseline in 20 days (Fig. 2e). In contrast, mice that received MPS IIIB lymphocytes increased their body weight 5% by the end of the observation period (i.e., 40 days post-transfer). This significant delay in weight gain paralleled the onset and progression of neurological impairment. Collectively, these data suggest that MPS IIIB lymphocytes cause neurological impairment and a sustained sickness response.

To evaluate the impact of adoptively transferred MPS IIIB lymphocytes on the nervous system of recipient mice, we used qRT-PCR to compare mRNA profiles of brains prepared from mice receiving wild-type or MPS IIIB lymphocytes at the peak of clinical disease (2 weeks post-transfer). We observed a significant up-regulation of markers of innate immunity and glial activation (i.e., CD45, CD68, GFAP) along with a significant increase in expression of multiple cytokines, including IFNγ, IL2, IL4, IL5, IL17, TNFα, IFNα and Ifi30 (Table 3). Western blotting for GFAP confirmed the astrocyte activation (data not shown). We also noted a modest increase in CD3e, CD4 and CD8a mRNA suggesting the presence of T-cell infiltrates in the CNS of mice receiving MPS IIIB lymphocytes (Table 3). Immunofluorescence staining of brain cryosections from recipient mice confirmed this hypothesis showing the presence of CD8+ T-cells in the brain and spinal cord of mice receiving MPS IIIB lymphocytes at 2 weeks (Fig. 3) but not at 4 weeks post transfer. The distribution of these T-cells was sporadic without evidence of profound clustering (as in EAE). Immunoflurescence for CD3 and CD4 was not successful due to the quality of the antibodies that did not even stain cells in spleen. We did not find T cells in the brain or spinal cord of mice receiving injections of wild-type lymphocytes.