Abstract
Persons with Down syndrome (DS, trisomy 21) have widespread cellular protein trafficking defects. There is a paucity of data describing the intracellular transport of IgG in the context of endosomal-lysosomal alterations linked to trisomy 21. In this study, we analyzed the intracellular traffic of IgG mediated by the human neonatal Fc receptor (FcRn) in fibroblast cell lines with trisomy 21. Intracellular IgG trafficking studies in live cells showed that fibroblasts with trisomy 21 exhibit higher proportion of IgG in lysosomes (~10% increase), decreased IgG content in intracellular vesicles (~9% decrease), and a trend towards decreased IgG recycling (~55% decrease) in comparison to diploid cells. Amyloid-beta precursor protein (APP) overexpression in diploid fibroblasts replicated the increase in IgG sorting to the degradative pathway observed in cells with trisomy 21. The impact of APP on the expression of FCGRT (alpha chain component of FcRn) was investigated by APP knock down and overexpression of the APP protein. APP knock down increased the expression of FCGRT mRNA by ~60% in both diploid and trisomic cells. Overexpression of APP in diploid fibroblasts and HepG2 cells resulted in a decrease in FCGRT and FcRn expression. Our results indicate that the intracellular traffic of IgG is altered in cells with trisomy 21. This study lays the foundation for future investigations into the role of FcRn in the context of DS.
Introduction
Down syndrome (DS, trisomy 21) is the most common survivable chromosomal aneuploidy in humans. The prevalence of DS in the US is approximately 1 per 700 live births, and there are ~200,000 people with DS living in the US 1. DS is caused by the presence of an additional whole or partial copy of chromosome 21 which results in genome-wide imbalances with a range of phenotypic consequences 2–5. The complex pathobiology of DS results in physical deficits and biochemical changes that can lead to multiple comorbid conditions 6. For example, some individuals with DS exhibit alterations in the immune system including abnormalities in the B- and T-cell compartments, abnormal immunoglobulin levels, and relatively poor immunoglobulin responses to certain vaccines 7–10. Individuals with DS are more susceptible to certain infections (e.g., infections in the upper respiratory tract). Children with DS are at a significantly higher risk (i.e., 8.7-fold increase) of contracting severe respiratory syncytial virus infections (RSV) 11. Prophylactic use of the IgG-based monoclonal antibody drug (mAb) palivizumab decreases the number of hospitalizations due to RSV infections in children with DS 12,13. The American Academy of Pediatrics and other pediatric associations (e.g., Canadian) still do not recommend prophylactic routine use of palivizumab in children with DS because of insufficient pharmacological data from this population 14,15. This gap of knowledge can be extended to the growing pipeline of mAbs that are being developed for the treatment of Alzheimer’s disease (AD) and acute myeloid leukemia, two prevalent comorbidities in persons with DS 6,16–18.
The human neonatal Fc receptor (FcRn) plays a key role in controlling the traffic and recycling of immunoglobulin G (IgG), mAbs, and albumin 19–22. This receptor binds to the Fc region of IgG molecules at acidic pH in early endosomes. FcRn-IgG complexes are protected from lysosomal degradation, and dissociate at more neutral pH at the plasma membrane during IgG recycling and transcytosis 23. FcRn is expressed in a variety of cell types and tissues, and contributes to the transport of IgG to target sites for the reinforcement of immunity 24. FcRn also acts as an immune receptor by interacting with and facilitating antigen presentation of peptides derived from IgG immune complexes 25. Whereas emerging roles for FcRn are becoming evident in conditions such as cancers and immune disorders, the potential contribution of FcRn to the complex pathobiology of DS remains to be defined 24–26.
Persons with DS have endosomal and lysosomal protein trafficking defects in various cell types 27,28. Endosomal-lysosomal abnormalities in individuals with DS are in part linked to the altered expression of the chromosome 21 gene APP 27–29. The extra copy of APP, the gene that encodes the Amyloid-beta precursor protein or APP, leads to Rab5 overactivation which results in endosomal-lysosomal protein trafficking and sorting defects including increased endocytic uptake, decreased lysosomal acidification, and increased protein misfolding28. The dynamics of FcRn-mediated endosomal sorting and trafficking are crucial to the salvage of molecules containing Fc-domains. There is a paucity of data describing the intracellular transport of IgG in the context of endosomal-lysosomal alterations linked to trisomy 21. The goal of this study was to examine the intracellular FcRn-mediated traffic and recycling of IgG in cells with trisomy 21.
Materials and Methods
Cell Culture
Human fibroblasts derived from donors with and without trisomy 21 were obtained from the Coriell Cell Repositories (Supplementary Table 1). Cells were cultured using MEM (Life Technologies), supplemented with 15% (v/v) fetal bovine serum in standard incubation conditions at 37 °C, 5% CO2, and 95% relative humidity.
IgG intracellular trafficking assays
1 × 104 cells per well were seeded into 96-well plates suitable for fluorescence microscopy and cultured for 24 h in growth medium. Growth medium was replaced with MEM supplemented with 0.05 μM LysoTracker® Red DND-99 (L7528, Life Technologies) and incubated for 30 min at 37 °C. After removal of LysoTracker, cells were supplemented with 0.25 mg/ml Alexa Fluor 633-human immunoglobulin G1 (hIgG1) and incubated for 60 min to allow IgG uptake. hIgG1 was removed, replaced with MEM, and cells were maintained in the incubator for up to 15 min before imaging. hIgG1 (Sigma-Aldrich, Cat# 400120, RRID: AB_437947) was previously labeled using an Alexa Fluor 633 Protein Labeling Kit (A20170, Molecular probes) following the manufacturer’s instructions. Incubations were performed in serum-free pre-warmed MEM without phenol red (51200038, Life Technologies).
Cell imaging was performed at 37 °C with 5% CO2 in a humidified incubator using a Dragonfly spinning disk confocal microscope (Andor Technology Ltd.) attached to a DMi8 base (Leica Microsystems). Images (16 bits, 0.096 μ per pixel) were obtained in sequential mode with a Zyla 4.2 PLUS sCMOS camera using a PlanApo 40 × 1.10 NA water immersion objective. Images from multiple fields (~10 fields/well) were taken, and independent incubations were performed in at least 3 different wells for each cell line.
IgG recycling assay
The IgG recycling assay was adapted from Grevys et al. 30. 2 × 105 cells per well were seeded into 24-well plates and cultured for 24 h in growth medium. Cells were washed twice with PBS, starved for 60 min at 37 °C in MEM, and incubated in pre-warmed MEM with 0.50 mg/ml hIgG1 (Sigma-Aldrich, Cat# 400120, RRID: AB_437947) for 60 min at 37 °C. The medium containing IgG was removed, and cells were washed three times with PBS. Pre-warmed MEM was added to the cells and incubated for 0 min (control), or 60 min at 37 °C to allow IgG recycling. The concentrations of IgG in cell lysates and supernatants after recycling were measured with an IgG Human ELISA Kit (88-50550, Invitrogen), following the manufacturer’s instructions.
Immunofluorescence
For studies in fixed specimens, fibroblasts (including cells expressing FcRn-GFP) were grown on glass coverslips, and incubated for 1h with 0.50 mg/ml hIgG1. After wash, cells were fixed in 4% paraformaldehyde in PBS for 20 min at 4 °C and permeabilized with 0.1% Triton X-100 and 200 mM glycine in PBS for 2 min at 4 °C. Samples were blocked in 3% bovine serum albumin (BSA)-PBS for 1 h, and then incubated for 2 h at room temperature with the following primary antibodies: rabbit anti-EEA1 (1:3000, Thermo Fisher Scientific Cat# PA1-063A, RRID:AB_2096819), rabbit anti-Rab11 (1:50, Thermo Fisher Scientific Cat# 71-5300, RRID:AB_2533987), and mouse anti-LAMP1 (1:100, Thermo Fisher Scientific Cat# 14-1079-80, RRID:AB_467426). After wash, samples were incubated with the following secondary antibodies for 1 h: Alexa 546-conjugated goat anti-rabbit IgG (1:1,000, Thermo Fisher Scientific Cat# A-11010, RRID: AB_2534077), and Alexa 647-conjugated goat anti-mouse IgG (1:1,000, Molecular Probes Cat# A-21235, RRID: AB_2535804). Controls for immunostaining specificity were included by replacing the primary antibody with a non-specific IgG isotype at the same final concentration (Figure S1). Samples were mounted onto glass slides using FluorSave (Calbiochem).
Images (8 bits) were obtained in sequential mode with a point scanning confocal microscope (Carl Zeiss, LSM 510 Meta) using a PlanApo 60 x 1.40 NA oil immersion objective. Images from multiple fields (~10 fields/condition) were taken. Identical microscope configuration and camera settings were maintained during image acquisition for conditions from the same experiment.
Quantitative image analysis
Image analysis was performed with the ImageJ software 31. Comparisons were performed by analyzing similar numbers of cells per condition with identical image processing parameters. Images were pre-processed for noisy pixel elimination and background subtraction using Gaussian smoothing followed by the rolling ball method in Fiji 32. Cellular regions of interest (ROIs) were created by segmentation from the differential interference contrast (DIC), bright field, or CellMask Plasma Membrane Stain channel. To quantify the average size and number of vesicles/mm2 per ROI, binary masks for each channel were obtained using automated global thresholding and the Analyze Particles approach (Particles size ≥ 0.019 μ2) followed by watershed transform to enable separation of contiguous vesicles. For colocalization analysis, Pearson’s correlation coefficient (PCC) was calculated for each ROI with the Colocalization test extension using Costes’ automated thresholding method within the Fiji software 31,32.
Cell transfections
For FCGRT and APP knock down, cells were transfected using Dharmafect 4 transfection reagent (T-2004-02, Dharmacon) following the manufacturer’s recommendations. Briefly, 2 × 104 cells per well were seeded into 24-well plates 24 h prior to transfection in antibiotic free media, and transfected with 5 nM siRNA. Cells were incubated at 37 °C in 5% CO2 for a total of 96 h, with replacement of transfection medium 24 h post-transfection. Non-Targeting siRNA Control Pool (NS-siRNA, D-001206-13-05), siRNA Pool targeting APP (M-003731-00-0005), and siRNA against FCGRT were obtained from Dharmacon. The siRNA against FCGRT was designed using the web-based software OligoWalk (FCGRT target sequence NM_001136019.2, Table S2)33.
For expression of proteins with fluorescent tags, cells were transfected using ViaFect transfection reagent (E4981, Promega) following the manufacturer’s recommendations. Briefly, 5 × 104 cells per well were seeded into 24-well plates 24 h prior to transfection in antibiotic free complete media, and transfected with 500 ng APP cDNA ORF Clone C-GFPSpark tag (HG10703-ACG, Sinobiological), or co-transfected with FCGRT cDNA ORF Clone C-GFPSpark tag (HG11604-ACG, Sinobiological) and B2M cDNA ORF Clone (HG11976-UT, Sinobiological). For control conditions, cells were transfected with an empty vector (PCMV6XL5, Origene). Cells were incubated at 37 °C in 5% CO2 for a total of 48 h.
Quantitative Real-time Polymerase Chain Reaction
Total RNA was isolated from cells using Trizol reagent following the manufacturer’s instructions (Thermo Fisher). FCGRT (alpha chain component of FcRn) and APP mRNA expression was analyzed with specific primers (Table S2). Total RNA (12.5 ng) was reverse transcribed and amplified with the iTaq Universal SYBR Green One-Step Kit (Bio-Rad). FCGRT, APP and the reference gene ACTB were amplified in parallel in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with the following cycling parameters: 50 °C for 10 min (reverse transcription), 95 °C for 1 min, followed by 44 cycles of 95 °C for 10 s, 60.5 °C for 20 s. Calibration curves were prepared to analyze linearity and PCR efficiency. qRT-PCR data were analyzed using the ΔΔCt method with CFX manager Software (Bio-Rad). The ΔCt method was utilized for determining the relative abundance of FCGRT and APP mRNA.
Immunoblotting
Cell lysates (20 μg) were denatured with NuPAGE LDS sample buffer containing NuPAGE sample reducing agent and protease inhibitor cocktail (Thermo Fisher Scientific), and boiled at 70 °C for 10 min prior to use. Proteins were separated by gel electrophoresis using NuPAGE Novex 4–12% Bis-Tris precast gels and transferred onto PVDF membranes using the iBlot Gel Transfer Device (Thermo Fisher Scientific). Membranes were blocked with 5% non-fat milk in 0.2% Tween 20-phosphate-buffered saline (PBS) for 1 h at room temperature and then probed with mouse monoclonal Anti-FcRn antibody (1:100, Santa Cruz Biotechnology Cat# sc-271745, RRID:AB_10707665), mouse monoclonal Anti-APP antibody (1:100, Thermo Fisher Scientific Cat# 13-0200, RRID:AB_2532993), or rabbit monoclonal Anti-APP antibody (1:100, Abcam Cat# ab133588, RRID:AB_2629851) overnight at 4 °C. Next, membranes were incubated with StarBright Blue 700 Goat Anti-Mouse IgG secondary antibody (1:2500, Bio-Rad, Cat# 12004159, RRID: AB_2884948), StarBright Blue 520 Goat Anti-Rabbit IgG secondary antibody (1:2500, Bio-Rad, Cat# 12005870, RRID: AB_2884949) and hFAB Rhodamine Anti-Tubulin Antibody (1:2500, Bio-Rad, Cat# 12004165, RRID: AB_2884950) for 1 h at room temperature. Immunoreactive bands were visualized in a ChemiDoc MP gel imaging system (Bio-Rad). Densitometric analysis was performed using Fiji (ImageJ) software 31.
Data processing and statistical analysis
Data processing was performed with Excel 2016 (Microsoft Office). Statistical analyses were performed with GraphPad Prism version 8. The D’Agostino & Pearson omnibus normality test was used to determine the normality of data sets. Comparisons between the means of two groups were performed with the Student’s t-test or Mann-Whitney’s U test for sets with normal and non-normal distributions, respectively. Spearman’s rank-order test was used for correlation analyses.
Results
Intracellular transport of IgG in the context of trisomy 21
First, optimal conditions for IgG uptake and intracellular trafficking were determined for fibroblast cell lines derived from donors with and without DS using live cell imaging and ELISA (Figure S2). After 1h of incubation with IgG, fibroblasts showed multiple IgG+ vesicles distributed intracellularly and actively transported in variable directions (Video 1). Cells with trisomy 21 exhibited intracellular IgG+ vesicles that were ~10% smaller in size (DS: 1.53 ± 0.30 μ2, NDS: 1.67 ± 0.36 μ2) and fewer in number (DS: 57.80 ± 16.52 vesicles/mm2, NDS: 64.17 ± 17.47 vesicles/mm2) in comparison to cells without trisomy 21 (Figure 1A and B). Co-localization analysis revealed that trisomic fibroblasts exhibited a ~11% increase in the distribution of IgG in lysosomes compared to diploid cells (PCC DS: 0.42 ± 0.16, PCC NDS: 0.38 ±0.17) (Figure 1A and D). The content of IgG in intracellular vesicles was reduced by ~9% in trisomic cells (DS: 184.0 ± 99.54 mean fluorescence/μ2, NDS: 203.6 ± 114.9 mean fluorescence/μ2) (Figure 1C). Cellular lysates from cells with and without trisomy 21 showed similar concentrations of IgG as measured by ELISA (DS: 24.72 ± 4.47 pg IgG/μg protein, NDS: 24.90 ± 9.17 pg IgG/μg protein). After 1h of IgG uptake followed by 1h of recycling, there was a non-significant ~55% decrease in IgG concentration in the supernatants from trisomic fibroblasts (DS: 15.29 ± 22.46 ng/ml, NDS 27.70 ± 44.45 ng/ml) (Figure 1 E).
Morphological features of compartments involved in the traffic of IgG in cells with trisomy 21
The number and size of vesicles positive for the following markers: a) Early Endosome Antigen 1 (EEA1, early endosomes), b) Rab11 (recycling endosomes), c) Lysosomal-associated membrane protein 1 (LAMP-1, lysosomes), and d) FcRn were determined in fixed cells using immunofluorescence and confocal microscopy (Figures 2 and 3). Lysosomes were also characterized in live cells with the marker lysotracker (Figures 1 and 2). Cells with trisomy 21 showed an increase in the number of Rab11+ vesicles (DS: 262.2 ± 104.9 vesicles/mm2, NDS: 203.2 ± 87.9 vesicles/mm2), and slightly bigger EEA1 (DS: 0.28 ± 0.04 μ2, NDS: 0.25 ± 0.05 μ2) and Rab11+ vesicles (DS: 0.31 ± 0.06 μ2, NDS: 0.25 ± 0.05 μ2). The size of LAMP1+ vesicles in fixed cells with trisomy 21 was smaller than in cells without trisomy 21 (DS: 0.31 ± 0.05 μ2, NDS: 0.34 ± 0.05 μ2). Confocal microscopy of live cells showed that lysosomes detected with lysotraker were ~28 % larger in trisomic fibroblasts in comparison to diploid cells (DS: 1.09 ± 0.41 μ2, NDS: 0.85 ± 0.37 μ2) (Figure 2).
On average, trisomic fibroblasts showed a ~23 % increase in the number of FcRn-GFP+ vesicles (DS: 269.1 ± 90.9 vesicles/mm2, NDS: 206.4 ± 80.9 vesicles/mm2) and a ~6 % increase in the size of FcRn-GFP+ vesicles in comparison to diploid cells (DS: 0.32 ± 0.08 μ2, NDS: 0.30 ± 0.05 μ2) (Figure 3A and B). The subcellular distribution of FcRn in early endosomes and lysosomes was similar in cells with and without trisomy 21 (Figure 3A and C). In cells with and without trisomy 21, the extent of colocalization of FcRn with the endosomal marker EEA1 was ~40 % (PCC DS: 0.40 ± 0.10, PCC NDS: 0.43 ± 0.12), and the extent of colocalization of FcRn with LAMP1 was ~10 % (PCC DS: 0.10 ± 0.05, PCC NDS: 0.09 ± 0.03).
FCGRT and APP expression in the context of trisomy 21
Trisomic fibroblasts showed higher FCGRT mRNA and FcRn protein expression in comparison to diploid cell lines derived from age- and sex-matched donors (Figure 4, Table S1). FCGRT mRNA expression was ~1.5 to 5-fold higher in trisomic cells, and on average, FcRn protein levels were ~3-fold higher in trisomic cells than in diploid cells (Figure 4A). Fibroblasts with trisomy 21 showed ~2-fold higher APP mRNA expression than diploid cells (DS: 2.14 ± 0.64 relative fold, NDS: 1.00 ± 0.34 relative fold) (Table S3). There was a significant positive correlation between the endogenous expressions of APP and FCGRT mRNA in diploid and trisomic cells (R2 = 0.97, Pearson r = 0.99, P < 0.001) (Figure 4B).
Impact of APP on the expression of FcRn
The impact of APP on the expression of FCGRT was investigated with two strategies. First, fibroblasts were transfected with an anti-APP siRNA pool (Figure 4C). APP knock down (APP mRNA expression <10%) resulted in a ~60% increase in the expression of FCGRT mRNA in the diploid cell line AG07095 (NDS control siRNA: 99.7 ± 6.6, NDS APP siRNA: 167.5 ± 22.6 % relative fold) and in the trisomy 21 cell line AG06922 (DS control siRNA: 99.7 ± 16.4, DS APP siRNA: 160.0 ± 6.22 % relative fold). The second strategy involved the overexpression of the APP protein in diploid cells. APP-GFP expression was confirmed by fluorescence microscopy and immunoblotting (Figure 4D and E). The specificity of APP and FcRn antibodies was examined by immunoblotting in lysates from non-transfected and transfected cells (Figure S3). Diploid AG07095 fibroblasts overexpressing APP-GFP showed a ~86 % decrease in the expression of FCGRT mRNA (NDS control: 100.0 ± 25.6, NDS APP-GFP: 14.3 ± 2.7 % relative fold), and a ~7 % decrease in FcRn protein expression (Figure 4D). These observations were extended by examining the interplays between APP and FcRn expression in HepG2 cells. HepG2 cells express relatively high basal levels of FcRn and APP as evidenced by immunoblotting (Figure S3). HepG2 cells overexpressing APP-GFP showed a ~40 % reduction in the expression of FCGRT mRNA (HepG2 control: 100.0 ± 13.3, HepG2APP-GFP: 60.8 ± 12.6 % relative fold), and a ~23 % decrease in FcRn protein expression (Figure 4E).
Impact of increased APP expression on the intracellular transport of IgG
Diploid fibroblasts over-expressing APP showed a ~20% increase in the distribution of IgG in lysosomes (PCC NDS control: 0.56 ± 0.14, PCC NDS APP-GFP: 0.67 ± 0.15) and a ~16% reduction in the intracellular content of IgG (Mean fluorescence NDS control: 28.54 ± 5.10, NDS APP-GFP: 23.85 ± 2.31), after 1h of incubation with IgG at 37 °C (Figure 5A, B and D). Diploid fibroblasts over-expressing APP-GFP showed no differences in the size (NDS control: 1.75 ± 0.47 μ2, NDS APP-GFP: 1.78 ± 0.49 μ2) and number (NDS control: 83.93 ± 22.50 vesicles/mm2, NDS APP-GFP: 79.18 ± 23.89 vesicles/mm2) of IgG+ vesicles in comparison to diploid fibroblasts not expressing APP-GFP (Figure 5C and D). The extent of colocalization between IgG+ vesicles and APP-GFP was ~40% (PCC: 0.43 ± 0.23) (Figure 5D).
Discussion
With the exception of neuronal phenotypes in DS, the impact of endosomal-lysosomal dysfunction on prominent receptor-mediated protein trafficking pathways has been scarcely documented 28,34,35. In this study, we analyzed the FcRn mediated-intracellular traffic of IgG in human fibroblasts with trisomy 21, a cellular model that recapitulates protein trafficking defects linked to DS 36,37.
FcRn is the only high affinity and pH specific receptor of IgG 23. Monomeric IgG internalized by fluid phase pinocytosis is protected from lysosomal degradation by pH-dependent binding to FcRn at endosomes (pH 5.5–6.0). FcRn then transports IgG in Rab11+ recycling endosomes back to the extracellular media or across cell layers in polarized cells 21,38. Our intracellular IgG trafficking studies showed that fibroblasts with trisomy 21 exhibit a higher proportion of IgG in lysosomes, decreased IgG content in intracellular vesicles, and a trend towards decreased IgG recycling in comparison to diploid cells (Figure 1). In general, recycling endosomes are tubulovesicular structures, and recent studies showed FcRn-mediated recycling of IgG in tubulovesicular transport carriers 21,39,40. Here, we documented ~10% decreases in size and number of IgG-containing intracellular vesicles in fibroblasts from donors with trisomy 21. In diploid cells, many of the IgG+ vesicles displayed a tubular-like shape with low overlapping to late endosomes/lysosomes. In contrast, IgG+ vesicles in trisomic cells were more puncta-like shaped, smaller than the tubular structures, and overlapped more frequently with late endosomes/lysosomes (Figure 1A). The increased expression of FcRn in trisomic cells did not result in increased IgG salvage from the degradative pathway (Figures 1D, E, and 4A, B). These observations suggest that increased FcRn expression in cells with trisomy 21 is a compensatory response for decreased FcRn activity. Fibroblast cell lines expressed relatively low levels of endogenous FcRn, and detection of FcRn expression by immunofluorescence was difficult (Figure S3). For this reason, an FcRn-GFP construct was used to analyze the subcellular distribution of FcRn 21,32. FcRn-GFP localized mainly in EEA1+ endosomes, and the distribution in sorting endosomes and LAMP1+ lysosomes was similar in trisomic and diploid cells (Figure 3 A, C). FcRn+ vesicles in fibroblasts with trisomy 21 were more numerous (~23% increase) and slightly bigger (~6% increase) than vesicles in diploid cells (Figure 3B). The subcellular localization of FcRn in fibroblasts was in line with previous studies documenting the distribution of FcRn in other cell types (transfected and non-transfected cells) 21,41,42. Our observations suggest that the sorting of IgG to recycling endosomes is decreased in cells with trisomy 21 with a consequent increase in lysosomal degradation.
Alterations in the morphology of endosomes have been described in primary fibroblasts, neurons, peripheral blood mononuclear cells, and lymphoblastoid cell lines from individuals with Down syndrome 36,37,43–45. Our comparative morphological analysis of IgG+ vesicles and subcellular compartments in fibroblasts with and without trisomy 21 revealed enlargement of subcellular organelles from the endosomal pathway, resembling the “traffic jam” previously described for DS and Alzheimer’s disease 45,46. We found that trisomic cells have larger EEA1+ endosomes (~12%), Rab11+ endosomes (~24%), and lysotracker-stained lysosomes (28%) than diploid cells (Figure 2). In agreement, other reports have noted a ~18% increase in the size of EEA1+ vesicles without significant differences in the number of this type of vesicles. Enlargement of lysosomes in fibroblasts from individuals with trisomy 21 has also been reported 45,47. Of note, the average size of LAMP1+ vesicles in fixed cells with trisomy 21 was smaller. It remains to be determined whether the observed differences reflect vesicular heterogeneity or result from microscopy approaches (i.e., fixed cells vs live cells), or a combination of both factors 48.
Several studies implemented receptor-mediated uptake approaches to examine endosomal defects in trisomic cells 37,45,47. There is limited information describing IgG traffic following internalization by fluid phase pinocytosis in the context of trisomy 21 21,49. Cataldo et al. documented increased endocytic fluid phase uptake of horseradish peroxidase in trisomic fibroblasts 37. We did not detect increases in intracellular IgG content or number and size of IgG+ vesicles during the IgG trafficking assays (Figure 1C, E). It is possible that under our experimental conditions, there is uptake of IgG followed by cellular recycling and re-uptake with no IgG accumulation. An alternative scenario involves increased IgG degradation during trafficking. This latter scenario is supported by our live cell imaging observations that showed increased distribution of IgG in lysosomes and decreases in number, size, and fluorescence levels of IgG+ vesicles in trisomic cells (Figure 1B, C, and E).
Evidence indicates that the increased expression of APP is linked to widespread cellular endosomal-lysosomal abnormalities in DS 27–29. The APP gene triplication contributes to the development of early-onset AD in DS 28. APP is processed in endosomes by the β-site APP cleaving enzyme 1 (BACE1) to form APP β-carboxyl-terminal fragment (APP-βCTF). In DS and AD, APP-βCTF pathologically over-activates Rab5 by recruiting APPL1, resulting in endosomal trafficking defects and abnormal endosome-mediated signaling 28,36. We observed that APP overexpression in diploid fibroblasts replicated the increase in IgG sorting to the degradative pathway observed for cells with trisomy 21 (i.e., ~20% increase in IgG into lysosomes and ~16% reduction in IgG content. Figures 1 and 5). In diploid fibroblasts overexpressing APP, the number and size of intracellular IgG vesicles remained unaltered, which suggests that other factors contribute to morphological alterations in trisomic cells in addition to increased APP expression44,45.
Analysis of endogenous expression of FCGRT and APP mRNA in fibroblasts derived from donors with and without trisomy 21 revealed substantial “inter-individual” variability in gene expression (APP range: ~1 to 5-fold, FCGRT range: ~1 to 9-fold). There was a positive correlation between APP and FCGRT mRNA expression levels in the group of diploid and trisomic cell lines (Figure 4B). This observation prompted us to further examine potential interplays between APP and FcRn expression. Notably, APP knock down increased the expression of FCGRT mRNA by ~60% in both diploid and trisomic cells (Figure 4C). Furthermore, overexpression of APP in diploid fibroblasts and HepG2 cells also resulted in a decrease in FCGRT and FcRn expression (Figure 4D and E). It is known that APP plays many roles and a growing amount of evidence suggests that APP may also act as a transcriptional regulator 50–52. Cleavage of APP-βCTF by γ-secretase results in the generation of Amyloid-β (Aβ) and APP intracellular domain (AICD) 53. Studies suggest that AICD regulates gene transcription, although the exact role that this fragment plays is still controversial because detection of endogenous AICD is difficult 52,54–56. It will be of interest to test whether the AICD fragment regulates the expression of FcRn. Points that merit further consideration are 1) whether the observed FCGRT-APP gene expression correlation extends to “inter-individual” comparisons involving various cell types and tissues from multiple donors, and 2) additional regulatory mechanisms that may impact FCGRT and FcRn expression in trisomic cells 57,58.
Our results suggest that the intracellular traffic of IgG is altered in cells with trisomy 21. This study lays the foundation for future investigations into the role of FcRn in the context of DS. For example, children with DS are susceptible to infections (e.g., recurrent respiratory infections) and many suffer from chronic immune dysregulation 59,60. FcRn plays an important role during the regulation of innate immune responses and immunosurveillance at mucosal sites 24. It is possible that alterations in IgG traffic extend to other cell types (e.g., cells of hematopoietic origin, epithelial cells, and endothelial cells) and could eventually limit the availability of IgG in specific tissues in individuals with DS. In addition, it is licit to hypothesize that augmented transport of IgG to the degradative pathway in dendritic cells with trisomy 21 could impact antigen presentation of peptides derived from IgG immune complexes (IgG IC) 25.
Conflict of Interest
All authors declare no competing interests.
Author Contributions
R.B. Cejas designed the study, performed experiments, analyzed data, and wrote the manuscript. M. Tamaño-Blanco performed experiments and analyzed data. J.G. Blanco conceptualized research, revised and edited the manuscript, provided resources and acquired funding. All authors reviewed the manuscript.
Acknowledgments
We acknowledge the excellent assistance and advise of Dr. Andrew McCall from the Optical Imaging and Analysis Facility at the-School of Dental Medicine, SUNY Buffalo. This study was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (award R21HD089053), the National Cancer Institute (award R21 CA245067), and the National Institute of General Medical Sciences (award R01GM073646).
Nonstandard abbreviations
- ACTB
- Actin beta
- AD
- Alzheimer’s disease
- AICD
- APP intracellular domain
- APP
- Amyloid-beta precursor protein
- APP-βCTF
- APP β-carboxyl-terminal fragment
- BACE1
- Beta-site APP cleaving enzyme 1
- BSA
- Bovine serum albumin
- DIC
- Differential interference contrast
- DS
- Down syndrome
- EEA1
- Early endosome antigen 1
- ELISA
- Enzyme-linked immunosorbent assay
- EV
- Empty vector
- FCGRT
- Fc fragment of IgG receptor and transporter
- FcRn
- Neonatal Fc receptor
- GFP
- Green fluorescent protein
- hIgG1
- Human IgG type 1
- IgG
- Immunoglobulin G
- LAMP1
- Lysosome-associated membrane glycoprotein 1
- mAbs
- Monoclonal Antibody drugs
- MEM
- Minimal essential medium
- mRNA
- Messenger RNA
- NDS
- No-DS
- NS-siRNA
- Non-Targeting siRNA
- PBS
- Phosphate-buffered saline
- PCC
- Pearson’s correlation coefficient
- PCR
- Polymerase chain reaction
- Rab11
- Ras-related protein Rab-11A
- RSV
- respiratory syncytial virus infections
- RNA
- Ribonucleic acid
- ROI
- Region of interest
- SD
- Standard deviation
- siRNA
- Silencing RNA