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Gaucher disease is a lysosomal storage disease resulting from insufficient enzyme acid β-glucosidase (glucocerebrosidase, GCase, EC 184.108.40.206) activity and the resultant accumulation of glucosylceramide. Macrophage (M) lineage cells are thought to be the major disease effectors because of their secretion of numerous cytokines and chemokines that influence other poorly defined immunological cell populations. Increases in several such populations were identified in a Gba1 mouse model (D409V/null; 9V/null) of Gaucher disease including antigen presenting cells (APCs), i.e., M, dendritic cells (DCs), neutrophils (PMNs), and CD4+T cells. FACS analyses showed increases in these cell types in 9V/null liver, spleen lung, and bone marrow. T-cells or APCs enhanced activations were evident by positivity of CD40L, CD69, as well as CD40, CD80, CD86, and MHCII on the respective cells. M, and, unexpectedly, DCs, PMNs, and T cells, from 9V/null mice showed excess glucosylceramides as potential bases for activation of APCs and T cells to induce Th1 (IFNγ, IL12, TNFα,) and Th17 (IL17A/F) cytokine production. These data imply that excess glucosylceramides in these cells are pivotal for activation of APCs and T cell induction of Th1 and Th17 responses and PMN recruitment in multiple organs of this model of Gaucher disease.
Gaucher disease (GD) is a common lysosomal storage disease that is caused by mutations in the GBA1 leading to decreased acid β-glucosidase (β-D-glucosyl-N-acylsphingosine glucohydrolase (EC 220.127.116.11; GCase) activity . This insufficient activity results from the detrimental effects on GCase’s catalytic function, stability, and/or trafficking of more than 300 mutations in the GBA1 gene [2–6]. The resultant excess lysosomal accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) substrates in macrophage (M) lineage cells of liver, spleen, lung, and bone marrow are a primary finding in these tissues. The incidence of Gaucher disease worldwide is 1/60,000, but reaches 1/850 in the Ashkenazi Jewish population [7, 8]. Based on the absence or presence and severity of neuronopathic involvement, Gaucher disease has been classified into three clinical phenotypes, non-neuronopathic (type 1), acute neuronopathic (type 2) and chronic neuronopathic (type 3) . A common feature between all three types is the accumulation of GC in the affected tissues. The cell types and mechanisms leading to consequent hepatosplenomegaly, thrombocytopenia, skeletal disease, anemia, and inflammatory signs are poorly understood [10, 11].
GC has a ceramide backbone with a β-D-glucopyranoside bound at the 1-hydroxyl position. The biosynthesis of GC is catalyzed by a UDP-glucose:ceramide glucosyltransferase [glucosylceramide synthase (GCS), EC 18.104.22.168] . GC is the precursor in the synthesis of 300–400 glycosphingolipids in different mammalian cell types [13–15]. These include ceramide and its degradation products that regulate cell proliferation, apoptosis, and modulation of cell signaling pathways [16–18]. These glycosphingolipids also have key roles in diabetes, cancer, kidney, and other common diseases [19–21]. Disruption of the balance between GC synthesis and degradation in Gaucher disease leads to inflammatory conditions and functions in different tissues .
Several cytokines have increased levels in Gaucher disease and have been implicated in pathophysiology of the disease including interleukin-1α (IL-1α), interleukin-1β (IL-β), IL-1 receptor antagonist (IL-1Ra), soluble IL2 receptor (sIL-2R), IL-6, IL-8, IL-10, IL-18, hepatocyte growth factor (HGF), macrophage colony-stimulating factor (MCSF), macrophage-inflammatory protein-1 (MIP-1), pulmonary and activation regulated chemokine (PARC), soluble CD14 (sCD14), transforming growth factor-beta1 (TGFβ1), and tumor necrosis factor-alpha (TNFα) [9, 22–24]. However, the associations of Gaucher disease with other immunological phenotypes as well as their state of activation and role in the production of T helper-1 (Th1), T helper-2 (Th2) and T helper-17 (Th17) cytokines are ill defined, but have basic and applied import. Interactions between APCs (i.e., M, DCs, PMNs) and T lymphocytes seem to be critical in Gaucher disease pathogenesis. These interactions involve several co-stimulatory molecular pairs, including CD28-B7 and, particularly, CD40-CD40L. In APCs, CD40/CD40L activation favors survival, production of cytokines, and expression of enzymes directly involved in the inflammatory response in many diseases [25, 26]. This plethora of effects reinforces the importance of the stimulatory and co-stimulatory molecule mediated signaling pathways in regulating immunity. Thus, activation of APCs and T cells could lead to increased inflammatory responses in Gaucher disease.
Here, the nature, types, and activation states of immunological cells in various organs from a Gaucher disease mouse model, 9V/null, were determined and correlated with GC species contents. These analyses broaden the scope of immunological cell type involvement in Gaucher disease and provide a basis for understanding the complexity of the disease phenotypes.
The following reagents were from BD Biosciences (San Jose, CA) or eBiosciences (San Diego, CA): Monoclonal antibodies (mAb) to CD11b-FITC (M1/70), CD11c-APC, F480-APC, F480-PE, Ly6G-APC (RB6-8C5), CD4-FITC, CD8-APC, CD40-PE, CD80-PE, CD86-PE, MHCII-PE, CD69-PE, CD40L-PE, CD44-PE, CD62L-FITC, and their corresponding isotypes antibodies (rat IgG2a-FITC, rat IgG2a-PE, rat IgG2a-APC, rat IgG2b-FITC, rat IgG2b-PE, rat IgG2b-APC, rat IgG2b-PECy5.5, rat IgG2a-Alexa647, Armenian hamster IgG-APC, Armenian hamster IgG2a- PE, Armenian hamster IgG2b-PE, Fc blocking antibodies, and ELISA kits for cytokines (IFNγ, TNFα, TGFβ, IL-12p40/70, IL12p70, IL23, IL-4, IL6, and IL-17A/E ). Proteome Profiler A was from R&D System (Minneapolis, MN), anti-Profiler A and Bio-Rad Molecular Imager® Gel Doc™ were from Bio-Rad (Hercules, CA). Anti- CD3 and CD28 antibodies were from Bio legend (San Diego, CA), Liberase Cl was from Roche (Indianapolis, IN). DNase and human Gaucher spleen GCs were from Sigma (St. Louis, MO). GMCSF and MCSF were from Peprotech (Rocky Hill, NJ). Anti-CD11c, anti-CD11b, anti-CD4, and anti-CD8 microbeads were from Miltenyi Biotec (Auburn, CA). C12-GC standards were from Matreya, LLC (Pleasant Gap, PA) or Avanti Polar lipids, Inc. (Alabaster, Alabama).
The D409V/null mice (9V/null) and WT controls were of the mixed background FVB/C57BL/6J/129SvEvBrd (50:25:25)  and were 24–28 weeks of age. Mice were maintained under pathogen-free conditions. Animal care was provided in accordance with National Institute of Health guidelines and was approved by Cincinnati Children’s Hospital Medical Center IACUC.
Spleens, livers, lungs, bone marrow and blood samples from WT and 9V/null mice were removed aseptically. Single cell suspensions from spleen were obtained by grinding and filtration through a 70-micron cell strainer. Similar suspensions from liver and lung were obtained from minced pieces that were treated with Liberase Cl (0.5mg/mL) and DNase (0.5mg/mL) in RPMI (45min, 37°C). Blood mononuclear cells were obtained after red blood cell (RBCs) lysis. For bone marrow cells, femurs, tibias, and humeri were flushed with sterile phosphate buffered saline (PBS), followed by RBC lysis (155 mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA), passage through a strainer, and pelleted by centrifugation at 350 g. Viable cells were counted using a Neubauer chamber and trypan blue exclusion. DCs, M, CD4+ T lymphocytes, and CD8+T lymphocytes were purified from single cell suspensions of liver, spleen, and lung using CD11c, CD11b, CD4 (L3T4), and CD8a (Ly2) microbeads according to the manufacturer’s protocol.
Bone marrow cells were used to generate M as previously described [27, 28]. Briefly, fresh bone marrow cells were stimulated with MCSF (10 ng/ml) in complete Dulbecco modified Eagle medium (FBS 10% + 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES and 1 mM sodium pyruvate). Cells were seeded in 6 well tissue culture plate and incubated at 37°C in a 5% CO2 atmosphere. Five days after seeding the cells, supernatants were discarded and the attached cells were washed with 10 ml of sterile PBS. Ten ml of ice-cold PBS were added to each plate and incubated at 4°C for 10 minutes. The macrophages were detached by gently pipetting the PBS across the dish. The cells were centrifuged at 200× g for 5 minutes and resuspended in 10 ml of above media. The cells were counted, seeded and cultivated in tissue culture plates 12 hours before any further experimental procedure.
DC was generated from mice bone marrow cells as previously discussed . Briefly, bone marrow was flushed from the long bones of the limbs and depleted of red cells with ammonium chloride. These bone marrow cells were plated in six-well culture plates (106 cells/ml, 3 ml/well) in RPMI 1640 medium supplemented with FBS 10% + 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES and 1 mM sodium pyruvate and 10 ng/ml recombinant murine GM-CSF At day 0,2,4, and 6 of culture, floating cells were gently removed and fresh medium was added. At day 7 of culture, nonadherent cells and loosely adherent proliferating DC aggregates were collected, counted, seeded and cultivated in tissue culture plates 12 hours before any further experimental procedure.
For identification of cellular phenotypes in organs, cells were suspended in PBS containing 1% bovine serum albumin. After incubation (15 min, 4°C) with the blocking antibody 2.4G2 (FcγRIII/I), cells were stained (45 min, 4°C) with antibodies for different cell types including 1) CD4 for T cells, 2) CD11b and F480 for M, 3) CD11b and CD11c for DCs, 4) CD11b and Gr1 for PMNs. Cells were also stained with the respective isotype antibodies as controls. Flow cytometeric analyses were performed on a FACS Calibur, where M were gated first by their typical FSC/SSC pattern based on F4/80 positivity and double stained for F4/80 and CD11b. Similarly, DCs were gated for CD11c positivity and double stained for CD11c and CD11b. PMNs were gated for Gr1 high positivity and stained for Gr1 and CD11b. The different APCs were also characterized for stimulatory and co-stimulatory molecules (CD40, CD80, CD86, MHCII). Flow cytometeric analyses of T lymphocytes were generated after gating lymphocytes from forward and side scatter and then identifying the CD4+ and CD8+T lymphocytes. CD4+ T cells were also stained for CD44, CD62L, CD40L, and CD69. A total of 106 events were acquired for each cells types of each organ. Absolute numbers of M DCs, PMNs and T cells in each organ of WT and 9V/null mice were determined by multiplying the total number of gated cells × percentage of CD11b+ and F480+ positive subsets for M, CD11chigh and CD11b+ positive subsets for DCs, CD11b+ and GR1high positive subsets for PMNs, and CD4+ and CD8+ positive subsets for T cells. FACS Calibur, LSRII flow cytometer (BD Biosciences, San Jose, CA), and FCS Express software (DeNovo) was used to analyzed these data.
WT (n=6) and 9V/null (n=6) mice were sacrificed and cardiac blood was collected in EDTA containing tubes. Neutrophil numbers in the circulation were determined using an automated hematology system (Hemavet 850, Drew Scientific, Oxford, CT).
Whole blood from WT (n=20) and 9V/null mice (n=20) was obtained by cardiac puncture. Serum was isolated after 1 hr. of incubation at RT followed by the dilution 1:10 with sterile PBS. Serum was used to identify IFNγ, IL-12p40/70, TNFα, TGFβ, IL-4, IL-6, and IL-17A/E by commercial ELISA kits and increased concentration of C-X-C chemokines i.e., keratinocyte-derived chemokine (KC) or chemokine (C-X-C motif) ligand 1 (CXCL1) and M-inflammatory protein-2 (MIP 2) or chemokine (C-X-C motif) ligand 2 (CXCL2) by using proteome profiler A and densitometery according to the manufacturer’s instructions. Bone marrow generated Ms were suspended in complete DMEM and DCs were suspended in complete RPMI medium to a concentration of 10 × 106 cells/ml. 200 μl per well of this cell suspension was placed in 96 well tissue culture plate and incubated for 48 h at 37° C with 5% CO2, The medium was then removed and used to measure KC and MIP2 by proteome profiler A and densitometry according to the manufacturer’s instructions. Duplicate assays were conducted thrice.
For T cell activation, anti-CD3 and CD28 antibodies (2 μg/ml) were dissolved in sterile PBS. These antibodies (200 μl/well) were coated onto 96-well plates, incubated overnight at 4°C, and washed twice in sterile PBS. Single cell suspensions (2×105 cells/well) from liver, spleen, and lung of WT and 9V/null mice were plated. Supernatants harvested 96 hrs. after stimulation with anti-CD3/CD28 antibodies were used to determine the concentrations of IFNγ, IL-12p40/70, IL12p70, IL23, TNFα, IL-17A/E, IL-6, and IL-4 and cells were used to measure the expression of CD40L by using FACS staining with antibodies to CD40 L.
To evaluated increased expression of CD40 and CD40L on 9V/null M, DCs, and CD4+T cells, WT and 9V/null bone marrow generated M and DCs were in vitro stimulated with IFNγ (50 ng/ml). Also, purified WT and 9V/null splenic CD4+T cells were stimulated with antibodies to CD3/CD28 for 96 hrs. Cells were harvested 96 hrs. after stimulation with IFNγ and anti-CD3/CD28 antibodies and used to measure expression of CD40 and CD40L by FACS staining.
A 1:2.5 ratio of dendritic cells (CD11c+ CD11b+) and T cells (CD4+ T cells) purified from WT and 9V/null liver, spleen, and lung were stimulated in the presence and absence of GC (100 ng/ml) for 96 hrs. in complete medium and the cells used for FACS staining with CD11b, CD11c, CD4, CD40, and CD40L markers. The respective supernatants were used to determine the concentrations of IFNγ, IL-12p40/70, IL12p70, IL23, TNFα, IL-17A/E, IL-6, and IL-4.
GCs were extracted from purified M, DCs, CD4+T cells, and CD8+T cells from liver, spleen, and lung, as well as PMNs, and bone marrow generated DCs and M of WT and 9V/null mice. GCs were quantified by ESI-LC-MS/MS using a Waters Quattro Micro API triple quadrapole mass spectrometer (Milford, MA) interfaced with Acquity UPLC system. Calibration curves were built for the GC species (C16:0, C18:0, C24:1) using C12-GC as standard. Quantification of GCs with various fatty acid chain lengths was realized by using the curve of each GC species with closest number of chain length. The total GCs in the liver, spleen, and lung were normalized to 1×106 cells.
One-tailed Students t-test (two groups) or analysis of variance (ANOVA) for multiple groups was used to determine significance (Prism Graph Pad™). Non-significant (ns), p>0.05; one asterisk, p<0.05; two asterisks, p<0.01; and three asterisks, p<0.001 are used in the figures.
M populations from liver, spleen, lung, and BM of WT and 9V/null were compared. Increased numbers of CD11b+ F4/80+ positive cells were observed in 9V/null liver (2-fold), spleen (6.1-fold), and lung (3.8-fold) (p<0.001) (Fig. 1A and Supplementary Fig. 1A), but not in bone marrow (p>0.05). Matching isotypes (Rat IgG2b and Rat IgG2a) for CD11b+ F480+ positive cells were negative (Supplementary Fig. 1B). Positivity for stimulatory and co-stimulatory (CD40, CD80, CD86, MHC-II) molecules was assessed with purified M (1×105) from liver, spleen, lung, and bone marrow. Significantly higher (p<0.001) numbers of CD40, CD80, CD86, and MHC-II positive cells were observed with M from 9V/null liver (Fig. 1B and Supplementary Fig. 1C) and lung (Fig. 1D and Supplementary Fig. 1E). 9V/null splenic M also had significantly (p<0.001) increased percentages of CD80 and CD86 co-stimulatory molecules, whereas CD40 and MHC II did not reach significance levels (Fig. 1C and Supplementary Fig. 1D). Similarly, 9V/null BM had significantly increased (p<0.001) percentages of CD40 and CD86, whereas CD80 and MHC II did not reach significance levels (Fig. 1E and Supplementary Fig. 1F). M purified from WT and 9V/null mice organs were analyzed for GC species, i.e., those with different fatty acid acyl chain compositions. Liver and splenic M from 9V/null mice showed (p<0.05-0.001) increases of GCs: 16-0, 22-0, 24-1 and 24-0 (Fig. 1F and and1G).1G). 9V/null lung M showed similar increases in 16-0, 24-1, and 24-0 (p<0.001) (Fig. 1H). Glycosphingolipids from MCSF generated 9V/null bone marrow M showed increased accumulation of GC species: 16-0 (p<0.05), 18-0 (p<0.05), 24-1 (p<0.001), and 24-0 (p<0.001) (Fig. 1I).
DC populations from the liver, spleen, lung, and BM of 9V/null were compared to WT. CD11chigh, CD11b+ DCs from 9V/null mice were increased in liver [15.1-fold (p<0.001)], spleen [2.5-fold (p<0.001)], lung [9.3-fold (p<0.001)], and bone marrow [1.7-fold (p>0.05)] (Fig. 2A and Supplementary Fig. 2A). There was no staining of identical isotypes (rat IgGb and rat Armenian hamster IgG) for CD11c+ CD11b+ positive cells in either organ (Supplementary Fig. 2B). Analyses of 9V/null DCs for stimulatory and co-stimulatory (CD40, CD80, CD86, MHC-II) molecules showed significantly increased positivity (p<0.001) in liver (Fig. 2B and Supplementary Fig. 2C), spleen (Fig. 2C and Supplementary Fig. 2D), lung (Fig. 2D and Supplementary Fig. 2E), and GMCSF generated bone marrow DCs (Fig. 2E and Supplementary Fig. 2F). Purified DCs, from WT and 9V/null mice tissues were also analyzed for GC species. GC species from 9V/null liver and splenic DCs showed increased accumulation: 16-0 (p<0.001) and 24-0 (p<0.001) (Fig. 2F and G). GCs from DCs of 9V/null lungs (Fig. 2H) and bone marrow (Fig. 2I) showed increased accumulation of 16-0, 22-0, 24-1, and 24-0 (p<0.001).
CD4+ T cell and CD8+ T cell phenotypes were evaluated in liver, spleen, lung, and bone marrow. Significantly increased CD4+ T cells were found in the liver (p<0.001), spleen (p<0.001), and lung (p<0.01) of 9V/null mice, but not in bone marrow (Fig. 3A and Supplementary Fig. 3A). None of these organs showed such significant (p>0.05), increased CD8+ T cells population in 9V/null mice (Fig. 3B). There was no staining of identical isotypes (rat IgG2b and rat IgG2a) for CD4+ T cells and CD8+ positive T cells in any of these organs (Supplementary Fig. 3B). To determine whether T cells are activated in 9V/null mice, CD4+ T cells and CD8+ T cells from liver, spleen, and lung were assessed for positivity with CD69 and CD40L. CD69 positive CD4+ T cells were increased (p<0.001) in spleen, but not in other organs (Fig. 3C and Supplementary Fig. 3C). CD40L positive CD4+ T cell numbers were increased (p<0.001) in liver, spleen, and lung of 9V/null mice (Fig. 3D and Supplementary Fig. 3D). However, CD8+ T cells did not show positivity (p>0.05) for CD69 and CD40L antibodies in any of these organs from 9V/null mice (Supplementary Fig. 3E–F). CD4+ T cells from these organs were also assessed for the presence of naïve and memory T cells by evaluating their positivity with CD44 and CD62L. None of the 9V/null organs had significant changes (p>0.05) in naïve (CD62L high CD44 int) or memory (CD44high CD62L int ) CD4+T cell populations (Supplementary Fig. 4A–L).
CD4+T cell GCs were analyzed using purified cells from liver, spleen, and lung of WT and 9V/null mice. In 9V/null liver, CD4+ T cells showed increased GC species: 16-0 (p<0.001), 18-0 (p<0.05), 20-0 (p<0.001), 22-0 (p<0.001), 24-1 (p<0.001), and 24-0 (p<0.001); the other species did not show any differences (Fig. 3G). In 9V/null spleen, CD4+ T cells showed increased GC species: 16-0, 20-0, 22-0, 24-1, 24-0 (p<0.001), and 18-0 (p<0.05); the other species i.e. 22-1, 26-1, and 26-0 did not show any differences (Fig. 3H). In 9V/null lung, CD4+ T cells showed increased GC species: 16-0 and 24-0 (p<0.001) and 24-1(p<0.05); the other species did not show any differences (p>0.05) (Fig. 3I). Additional analyses showed that the total GCs in liver, spleen, and lung CD8+T cells were 6–29 fold increased compared to WT.
APCs and T cells of 9V/null mice were increased in numbers and had excess GC storage. Also, enhanced positivity was present for stimulatory and co stimulatory molecules. Thus serum levels of cytokines were evaluated in 9V/null mice. The cytokines i.e., IFNγ (Fig. 4A), IL-12p40 (Fig. 4B), TNFα (Fig. 4C), IL-17A/F (Fig. 4D), IL-6 (Fig. 4E), and TGBβ (Fig. 4F) were increased (p<0.01-p<0.001) in 9V/null sera. In contrast, IL-4 was not (Fig. 4G (p>0.05).
Increased positivity of 9V/null APCs and CD4+T cells for stimulatory and co-stimulatory molecules suggested their joint cooperation to cause T cell activation and induction of Th1 and Th17 cytokine production in 9V/null mice. To explore this, mononuclear cells from lung, liver, and spleen were stimulated with the CD3/T cell receptor (TCR) complex and the CD28 co-stimulatory molecule. In general, T cell initiation requires two signals from APCs. The first signal comes from ligation of T cell receptor and the major histocompatibility complex (MHC) antigen presented on the surface of APCs. The second signal is via additional co-stimulatory molecules, including the interaction between the CD28 family of T cells and B7 molecules which is CD80 (B7-1) and 86(B7-2) expressed on the APCs responsible for activation of T cells effector functions [27, 29]. T cells incubated in medium alone produced no cytokines (data not shown). Crosslinking 9V/null liver, spleen or lung cells with antibodies to CD28/CD3 led to the increased production of IFNγ, TNFα, IL-12p40, IL12p70, IL23, IL-6, IL-17A/F (p<0.001–p<0.01) (Figs. 5A–G). In contrast, the Th2 cytokine IL-4 was not increased (Fig. 5H).
Since 9V/null mice have increased CD40 and CD40L on respective APCs and T cells, in vitro stimulation of APCs and T cells were assessed for their ability to amplify their CD40 and CD40L positivity. To evaluate this, IFNγ was used to stimulate M and DCs. Antibodies to CD3 and CD28 were used to stimulate splenic CD4+T cells. Also, GC was used to stimulate DC and T cells in co-culture experiments. Bone marrow generated M and DCs of 9V/null mice showed increases (p<0.010–p<0.05) in the CD40 expression, which was maximally stimulated (p<0.001) by IFNγ (Fig. 6A–B). Unstimulated 9V/null CD4+T cells showed little increase (p>0.05) in CD40L expression, but this was markedly enhanced (p<0.001) by stimulation with antibodies to CD3 and CD28 (Fig. 6C). Unstimulated WT and 9V/null DC-T cell co-cultures did not show differences (p>0.05- p<0.05) in the positivity of CD40 on DCs and CD40L on CD4+T cells. Also, the in vitro addition of GC to WT and 9V/null DC-T cell co-cultures led to increased percentages (p<0.001-p<0.001) of CD40 positive DC (CD11c+CD11b+) and CD40L positive CD4+ T cells from several tissues (Figs. 6D–G).
CD40-CD40L interaction between APCs and T cells causes the development of Th1 and Th17 immune responses [28, 30]. 9V/null mice APCs and T cells showed spontaneous as well as IFNγ, CD3/CD28 and GC induced increased positivity of CD40 and CD40L, as well as increased production of Th1 and Th17 cytokines. The effects of in vitro addition of GC on DCs and T cells on the induction of Th1 and Th17 cytokine production in 9V/null mice were evaluated. GC addition led to the production of IFNγ (Fig. 7A), TNFα (Fig. 7B), IL-12p40 (Fig. 7C), IL12p70 (Fig. 7D), IL23(Fig. 7E), IL-6 (Fig. 7F), and IL17A/F (Fig. 7G), and decreased generation of IL-4 (Fig. 7H) (p<0.001–p<0.01) in DC-T cell co-culture supernatants of 9V/null cells.
IL-17 has been implicated in induction of mediators that promote granulopoiesis and selective tissue recruitment of PMNs [31–33]. 9V/null CD3 coating as well as DC-T cell co-cultures experiment showed increased concentrations of IL17. Thus, liver, spleen, lung, and bone marrow PMNs were determined: PMNs gated on Gr1high and CD11b positivity showed modest increased percentages in liver [2-fold, (p<0.01), spleen (1.5-fold, p<0.01), and bone marrow (1.3-fold, p<0.001] (Supplementary Fig. 5A–B). This was not the case in the lung (p>0.05). Matching isotypes (rat IgG2bFITC and rat IgG2bAPC) for CD11b+ + Gr1+ PMNs were negative (Supplementary Fig. 5C). Blood counts and FACS staining confirmed increased numbers of PMNs [2–4 fold, (p<0.001)] in blood of 9V/null mice (Supplementary Fig. 5D–E). In addition with the IL17, increased concentration of C-X-C chemokines i.e., KC or CXCL1 and MIP 2 or CXCL2 in sera (p<0.01-p<0.001) and culture supernatant of both M (p<0.01) and DC (p<0.001) implicate the contribution of these elements in the recruitment of PMNs into GCase deficient tissues (Supplementary Figs. 5F–H). GC species from WT and 9V/null mouse bone marrow derived PMNs showed significantly less increases of 16-0, 22-0, 24-1, and 24-0 GCs (p<0.001) than those in 9V/null lung M and DCs The other GC species showed minor differences (p>0.05) (Supplementary Fig. 5I).
To gain insight into the pathogenesis of Gaucher disease, the present studies characterized the nature and activation states of immune-related cells isolated from several involved visceral organs. Increased frequencies of M, PMNs, DCs, T cells, and B cells (unpublished data) were shown in 9V/null mice. These immunological cells were associated with excess of GC, increased production of Th1/Th17 cytokines, and differential positivity for stimulatory and co-stimulatory molecules in several visceral tissues of 9V/null mice. This suggests that excess GC induces APCs and T cell stimulatory and co-stimulatory molecules, respectively, with consequent production of Th1, Th17 cytokines and KC/CXCL1, MIP2/CXCL2 chemokines to cause PMN recruitment to propagate the disease state.
Ms have been the major focus of the pathophysiology of Gaucher disease because of their unique histologic appearance and their massive GC contents. This excessive substrate accumulation leads to inflammatory responses and visceral organ abnormalities by undefined mechanisms . However, other immunological cells and their activation states have been incompletely characterized. Here, the repertoire of immune cells with excess GC accumulation is expanded to also include DCs, PMNs and CD4+T cells. Furthermore, the GC species accumulation in M, DCs, and CD4+T cells from liver and lung, i.e., tissues with preferential C16 or C24 contents, respectively, and increases in stimulatory and co-stimulatory molecules (CD40, CD80, CD86, MHC-II, CD40L, and CD69) were different compared with such cells from spleen and bone marrow. 9V/null M, DCs, and CD4+T cells showed further increased expression of CD40 and CD40L upon activation with IFNγ, antibodies to CD3/CD28, and GC stimulation and similar cytokines profiles sera vis-à-vis increases of IFNγ, IL-12p40, IL12p70, IL23, TNFα, TGFβ, IL6, and IL-17. The enhanced positivity for stimulatory and co-stimulatory molecules of 9V/null APCs and T cells as well as their enhanced production of IL-12/IFN-γ and IL-6/IL-17 support their activation in 9V/null model mice. These results implicate a significant activation of major components of the immune system as a result of defective GCase activity and accumulation of different GC species in many cell types.
CD3 and anti-CD28 antibodies stimulated mononuclear cells from 9V/null liver, spleen, and lung showed increased secretion of APC-derived cytokines, i.e., IL-12 or IL-6, indicating the involvement of CD40-mediated activation of APCs and their interaction with CD40L upregulated T cells. APCs and their involvement in T cell activation and co-stimulation, via CD3/CD28 ligation, were evaluated by in vitro analyses of GC effects on induction of DCs and T cell Th1 and Th17 cytokine production. Indeed, GC in DC-T cell co-cultures induced production of T cell and APC-derived cytokines (IFNγ, IL-12p40, IL12p70, IL23, TNFα, TGFβ, IL6, and IL-17) and increased positivity for CD40 and CD40L. These results indicate APC and T cell interactions are important for cytokine-mediated inflammation in 9V/null mice.
In addition, the unexpected increases in Th1 and Th17 effector cells suggest their cooperativity in an activation of the innate immune response as indicated by a few previous studies of WT cells [34, 35]. In particular, the enhanced numbers of GC loaded neutrophils in various visceral tissues of the 9V/null mice was unexpected since this is not a major component of Gaucher disease histology. These results indicate that the autocrine effect of IL-17 on neutrophil recruitment is significant and is differentially expressed in liver, spleen, bone marrow, and blood. Their role in Gaucher disease propagation will require further investigation. These results do support a differentiated cellular metabolism of various GC species in different cell types and organs with potentially differing effects on APCs and T cell interactions and enhancement of the IL-12p40, IL12p70, IL23, IFN-γ as well as TNFα, IL-6, and IL-17 pathways in GCase defective mice.
Increase of damage associated molecular patterns (DAMPs) including endogenous alarm signals as well as pathogen associated molecular patterns (PAMPs) have been recognized in many immunological cells including DCs, M, and PMNs which trigger innate and adaptive immune inflammation in several diseases [36–40]. Our results indicate that presence of excess GC species, particularly C16 and C24 causes the upregulation of stimulatory and co stimulatory molecules (CD40, CD80, CD86, MHC-II, CD40L, CD69) on corresponding APCs (i.e., M, DCs) and T cells of 9V/null mice. This then leads to T cell activation, and Th1/Th17 responses. These immune responses correlate with excess accumulation of C16 and C24 species of GC, thereby potentially implicating these lipids as damage associated molecular patterns that likely trigger an enhanced inflammatory status in Gaucher disease.
This concept is supported by the GC mediated activation of 9V/null CD4+T cells by ex vivo experiments. This process is mediated by 1) ligation of T cell receptor and the MHC-II present on the APCs, 2) interaction between CD28 family of T cells and B7 molecules, i.e. CD80 (B7-1) and 86(B7-2), expressed on APCs and more importantly, 3) the ligation of APC CD40 and T cell CD40L. The latter has been found in several animal models of chronic inflammation, e.g. arthritis, graft versus host disease, transplant rejection, lung inflammation, and multiple sclerosis [41–43]. Here, GC added to cultures of 9V/null DC and CD4 T cells led to increased percentages of CD40 positive DCs and CD40L positive CD4+T cells, and increased Th1 and Th17 cytokines(IFNγ, IL-12p40, IL12p70, IL23, TNFα, TGFβ, IL6, and IL-17) relative to IL-4.
IL-12 and IL-23 are critical for development of naïve and memory T cells, respectively [44–46]. Despite increased secretion of IL12 and IL23 both naïve and memory CD4+T cells percentages were similar to WT in all 9V/null tissues. Much higher levels of IL-23, compared to IL-12, were detected in both anti- CD3/CD28 antibodies coated cells and GC stimulated DC- CD4+T cells of 9V/null mice. This observation strongly supports the predominant role of IL-23 in mediating Th17 cell response. Notably, the GC-mediated DC-T cell interaction in promoting the Th1 and Th17 responses in 9V/null mice may also relate to its efficiency in inducing the CD40L expression, as CD40-CD40L crosstalk is important to the Th1 and Th17 responses [30, 47–50]. These results support GC as promoting the engagement of CD40 through T cell CD40L; This is a critical step for T cells activation and up-regulation of Th1 and Th17 immune responses [30, 43, 51, 52]. IL-12 cytokine initiates Th1 responses, whereas IL-6 and IL23 lead to Th17 responses [53–57]. The increases in IFNγ, IL12p40, IL12 p70, IL23, IL6, and TGFβ, observed in the 9V/null mice implicate excess GC in Th1 and Th17 immune stimulation.
A model is suggested in which excess GC (potentially of particular species) in APCs, i.e., M, and DCs, and CD4+T cells causes their activation and positivity for stimulatory and co-stimulatory molecules (CD40, CD80, CD86, MHCII, CD69, CD40L). These APCs through the CD40 and B7 family molecules and T cells through the CD40L and CD28 molecules (Supplementary Fig. 6A–B) interact and trigger enhanced responses of the Th1 and Th17 family cytokines (Supplementary Fig. 6C). In addition, with the production of IL17 by the combined activity of APCs and T cells, initial M and DC activation resulting from excess GC directly triggers a vicious cycle for the release of PMN attracting chemokines, i.e. KC/CXCL1 and MIP2/CXCL2, which causes the PMN migration into the different visceral organs (Supplementary Fig. 6D). These results highlight the importance of GC accumulation in APC and CD4+T cells in the initiation and propagation of various stimulatory and co-stimulatory molecules as well as enhanced Th1 and Th17 cytokines in 9V/null mice
The present data focuses attention on the here-to-fore unappreciated contributions of many immunological cells, the nature of their excess GCase substrates, and the induced inflammatory state in Gaucher disease. The enhanced inflammatory state in 9V/null mice is linked to excesses of several tissue specific GC, C16-0 and C24-0 species. However, important unresolved questions remain about how these GC species and/or glucosylsphingosine mechanistically affect the different immunological cells in Gaucher disease leading to the induction of Th1 and Th17 cytokine and recruitment of other cell types. These findings should foster the dissection of the essential functions and complex interactions immune perturbations in Gaucher disease.
Single cell suspensions prepared from different organs were analyzed by FACS. M that were positive CD11b+ and F480+ antibodies (A) and negative for matching isotypes i.e., Rat IgG2a and Rat IgG2b (B)in various tissues. (C–F) M from various tissues were purified using CD11b microbeads and stained for CD40, CD80, CD86, MHC-II and CD11b antibodies. WT (top) and 9V/null (bottom) mice. Data are from three independent experiments (mean±SD).
Single cell suspensions prepared from different organs were analyzed by FACS using double positivity for CD11c+ CD11b+ (A) and negative for their corresponding isotypes, Hamster IgG and Rat IgG2b (B). (C–F) DCs were purified using CD11c microbeads and gated by their higher positivity with CD11c and staining for CD40, CD80, CD86, MHC-II and CD11b from WT (top) and 9V/null mice (bottom). Data are from three independent experiments (mean±SD).
Single cell suspensions prepared from different organs were analyzed by FACS using CD4+T cells, CD8+ T cells (A), and negative for their corresponding isotypes, Rat IgG2b and Rat IgG2a (B). (C–D) CD4+T cells and (E–F) CD8+T cells from various tissues were purified using CD4 (L3T4) and CD8a (Ly2) T cell isolation kit stained for CD69 and CD40L antibodies. WT (left) and 9V/null (right) mice. Data are from three independent experiments (mean±SD).
Purified CD4+ T cells from liver, spleen, and lung cells of WT(filled column) and 9V/null mice (open column) were stained with antibodies to CD3, CD4, CD62L and CD44.(A–F) Absolute percentage of CD3+CD4+ CD62 Lhigh CD44int naïve T cells(A–C) and CD3+CD4+ CD44high CD62Lint memory T cells(D–F) are shown from various tissues (ns, not significant, p>0.05).(G–L) Dot plots were from one representativeexperiments which show percentage of naïve (CD62Lhigh CD44int) and memory (CD44high CD62L int) CD4+T cells in liver (G–H), spleen (I–J), and lung( K–L) of WT and 9V/null mice.
Single cell suspensions were from liver, spleen, lung, and bone marrow, and analyzed by FACS. PMNs were gated by Gr1 high positivity and stained GR-1 and CD11b markers. (A) Absolute numbers of Gr1high + CD11b+ positive PMNs in various tissues (ns, not significant; **, p<0.01; ***, p<0.001). Distribution of PMNs positive for Gr1+ CD11b+ (B) and negative for their corresponding isotypes, Rat IgG2b and RatIgG2b (C) in various tissues. (D–E) In some experiments, blood was collected and PMNs were determined using an automated system (Hemavet 850, Drew Scientific, Oxford, CT) and FACS as discussed in the material and method. (F–H)PMN chemokines, i.e. KC and M inflammatory protein-2 (MIP2)in sera, culture supernatant of M and DC of WT and 9 V/null mice were determined by Proteome Profiler A and densitometry (mean ± SD). (I) GC species in bone marrow-derived PMNs (ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001). WT (filled column), 9 V/null mice (open column), and data are from three independent experiments for all experiments (mean±SD) and group comparisons were by ANOVA.
(A–B) increase GC accumulation in 9V/null mice APCs (M, DCs), and CD4+T cells induces the expression of CD40, CD80, CD86, MHCII molecules on APCs, as well as CD69, and CD40L molecules on CD4+ T cells. (C) These stimulatory and co-stimulatory molecules cause the interaction between these two cells to induce Th1 and Th17 cytokines. (D) In addition with Th17 cytokines produced by combined activity of APCs and T cells initial M and DC activation due to excess accumulation of GC trigger the release of C-X-C chemokine i.e.; keratinocyte-derived chemokine (KC) orchemokine (C-X-C motif) ligand 1 (CXCL1) and M-inflammatory protein-2 (MIP 2) or chemokine (C-X-C motif) ligand 2 (CXCL2) which causes the recruitment of PMNs into Gaucher disease visceral organs.
The authors thank Professor Jörg Köhl for helpful discussion, and Stuart Tinch, Venette Inskeep, and Brian Quinn for their excellent technical assistance. This work is supported by a NIH grant (DK 36749) to GAG.
Disclosure/conflict of interest: The authors declare no competing financial interests.
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