Scientific Papers

NOD1 deficiency ameliorates the progression of diabetic retinopathy by modulating bone marrow–retina crosstalk | Stem Cell Research & Therapy

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Animals

Ethics approval for all animal procedures was obtained by the Animal Research Committee at Chongqing Medical University, and all procedures were conducted in accordance with established standard protocols for animal care. The animal studies presented in this manuscript were reported in compliance with the ARRIVE guidelines. In general, distinct genotypes of mice were utilized for this study: C57BL/6J wild type (WT, genotype: NOD1+/+:Ins2+/+), Akita (genotype: NOD1+/+:Ins2+/Akita), NOD1 knockout (NOD1−/−, genotype: NOD1−/−:Ins2+/+), and NOD1 knockout Akita double mutant (NOD1−/−-Akita, genotype: NOD1−/−:Ins2+/Akita). C57BL/6J WT and homozygous NOD1−/− mice were purchased from Cyagen Biosciences. Male heterozygous diabetic Akita mice were obtained from Shanghai Model Organisms Center. The mice were carefully bred and nurtured at the animal facilities of both Cyagen Biosciences and Chongqing Medical University. Male Akita mice were crossed with female NOD1−/− mice to generate the F1 generation, which was then bred with NOD1−/− mice to obtain NOD1−/−-Akita double mutants. Given that male Akita mice manifest a markedly more severe diabetic phenotype than do female mice [15], male mice were exclusively employed for this study. We used resource equation to calculate the sample size of mice. Based on the “3R” principle of animal experiments, the number of each group was finally determined.

Body weights and random blood glucose levels were recorded every 2 weeks. Diabetic mice were examined at the 6-month mark after diabetes onset. Glycated hemoglobin was quantified using the A1CNow + kit (Bayer HealthCare, Sunnyvale, CA, USA) at the conclusion of the study. Upon completion of the study, mice were anesthetized and humanely euthanized through inhalation of an overdose of isoflurane, followed by cervical dislocation.

Cell-based NOD1 activity reporter assay

HEK-Blue-mNOD1 HEK293 cells containing the murine NOD1 gene along with a reporter gene for secreted embryonic alkaline phosphatase were procured from InvivoGen (cat#: hkb-mnod1, San Diego, CA, USA). These cells were seeded and cocultured with HEK-Blue detection medium (cat#: hb-det2, InvivoGen, San Diego, CA, USA) alongside murine or human serum samples within a 96-well plate. Following 24 h of incubation, NOD1 stimulation was assessed utilizing a spectrophotometer at 630 nm.

Optical coherence tomography (OCT)

OCT scans were performed using the InVivoVue imaging system (Bioptigen, Inc., NC, USA) to monitor retinal morphological changes in vivo. Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg). After dilation of the eyes with a 1% tropicamide solution, multiple high-resolution horizontal and vertical images were acquired per lesion, as detailed previously [14]. OCT imaging was performed in high-definition circular scanning mode. Total retinal thickness was defined as the distance from the distal edge of the retinal pigment epithelium (RPE) layer to the proximal edge of the nerve fiber layer (NFL). The retinal layers were further divided into the inner retinal layer (IRL) (from the NFL to the outer edge of the inner core layer (INL)) and the outer retinal layer (ORL) (from the INL terminal to the RPE). Retinal thickness measurements were taken at various distances from the optic disk (OD)—specifically at intervals of 150, 300, 600, and 900 µm in dorsal–ventral and nasal–temporal sectors as previously reported [16].

Electroretinography (ERG)

The electrical responses of mouse retina were measured by ERG using a UTAS- E 2000 ERG system (LKC Technologies, MD, USA) as previously described with minor modifications [15]. After adaptation to the dark overnight, the mice were anesthetized with pentobarbital sodium (40 mg/kg), the pupils were dilated with 2.5% phenylephrine hydrochloride and 1% atropine sulfate ophthalmic solution, and the mice were placed on a temperature-regulated heating pad during recording periods. The recording ring electrode was placed in the center of the corneal surface, the grounding electrode was attached to the tail root, and the negative electrode was placed in the mouth of each mouse. The scotopic responses were assessed through 10 ascending stimulus intensities of light (0.0001, 0.001, 0.01, 0.1, 1, 3 cds/m2). After 3 min of adaptation to brightness, photopic responses were assessed utilizing 3 increasing light intensities (0.1, 1, 3 cds/m2). The light intensity was the average of three flashes 30 s apart. The next flash stimulus was used for OPS. The A-wave amplitude was measured from the first lowest peak to the prestimulus baseline after the flash began, and the B-wave amplitude was measured between the first lowest peak and the first peak. The oscillating potentials (OPs) were automatically filtered by ERG software. The latency and amplitude of OP1-3 were measured. For the flicker ERG, standard flash luminance with frequency (12 Hz) was obtained without any background illumination. The amplitude of flicker ERG is described as the lowest point of the waveform to the highest point.

Acellular capillary quantification

The retinal vasculature was prepared through trypsin digestion, as previously described [17]. Briefly, retinas were fixed overnight using 4% paraformaldehyde, followed by incubation in 3% trypsin 250 (BD Biosciences, San Jose, CA, USA) on a gentle shaker at 37 °C. After each 30-min trypsin incubation interval, the retinas were rinsed with PBS until the internal limiting membrane was digested, leaving only the retinal vasculature. Subsequently, the retinal vasculature was meticulously mounted onto a glass slide and stained using periodic acid-Schiff’s base (PAS)-hematoxylin (Sigma-Aldrich, MO, USA). A Leica DM300 microscope was employed to capture retinal images, with acellular capillaries quantified across 10 randomly selected fields by two independent blinded investigators.

Hematoxylin–eosin (HE) Staining

The right eyeball of mice was extracted and fixed in FAS eyeball solution (Servicebio, G1109) at room temperature for 24 h. Following fixation, the tissue was dehydrated using graded ethanol solutions and subsequently embedded in paraffin wax. Horizontal sections, 4 μm thick, were obtained from the optic nerve head using a microtome. These sections were then stained using standard H&E staining for morphological examination. The stratification of the entire retina, including the inner and outer layers, aligned with that observed in OCT imaging. Thickness measurements were taken using Image-pro Plus software.

Bone marrow transplantation

Chimeric mice were established by bone marrow transplantation with C57BL/6J WT and Akita mice, following a previously outlined method with slight adaptations [10, 18]. Briefly, 8-week-old male recipient mice were lethally irradiated at a total dose of 11 Gy, after which they were transplanted with 5 × 106 bone marrow cells from either NOD1+/+ or NOD1−/− donors of the same age. Four distinct groups were established: WT → WT, NOD1−/− → WT, Akita → Akita, and NOD1−/−-Akita → Akita. These groups were maintained until the study endpoint, which was set at 6 months after diabetes onset.

Flow cytometry

Bone marrow cells were collected from mouse femurs and lysed with ammonium chloride solution (ACS). Retinal samples from six mice within the same group were pooled and digested with collagenase D (2 mg/mL) in a 37 °C incubator for 45 min, adhering to established protocols [19]. Isolated cells were then stained with primary antibody cocktails.

For bone marrow HSPC populations, the following antibodies were used as previously described [8]: anti-mouse CD127 (BD Biosciences, Cat# 562419); anti-mouse lineage antibodies (Biolegend, Cat# 133311); anti-mouse Sca-1 (Biolegend, Cat# 108114); anti-mouse c-Kit (Biolegend, San Diego, CA, Cat# 105806); anti-mouse CD135 (eBioscience, San Diego, CA, Cat# 46-1351-82); anti-mouse CD34 (Biolegend, Cat# 119308) and anti-mouse CD16/CD32 (eBioscience, Cat# 14–0161–82). For bone marrow immune cells and monocytes, the following primary antibodies were used: anti-mouse CD45 (BD Biosciences, Cat# 560510); anti-mouse NK 1.1 (BD Biosciences, Cat# 551114); anti-mouse CD3 (BD Biosciences, Cat# 560771); anti-mouse CD115 (Biolegend, Cat# 135513); anti-mouse Ly6G (BD Biosciences, Cat# 551461); anti-mouse Ly6C (Biolegend, Cat# 128006); and anti-mouse CD11b (Invitrogen, Cat# RM2817).

For retinal cells, the antibody cocktails comprised anti-mouse CD45 (BD Biosciences, Cat# 560510), anti-mouse CD11b (Invitrogen, Cat# RM2817), anti-mouse Ly6G (BD Biosciences, Cat# 551461), anti-mouse Ly6C (Biolegend, Cat# 128006), and F4/80 (Biolegend, Cat# 123116). The samples were stained with the primary antibody cocktails at 4 °C for 30 min in the dark, followed by staining with Flexible Viability Dye for an additional 30 min at 4 °C. Cells fixed with 1% PFA were subsequently analyzed using the LSR II flow cytometer and assessed using FlowJo software (V10).

Colony-forming unit assay

ACS-lysed bone marrow cells were plated in MethoCult™ GF M3434 (STEMCELL Technologies) in accordance with the manufacturer’s instructions. A 1.1 ml MethoCult mixture was dispensed into a 35-mm petri dish. The final concentration of bone marrow cells in each dish for every group was 5 × 103.

Quantitative RT‒PCR

Retinal RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) and quantified using a Nanodrop (Thermo Scientific, Wilmington, DE). Subsequently, cDNA was synthesized with the iScript™ cDNA synthesis kit (Bio-Rad, Pleasanton, CA) following the manufacturer’s instructions. Quantitative PCR was conducted using SYBR Green with predesigned primers (Qiagen, cat#: 249900) on a StepOne Plus Real-Time PCR System (Life Technologies).

Isolation and culture of bone marrow-derived macrophages (BMDMs)

Mouse femur bone marrow was flushed using PBS. The extracted bone marrow cells were cultured and differentiated into BMDMs as previously described [20]. BMDMs from WT → WT, NOD1−/− → WT, Akita → Akita, and NOD1−/−-Akita → Akita chimeras were treated with either 50 μg/mL NOD1 ligand, PGN (Cat#: 77140, Sigma‒Aldrich, St Louis, MO), or an equivalent volume of vehicle for 24 h.

ELISA

The expression of neutrophil elastase (NE) and myeloperoxidase (MPO) in the retina was measured using ELISA kits from R&D Systems. Pooled retina samples (5 retinas per sample) were prepared at a 1:1000 dilution for analysis. CXCL1 and CXCL2 secretion in BMDM culture supernatant was quantified using R&D Systems ELISA kits. Absorbance readings for test samples and standards were recorded by a microplate reader at 450 nm and subsequently adjusted with readings at 540 nm, following the manufacturer’s instructions.

Human blood samples

This study was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University. Sample collection was conducted in strict accordance with the tenets of the Declaration of Helsinki and the ARVO statement on human subjects. Peripheral blood was collected from age- and sex-matched groups: healthy controls (HC, n = 12), individuals with diabetes without microvascular complications (DM-NC, n = 8), and individuals with diabetes with nonproliferative diabetic retinopathy (NPDR, n = 8). The diagnosis of DR was established by proficient ophthalmologists based on fundus images.

Data analysis and statistics

All results were statistically analyzed by GraphPad Prism software (version 8.3.0). Student’s t test was utilized for pairwise comparisons, and one-way or two-way ANOVA was employed for multiple comparisons, followed by appropriate post hoc tests. For nonnormally distributed data, the nonparametric Mann‒Whitney or Kruskal‒Wallis tests were utilized for statistical analysis. P values less than 0.05 (p < 0.05) were considered statistically significant. All data are presented as the mean ± standard deviation (SD).

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