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Increased maternofoetal transfer of antibodies in a murine model of systemic lupus erythematosus, but no immune activation and neuroimmune sequelae in offspring
Department of Biomedicine, Aarhus University, DenmarkDANDRITE, Danish Research Institute of Translational Neuroscience, Aarhus University, 8000, Aarhus C, Denmark.
Increased maternofoetal transfer of antibodies in offspring born to autoimmune mothers.
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No elevated anti-dsDNA antibody levels in offspring born to autoimmune mothers.
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No significant changes in microglia morphology in all embryo transfer offspring.
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Absence of significant anti-dsDNA antibody levels in lupus prone BXSB/MpJ females.
Abstract
Maternally transferred autoantibodies can negatively impact the development and health of offspring, increasing the risk of neurodevelopmental disorders. We used embryo transfers to examine maternofoetal immune imprinting in the autoimmune BXSB/MpJ mouse model. Anti-double-stranded DNA antibodies and total immunoglobulins were measured, using allotypes of the IgG subclass to distinguish maternally transferred antibodies from those produced endogenously. Frequencies of germinal center and plasma cells were analysed by flow cytometry. Microglial morphology in offspring CNS was assessed using immunohistochemistry. In contrast to prior findings, our results indicate that BXSB/MpJ mothers display a mild autoimmune phenotype, which does not significantly impact the offspring.
Systemic lupus erythematosus (SLE) is a severe, multisystem autoimmune disease, characterized by the production of a range of autoantibodies. Among these, antibodies towards double-stranded DNA (dsDNA), especially of the immunoglobulin (Ig) G isotype, are robust diagnostic markers of SLE, and often correlate with disease severity (
Anti-dsDNA antibody isotypes in systemic lupus erythematosus: IgA in addition to IgG anti-dsDNA help to identify glomerulonephritis and active disease.
). However, diagnosis and accurate clinical assessment of SLE at an early stage of disease remains a challenge due to clinical and serological heterogeneity (
). Maternal SLE can have severe negative consequences for the offspring, the most well-described of which is neonatal lupus, a condition characterized by congenital heart-block caused by placental transfer of anti-Ro antibodies (
Serum biomarkers of inflammation, fibrosis, and cardiac function in facilitating diagnosis, prognosis, and treatment of anti-SSA/Ro-associated cardiac neonatal lupus.
Binding of anti-SSA antibodies to apoptotic fetal cardiocytes stimulates urokinase plasminogen activator (uPA)/uPA receptor-dependent activation of TGF-beta and potentiates fibrosis.
). In recent years, it has become clear that activation of the maternal immune system may also predispose offspring to a range of neuropsychiatric diseases (
Maternal immune activation (mIA) has been suggested to affect the offspring through the action of cytokines, transfer of autoantibodies, as well as maternofoetal microchimerism (
). During pregnancy, a natural maternal transfer of IgG antibodies to the foetus occurs across the placenta via the neonatal Fc receptor (FcRn), whereas class A immunoglobulins (IgA) are transferred via breast milk in the postnatal period (
Isolation from human placenta of the IgG transporter, FcRn, and localization to the syncytiotrophoblast: implications for maternal-fetal antibody transport.
). These maternally acquired antibodies are essential for neonatal immunity. However, transfer of pathogenic autoantibodies from SLE mothers to their foetuses may manifest in damage to different areas of the foetal anatomy such as the central nervous system and heart (
Most of the available murine models of SLE have a complex genetic background, making it difficult to separate inherited genetic factors from the maternal immune environment, and thus hampering the understanding of each component in isolation. This can be circumvented by embryo transfer, a method allowing for an uncoupling of genetic and environmental factors by transferring fertilized embryos from a female of one genetic background to a female belonging to another genetically distinct background. If appropriately designed, maternofoetal transfer of antibodies or cells can be detected by the presence of selected genetic markers from the mother found in the genetically distinctive offspring.
To study the impact of maternal autoimmunity on the offspring, we leveraged a murine model of SLE in which dams ostensibly develop autoantibodies, namely the BXSB/MpJ model (
). However, females of this strain experience an attenuated form of SLE, while males experience an accelerated form of the disease, due to the presence of an autoimmune accelerator locus on the Y-chromosome denoted Yaa. The accelerator locus originated from a translocation of a duplicated X-linked telomeric segment, including the gene coding for the endosomal RNA-sensor Toll-like receptor 7 (TLR7), which in males acts in synergy with possible autosomal autoimmune induction genes (
). As healthy control recipients, we used C57BL/6J dams. Hence, fertilized ova of normal, healthy DBA/2JRj mice were transferred into pseudopregnant dams of either the autoimmune BXSB/MpJ or non-autoimmune C57BL/6J strain. Naturally born BXSB/MpJ, C57BL/6J and DBA/2JRj offspring were included as additional controls.
In the present study, we focused on levels of immunoglobulin isotypes and anti-dsDNA antibodies as markers of autoimmune disease. The IgG class of immunoglobulins is in mice divided into IgG1, IgG2a, IgG2b, and IgG3 subclasses. Of these, IgG2a is the most important subclass in Th1 restricted responses associated with antiviral immunity or autoimmunity (
). We exploited the observation that in inbred lines of mice, the IgG2a subclass is segregated into two antigenically distinct allotypes termed IgG2A and IgG2C (
). Thus, whereas DBA/2JRj mice carry the IgG2A allotype, BXSB/MpJ and C57BL/6 J mice carry IgG2C instead. Hence, maternofoetal transfer can be determined by tracing the immunoglobulin allotype IgG2C, whereas the allotype IgG2A allows for assessment of endogenous antibody production in the offspring. We use the nomenclature of IgG2 non-capital “a” to signify the isotype, and capital “A” and “C” when referring to the allotypes, throughout.
We hypothesized that mIA causes maternofoetal transfer of anti-dsDNA and other antibodies, which may alter offspring development.
The aim of this study was to investigate the maternofoetal transfer of anti-dsDNA and IgG2C antibodies, assess cellular immune activation, and evaluate the effect on microglia morphology and activation in both the striatal and hippocampal cortex of embryo transfer offspring from the murine SLE model BXSB/MpJ.
2. Materials and methods
2.1 Mice
BXSB/MpJ (strain no. 000740) were purchased from The Jackson Laboratory, while DBA/2JRj and C57BL/6 JRj were purchased from Janvier Labs. The 564Igi line was kindly made available by Thereza Imanishi-Kari (Tufts University, Medford, Massachusetts), and obtained via the laboratory of Michael C. Carroll (Boston Children's Hospital, Brookline, Massachusetts) (
Mice were bred and maintained in the special pathogen free (SPF) animal facility at the Department of Biomedicine, Aarhus University. Both male and female offspring were used in the study. Mice were housed in a standard 12 h light/dark cycle with food and water ad libitum. The use of animals complied with the European Community guidelines and the Institutional guidelines of Aarhus University, and all experiments were approved by the Danish Animal Experimentation Inspectorate (protocol no.: 2017-15-0201-01319 and 2020-15-0201-00462).
2.2 Embryo transfers
DBA/2JRj donors were superovulated with fertility hormones, 5 IU of pregnant mare serum gonadotropin (PMSG) (PMSG-Intervet 5000 iu Powder and solvent for solution for injection, MSD Animal Health) and 5 IU of human chorionic gonadotropin (hCG) (Suigonan VET. powder and solvent for solution for injection, MSD Animal Health), injected at an interval of 46–48 h. After hCG injection, females were mated with proven fertile DBA/2JRj males overnight and checked for vaginal plug the following morning. Embryos at the two-cell stage, 1.5-day post-coitum (pc), were collected the day after plugs were detected, by taking out oviducts and flushing them with M2 media. The embryos were aseptically transferred to the oviduct of 0.5-day pc recipients through a dorsal incision. Recipients were females of proven fertility made pseudopregnant by mating the previous day to a vasectomized RjOrl:SWISS male of proven infertility. The following morning plug-positive females were used as recipients.
Female BXSB/MpJ recipients were 15–29 weeks of age at the time of embryo transfers, while C57BL/6 J recipients were 12–18 weeks. DBA/2JRj embryo donors were 3–8 weeks old at the time of mating.
Respectively, 80 and 51 fertilized DBA/2JRj embryos were transferred to ten BXSB/MpJ mothers and five C57BL/6 J mothers. Ten naturally reared DBA/2JRj mice were used as controls. All embryo transfers and controls were separated into five cohorts.
The transfer of DBA/2JRj embryos into BXSB/MpJ mothers resulted in 18 offspring, while transfer of DBA/2JRj embryos into C57BL/6 J mothers resulted in twelve offspring.
At weaning (28 days) the mice were housed five in each cage with food and water ad libitum. Embryo transfer offspring were between six and seven weeks of age when analysed and DBA/2JRj controls were six weeks. Adult control mice were between nine and 21 weeks of age and included eight DBA/2JRj, six C57BL/6 J, and four BXSB/MpJ mice.
2.3 Tissue processing
Animals were anaesthetized with isoflurane (Attane vet. 1000 mg/g, 055226) and bled retroorbitally to obtain blood samples for serological analysis (TRIFMA) (Fig. S1). They were then injected intraperitoneally with 100 μl of pentobarbital solution (Euthasol vet. 400 mg/ml, 450,009, diluted 1:10 in phosphate-buffered saline (PBS), PBSx10 (Lonza, BE17-515Q), w/o calcium, magnesium, or phenol red, diluted 10× with milliQ water). Following confirmation of terminal anaesthesia, an incision was made in the peritoneal wall. The spleen was retracted, clamped with a peang, and excised. A piece of the spleen was placed in optimal cutting temperature (O.C.T) medium (Tissue-Tek, Sakura Finetek, 4583) for cryopreservation, and the rest was placed in fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% v/v fetal bovine serum (Life Technologies, 10,270,106) and 1 mM ethylenediaminetetraacetic acid (EDTA) (Merck, 1.08418.0250)). The mesenteric and inguinal lymph nodes were harvested and placed in FACS buffer. The sternum was opened to allow access to the heart for cardiovascular perfusion. Mice were perfused with 10–15 ml PBS followed by 5–10 ml 4% paraformaldehyde (PFA) solution (4% w/v PFA in 0.1 M sodium phosphate buffer, pH 7.4), using a peristaltic pump.
Afterwards, the head was detached using scissors, and the skull exposed. The skull was cut along the sagittal suture and carefully removed with surgical forceps. A small spatula was used to separate the brain from the underlying tissue and cranial nerves connected to the skull. The brain was carefully extracted and placed in 4% PFA solution and stored at 4 °C. The following day, brain-hemispheres were separated, and one hemisphere was transferred to vials containing a 30% sucrose solution (30% w/v sucrose in 0.02 M sodium phosphate buffer, pH 7.4) and kept at 4 °C until cryosectioning, while the other hemisphere was frozen in O.C.T medium.
Prior to cryosectioning, the caudal part of the cerebellum was removed to ensure the brain orientation for coronal sectioning. Brains were then snap-frozen in O.C.T. medium and mounted onto a mould at −20 °C, and finally cut into 40 μm thick sections on a CryoStar NX70 cryostat. Sections were transferred to 24-well plates (6–12 series of sections/brain) and stored at −20 °C in antifreeze solution (30% v/v ethylene glycol (VWR, 24407361), 30% v/v glycerol (VWR, 24387361), 40% v/v water, 13 mM monosodium phosphate, and 39 mM disodium phosphate).
2.4 Sera
Coagulated blood samples were centrifuged at 3000 g for 10 min. at room temperature (RT), followed by 20,000 g for 3 min. at 4 °C. Serum was frozen at −20 °C until further analysis.
2.5 Anti-dsDNA autoantibody measurements
FluoroNunc Maxisorp 96-well microtiter plates (Nunc, Denmark) were coated with 100 μg/ml salmon sperm dsDNA (Invitrogen, Cat.: AM9680) and then incubated overnight at 4 °C. After incubation, wells were blocked with 1% w/v bovine serum albumin (BSA) (Sigma-Aldrich, Cat.: A4503) in tris-buffered saline (TBS) (Fisher Scientific, Cat.: BP2471–500) containing 0.09% w/v sodium azide (Ampliqon), incubated 1 h at RT and subsequently washed three times with TBS/Tw (TBS with 0.05% v/v Tween 20 (Merck, Cat.: 8.17072.1000)).
Serum samples, standards and quality controls were resuspended on a vortex mixer and diluted in TBS/Tw containing 5 mM EDTA and 0.1% w/v BSA (Sigma-Aldrich, Cat.: A4503) and added to wells. Each dilution was added in duplicate wells, incubated 1 h at 37 °C and subsequently washed three times with TBS/Tw. Wells were incubated 1 h at 37 °C with biotinylated Goat anti-mouse antibody, diluted in TBS/Tw to 1 μg/ml, according to the desired antibody or allotype measured: Goat anti-mouse IgG2A (Southern Biotech, Cat.: 1083–08), Goat anti-mouse IgG2C (Southern Biotech, Cat.: 1077–08) or Goat anti-mouse Ig (Southern Biotech, Cat.: 1010–08). Wells were subsequently washed three times with TBS/Tw. Next, wells were incubated 1 h at RT with Eu3+-labelled streptavidin (PerkinElmer, Cat.: 1244–360) diluted to 0.1 μg/ml in TBS/Tw containing 25 μM EDTA, and subsequently washed three times with TBS/Tw. Enhancement buffer (Ampliqon) was added to wells which were then shaken for approximately 5 min on a plate shaker and afterwards analysed on a Victor X5 Multilabel Plate Reader (PerkinElmer).
For the anti-dsDNA IgG2A assay, we used a serum pool from 564Igi heterozygous mice as standard (defined at 1000 mU) and three dilutions of a serum pool from 564Igi hetero- and homozygous mice as internal controls. In the anti-dsDNA IgG2C assay, we used a serum pool from BXSB/MpJ males as both standard serum (defined at 1000 mU) and internal controls. The anti-dsDNA total Ig assay had as standard a serum pool from 564Igi heterozygous mice (1000 mU) and a serum pool from 564Igi hetero- and homozygous mice as internal controls.
2.6 Total immunoglobulin measurements
FluoroNunc Maxisorp 96-well microtiter plates were coated with anti-mouse antibodies, depending on the desired antibody or allotype measured: Goat anti-mouse IgG2A (Southern Biotech, Cat.: 1083–01) 1 mg/ml, Goat anti-mouse IgG2C (Southern Biotech, Cat.: 1077–01) 1 mg/ml or Rabbit anti-mouse Ig (DAKO, Cat.: Z0259) 2 mg/ml. Goat anti-mouse IgG2A and Goat anti-mouse IgG2C were diluted to 1 μg/ml in PBS, while Rabbit anti-mouse Ig was diluted to 0.125 μg/ml in PBS. Coated plates were incubated o/n at 4 °C. After incubation, wells were blocked with human serum albumin ((CSL Behring, Cat.: 109697), 1 mg/ml in TBS (Fisher Scientific, Cat.: BP2471–500) containing 0.09% sodium azide w/v), incubated 1 h at RT and subsequently washed three times with TBS/Tw.
Serum samples, standards and internal controls were resuspended on a vortex mixer and diluted in TBS/Tw containing 100 μg/ml of heat-aggregated human Ig (hIg Gammanorm, Octapharma, Cat.: 478393) and added to wells; each dilution was added in duplicate wells, incubated o/n at 4 °C or 2 h at RT, and subsequently washed three times with TBS/Tw. Wells were next incubated 2 h at RT with biotinylated Goat anti-mouse antibody, diluted in TBS/Tw to 1 μg/ml, according to the desired antibody or allotype measured: Goat anti-mouse IgG2A (Southern Biotech, Cat.: 1083–08), Goat anti-mouse IgG2C (Southern Biotech, Cat.: 1077–08) or Goat anti-mouse Ig (Southern Biotech, Cat.: 1010–08). Wells were subsequently washed three times with TBS/Tw.
Next, wells were incubated 1 h at RT with Eu3+-labelled streptavidin (PerkinElmer, Cat.: 1244–360) diluted to 0.1 μg/ml in TBS/Tw containing 25 μM EDTA, and subsequently washed three times with TBS/Tw. Enhancement buffer was added to wells which were then shaken for approx. 5 min on a plate shaker and afterwards analysed on a Victor X5 Multilabel Plate Reader (PerkinElmer).
Standards and internal quality controls differed, depending on the antibody or allotype measured. The standards used in the different assays were Mouse IgG2A (Southern Biotech, 5300–01), Mouse IgG2C (Southern Biotech, 5300-01B), and Mouse IgG (Lampire, 7,404,304), respectively. The internal control in both the assay measuring IgG2C and the assay measuring total Ig was a C57BL/6 J serum pool, while the IgG2A assay included a DBA/2JRj serum pool as internal control.
2.7 Flow cytometry
The spleen and lymph node samples were mechanically dissociated with a pestle, filtered through a 70-μm filter and the cells were recovered by centrifugation (200 g at 4 °C for 5 min.). The pelleted cells from the lymph node samples were resuspended in FACS buffer. The pelleted splenocytes were resuspended in red blood cell (RBC)-lysis buffer (155 mM NH4Cl, 12 mM NaHCO3 and 0.1 mM EDTA (pH 7.3) in MilliQ water) to initiate lysis of the erythrocytes. After 3–5 min. The lysis reaction was terminated by addition of FACS buffer, and the cells were centrifuged (200 g at 4 °C for 5 min.) followed by resuspension in FACS buffer. All cell samples were then plated and pre-incubated for 10 min. With Fc block (Purified Rat Anti-Mouse CD16/CD32 (BD Pharmingen, 553,142)) prior to addition of antibodies. Subsequently, they were added stains for viability (Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific, 65–0865-14)), hematopoietic cells (BV650 Rat Anti-Mouse CD45 (BD Biosciences, 563,410)), B cells (Pacific Blue Rat Anti-Mouse CD45R/B220 (BD Biosciences, 558,108)), plasmablasts/cells (PE Anti-Mouse CD138 (Nordic Biosite, 142,504)), germinal centres (PE/Cyanine7 Anti-Mouse CD38 (Nordic Biosite, 102,718) and Purified NA/LE Hamster Anti-Mouse CD95 antibody (BD Biosciences, 554,254) in-house conjugated to an iFluor 647 Succinimidyl Ester (AAT Bioquest, 1031)), and CD4- (PerCP Rat Anti-Mouse CD4 (BD Biosciences, 553,052)) and CD8-positive (PerCP/Cyanine5.5 Anti-Mouse CD8a (Nordic Biosite, 100,734)) T cells. Afterwards, FACS buffer was used for washing, followed by resuspension, and the cells were analysed on an LSR Fortessa (BD Biosciences) equipped with 4 lasers (405 nm, 488 nm, 561 nm, and 633 nm) and 18 detectors. Data were analysed using FlowJo v. 10.8 (BD Biosciences). For one of the cohorts, it was necessary to perform a time gating to exclude part of the acquisition diagram, due to a flow rate issue.
2.8 Immunohistochemistry with DAB development
Brain sections were transferred to net wells and washed 4 × 5 min. in PBS. Sections were quenched in a solution containing 3% v/v H2O2 (Merck 1.07209.0250), 10% v/v methanol, and 80% v/v PBS for 10–20 min. at RT and washed 3 × 5 min. in PBS. Sections were blocked with 5% v/v goat serum (BioRad, C07SA containing 0.09% sodium azide w/v (Ampliqon)) in 0.25% v/v Triton-X100 in PBS 1 h at RT before being transferred to glass vials. Next, sections were incubated with primary antibody (1:400 (Rat anti-mouse MHCII, eBioscience, 14–5321) in 2.5% v/v goat serum and 0.25% v/v Triton-X100 in PBS) overnight at RT. Sections were transferred to net wells, washed 4 × 5 min. in PBS and blocked (1% v/v goat serum in 0.25% v/v Triton-X100 in PBS) for 10 min. at RT. Sections were incubated with secondary biotinylated antibody (1:200 (Biotinylated goat anti-rat IgG, Vector Laboratories, BA-9401) in 1% v/v goat serum and 0.25% Triton-X100 in PBS) for 2 h at RT and washed 4 × 5 min. in PBS. Next, sections were incubated with an avidin-biotin-peroxidase complex (2% v/v ABC kit (Vectastain ABC kit at 4 °C, Vector Lab. Elite PK-6100 standard) in PBS) for 1 h at RT. Sections were then washed 3 × 5 min. and 2 × 10 min. in PBS.
For visualization, 3,3′-diaminobenzidine (DAB) (2% v/v DAB (Sigma-Aldrich, D5637) in PBS) was added and development reaction started by addition of H2O2 (1% v/v H2O2 in PBS) and ended by adding excess PBS to net wells. Afterwards, sections were transferred to glass vials containing PBS and stored at 4 °C. If sections were not mounted within three days, 10 μl sodium azide was added to the glass vials.
2.9 Immunofluorescence
A “dilution buffer” was made with 0.25% v/v Triton-X100 in PBS. Two to three striatal and hippocampal sections were chosen per brain and transferred to glass vials. Sections were washed 4 × 5 min. in PBS and blocked (5% v/v goat serum in 0.25% v/v Triton-X100 in PBS) 1 h at RT. Next, sections were incubated with primary antibodies (1:1000 (Rabbit anti-Iba1, Wako Pure Chemicals Industries, 019–19,741 and Rat anti-mouse CD68, BioRad, MCA1957)) in 2.5% v/v goat serum and 0.25% v/v Triton-X100 in PBS) overnight at RT.
The next day, sections were washed 4 × 5 min. in PBS and blocked (1% v/v goat serum in 0.25% v/v Triton-X100 in PBS) for 10 min. at RT and shielded from light in downstream steps. Sections were incubated in the dark with secondary antibodies coupled to fluorophore (1:400 (Goat anti-rabbit Alexa Fluor 488, Thermo Scientific, A-11008 and Goat anti-rat Alexa Fluor 647, Thermo Scientific, A-21247) antibody and 1:2000 DAPI in 1% v/v goat serum and 0.25% v/v Triton-X100 in PBS) for 2 h at RT. Afterwards, sections were washed 4 × 5 min. in PBS and mounted onto coated slides, then cover-slipped. Sections were stored in the dark at 4 °C until analysis.
2.10 Cresyl violet staining
Prior to mounting of brain sections, glass slides were coated with a solution of 0.05% w/v chromium potassium sulphate and 0.5% w/v gelatine in distilled water. Brain sections were mounted on coated slides and dried overnight. Nissl staining with cresyl violet was used for visualization of neuronal structure. Next, sections were dehydrated in ethanol and xylene and coverslipped.
2.11 Microscopy and image analysis
For the microglia quantification, adjacent serial sections were used. The MHCII-positive microglia were counted under a light microscope (Leica DMi1), with a MHCII-positive cell defined by a nuclear stain and MHCII covering the surroundings. The objective lenses used were 20× and 40× with a 0.30 and 0.50 numerical aperture, respectively.
Immunofluorescence staining of sections for Iba-1 were imaged on a Zeiss LSM800 Confocal Microscope and z-stacks (12 slices with a 1.5 μm interval) from the cortical regions were acquired. The objective lens used was 40× with a 0.95 numerical aperture. Samples were excited with a 405 nm laser for DAPI, a 488 nm laser for the Alexa Fluor 488, and a 640 nm laser for the Alexa Fluor 647, and emission was detected in the range 400–605 nm, 400–650 nm, and 645–700 nm, respectively. Image analysis was done in Fiji (ImageJ) v. 2.3.0. Background signal was adjusted based on averaging background values from four areas negative for signal in each channel and subtracting the mean values from the final image. Microglia morphology was analysed using an established protocol from Young and Morrison (
) (Fig. S2). Maximum intensity z-projections were converted to 8-bit greyscale and the contrast was enhanced through adjustment followed by the Unsharp Mask filter and then binarized. Images were then analysed using the Skeleton Plugin from ImageJ. Based on the data provided from the Skeleton analysis, and on a 0.5 cut-off value, the summarized process lengths and endpoints were normalized to the number of microglial somas in each z-stack to calculate process length/cell and endpoints/cell.
For CD68 expression in microglia, Iba1 signal was thresholded automatically and analysed to create a ROI mask for each slice in the z-projection of microglial cells (Fig. S3). To remove pixel noise and any artefacts, structures below 0.2 μm2 were excluded from the masks during analysis.
The ROI masks were applied to measure CD68 expression in their corresponding slice of the z-projection. From there, the mean fluorescence intensity was calculated using the mean grey values from each slice in the z-projection.
2.12 Statistical analysis
Statistical analyses were performed using GraphPad Prism v. 8.0.2 (1994–2021 GraphPad Software, LLC.). Normality was assessed by Shapiro-Wilk's test and QQ plot, and homoscedasticity was confirmed using Bartlett's test. In case of non-normality or heteroscedasticity, data was log-transformed, or a nonparametric test (Kruskal-Wallis test) was performed.
The statistical significance of the results was assessed by one- or two-way analysis of variance (ANOVA) with either Tukey's or Holm-Sidak's post-test for multiple comparisons in GraphPad Prism v. 8.0.2.
All data are expressed as means ± SEM, with p < 0.05 considered significantly different.
3. Results
To uncouple the genetic and environmental factors in autoimmune maternofoetal effects on offspring, we employed embryo transfers (Fig. S4). The transfer of DBA/2JRj embryos into BXSB/MpJ mothers resulted in 18 offspring, while transfer of DBA/2JRj embryos into C57BL/6 J mothers resulted in twelve offspring, while controls comprised ten naturally reared DBA/2JRj mice. Adult controls included eight DBA/2JRj, four BXSB/MpJ and six C57BL/6 J mice.
To decipher the degree of maternal antibody transfer to offspring, we leveraged the fact that BXSB/MpJ and C57BL/6 J mothers produce the IgG2C allotype, whereas DBA/2JRj produce the IgG2A allotype, thus allowing us to discriminate maternally derived versus endogenously produced Ig of the IgG2a isotype.
3.1 IgG2A vs. IgG2C is a reliable predictor of genetic origin of the IgG2a isotype
We first sought to verify that our assays reliably discriminated IgG2A and IgG2C, and that these allotypes were reliable predictors of the genetic origin of immunoglobulin of the IgG2a isotype.
To this end, we measured total IgG2A versus IgG2C in females of the background strains employed. As expected, high levels of IgG2A were found in the DBA/2JRj controls, whereas, in comparison, only background levels of IgG2A were found in C57BL/6 J and BXSB/MpJ controls (Fig. 1A ). The highest levels of IgG2C were observed in both BXSB/MpJ and C57BL/6 J controls, while only background levels were observed in DBA/2JRj (Fig. 1B). Additionally, the total level of immunoglobulins measured was comparable between all three groups (Fig. S5A).
Fig. 1Total IgG2a levels of either the IgG2A or IgG2C allotype in BXSB/MpJ, C57BL/6 J and DBA/2JRj dams.
(A) Total IgG2A in BXSB/MpJ (n = 4), C57BL/6 J (n = 6), and DBA/2JRj (n = 8), controls. Significantly higher levels of IgG2A were observed in DBA/2JRj controls compared to both C57BL/6 J (p = 0.0013) and BXSB/MpJ (p = 0.0036).
(B) Total IgG2C in BXSB/MpJ (n = 4), C57BL/6 (n = 6), and DBA/2JRj (n = 8) controls. IgG2C levels in BXSB/MpJ and C57BL/6 J controls were significantly greater compared to DBA/2JRj (p = 0.0292 and p = 0.0005, respectively).
Statistical significance based on multiplicity-adjusted p-value determined using one-way ANOVA with Tukey's test for multiple comparisons. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
3.2 IgG2C is transferred maternofoetally, and to a higher extent from BXSB/MpJ than C57BL/6 J mothers
We next sought to determine the degree to which immunoglobulin of the IgG2a isotype was transferred from mother to offspring.
IgG2A antibody levels in embryo transfers were comparable and without statistical significance between all groups independent of the maternal background and the sex of the offspring (Fig. 2A ). This was not surprising, because all embryo transfer offspring were of the DBA/2JRj background, and hence had a similar genetically encoded capacity to produce IgG2A.
Fig. 2Total IgG2a levels of either the IgG2A or IgG2C allotype in offspring resulting from DBA/2JRj embryo transfers to BXSB/MpJ or C57BL/6 J mothers.
(A) Total IgG2A in BXSB/MpJ (n = 16) and C57BL/6 J (n = 12) ET offspring and DBA/2JRj controls (n = 10). All groups displayed similar levels of endogenous IgG2A.
(B) Total IgG2C in BXSB/MpJ (n = 16) and C57BL/6 J (n = 12) ET offspring and DBA/2JRj controls (n = 10). ET offspring born to BXSB/MpJ mothers exhibited the greatest level of IgG2C compared to offspring born to C57BL/6 J (p < 0.0001) and DBA/2JRj (p < 0.0001) mothers. Levels of total IgG2C were significantly greater in offspring born to C57BL/6 J mothers compared to DBA/2JRj controls (p = 0.0003).
Statistical significance based on multiplicity-adjusted p-value determined using one-way ANOVA with Tukey's test for multiple comparisons. *** = p < 0.001, **** = p < 0.0001.
Conversely, DBA/2JRj mice should not have the capacity to produce IgG2C, and the presence of this allotype could only result from maternofoetal transfer. In line with this, we observed only baseline levels in DBA/2JRj controls but noted considerable levels in the embryo transfer offspring. The greatest level of IgG2C antibodies was observed in embryo transfer offspring from BXSB/MpJ mothers, and this was significantly greater than the levels found in offspring born to C57BL/6 J mothers (Fig. 2B). This finding demonstrated a disproportionately high degree of maternofoetal transfer of IgG2C from the BXSB/MpJ mothers to their offspring. The total level of immunoglobulins in embryo transfer offspring of C57BL/6 J mothers was also depressed compared to that of embryo transfer offspring of BXSB mothers and, surprisingly, conventionally reared DBA/2JRj controls (Fig. S5C).
3.3 No significant level of anti-dsDNA autoantibodies of IgG2A and IgG2C allotypes in embryo transfer offspring
The IgG2a isotype is produced in connection with TH1 responses directed against intracellular pathogens such as viruses but is also found upregulated in autoimmunity. Hence the observation of significantly elevated IgG2C levels in embryo transfer offspring from BXSB/MpJ mothers could be indicative of an autoimmune transfer. Furthermore, if the autoimmune uterine environment imprinted an autoreactive phenotype upon the offspring, this could manifest through increased production of endogenous IgG2A autoantibodies. To further investigate these possibilities, we tested whether embryo transfer offspring of BXSB/MpJ mothers displayed an elevated level of anti-dsDNA antibodies of the two IgG2a allotypes. Few embryo transfer offspring born to BXSB/MpJ (3 out of 15 offspring) did appear to harbour slightly elevated levels of anti-dsDNA IgG2A compared to C57BL/6 J maternal embryo transfers and DBA/2JRj controls (Fig. 3A ), while a baseline level of anti-dsDNA IgG2C was measured within all groups with a weak and non-significant trend towards increased levels in BXSB/MpJ and to a lower extent C57BL/6 J embryo transfer offspring (Fig. 3B). Also, both BXSB/MpJ and C57BL/6 J embryo transfers exhibited slightly greater levels of total anti-dsDNA Ig compared to DBA/2JRj controls (Fig. S5D).
Fig. 3Levels of anti-dsDNA antibodies of either the IgG2A or IgG2C allotype in offspring resulting from DBA/2JRj embryo transfers to either BXSB/MpJ or C57BL/6 J mothers.
(A) Anti-dsDNA IgG2A in BXSB/MpJ (n = 15) and C57BL/6 J (n = 12) ET offspring and DBA/2JRj controls (n = 10). Anti-dsDNA IgG2A was observed in a subset of BXSB/MpJ ET offspring with a statistical difference compared to C57BL/6 J ET offspring (p = 0.0445).
(B) Anti-dsDNA IgG2C in BXSB/MpJ (n = 16) and C57BL/6 J (n = 10) ET offspring and DBA/2JRj controls (n = 8). Levels of anti-dsDNA IgG2C were comparable between all groups, and no statistically significant differences were observed.
Statistical significance was tested by one-way ANOVA with Tukey's test for multiple comparisons. * = p < 0.05.
ET = embryo transfer, AU = arbitrary unit, n = sample size.
3.4 Slightly increased plasmablast/cell levels in spleens of offspring from BXSB/MpJ compared to C57BL/6 J embryo transfers
Additionally, inguinal, and mesenteric lymph nodes and spleens were harvested and analysed by flow cytometry to determine the frequencies of selected immune cell populations in embryo transfer offspring of both BXSB/MpJ and C57BL/6 J mothers, and age-matched DBA/2JRj controls (Fig. 4). The approach was to first gate on lymphocytes followed by single cells, live cells, and subsequently CD45-positive cells. B cell, CD4 and CD8 T cell, and CD138-positive cell frequencies were based on gating on the CD45-positive population, while germinal center (GC) B cell frequency was determined by gating on the B cell population (Fig. S6).
Fig. 4Immune cell population frequencies in DBA/2JRj embryos transferred to either BXSB/MpJ or C57BL/6 J mothers.
Percentages of (A) B cells, (B) Germinal center B cells, (C) CD4 T cells, (D) CD8 T cells, and (E) Plasmablasts/cells in inguinal and mesenteric lymph nodes and spleen from BXSB/MpJ and C57BL/6 J embryo transfer offspring and DBA/2JRj controls.
(A) Compared to DBA/2JRj controls, ET offspring of both BXSB/MpJ and C57BL/6 J mothers displayed slightly elevated levels of B cells in the inguinal lymph nodes (p = 0.0421 and p = 0.0152, respectively) and spleen (p = 0.0081 and p = 0.0107, respectively).
(B) No significant differences in germinal center B cell levels were observed between the groups.
(C) Increased levels of CD4 T cells were observed in inguinal lymph nodes of DBA/2JRj controls compared to ET offspring of C57BL/6 J mothers (p = 0.0089) and in the spleen compared to ET offspring of both BXSB/MpJ and C57BL/6 J mothers (p = 0.0454 and p = 0.0021, respectively).
(D) CD8 T cell levels were slightly elevated in the inguinal lymph nodes of DBA/2JRj controls compared to ET offspring born to BXSB/MpJ mothers (p = 0.0154).
(E) In comparison to ET offspring born to C57BL/6 J mothers, slightly increased levels of plasmablasts/cells in the mesenteric lymph nodes were observed in ET offspring to BXSB/MpJ mothers (p = 0.0352). Also, greatly elevated levels were detected in both ET offspring to BXSB/MpJ mothers and DBA/2JRj controls compared to ET offspring to C57BL/6 J mothers (p < 0.0001 for both groups).
Statistical significance reported as multiplicity-adjusted p-value following two-way ANOVA with Tukey's test for multiple comparisons.
* = p < 0.05, ** = p < 0.01, **** = p < 0.0001.
In all graphs: Inguinal lymph nodes (n = 18 for BXSB/MpJ ET offspring, n = 11 for C57BL/6 J ET offspring; n = 8 for DBA/2JRj controls), mesenteric lymph nodes (n = 18 for BXSB/MpJ ET offspring, n = 6 for C57BL/6 J ET offspring; n = 10 for DBA/2JRj controls), and spleen (n = 18 for BXSB/MpJ ET offspring, n = 7 for C57BL/6 J ET offspring; n = 8 for DBA/2JRj controls).
ET = embryo transfer, IngLN = inguinal lymph nodes, MesLN = mesenteric lymph nodes, GC = germinal center, n = sample size.
There was a slightly higher level of B cells in the inguinal lymph nodes and spleens of both groups of embryo transfers, compared to DBA/2JRj controls (Fig. 4A). No significant differences in GC B cell levels were observed between groups (Fig. 4B). Slightly greater levels of both CD4 (Fig. 4C) and CD8 (Fig. 4D) T cells were seen in the DBA/2JRj controls compared to either one or both embryo transfer groups, together accounting for the relative decrease in observed B cell frequencies. Interestingly, and in spite of the lack of differences in GC B cells, the frequencies of splenic plasmablasts/cells were considerably higher in BXSB/MpJ embryo transfer offspring compared to C57BL/6 J embryo transfer offspring, but on par with those of conventionally reared DBA/2JRj controls (Fig. 4E). In addition, there was a similar, albeit less dramatic, effect in the mesenteric lymph nodes, which only reached statistical significance for the BXSB/MpJ embryo transfer group.
3.5 No significant level of autoantibodies of IgG2C allotype in BXSB/MpJ females
Given the reportedly well-established autoimmune phenotype of the BXSB/MpJ model, we had expected to observe a transfer of anti-dsDNA antibodies into the embryo transfer offspring from BXSB/MpJ mothers. To further investigate why this did not occur, we measured the level of anti-dsDNA in females from the BXSB/MpJ, C57BL/6 J, and DBA/2JRj strains.
Surprisingly, comparable, and only background levels of IgG2A anti-dsDNA and IgG2C anti-dsDNA were detected across the groups (Fig. 5A and B ). This observation was also evident when comparing levels of total anti-dsDNA Ig between all groups (Fig. S5B). Moreover, we measured levels of IgG2C anti-dsDNA in BXSB/MpJ females of different ages (9, 21, 34, 46, and 59 weeks), including the 16 weeks old females from Fig. 1, Fig. 5, and in 13 weeks old BXSB/MpJ males as controls. Although there was a trend towards an increase with age, we did not observe a statistically significant difference from the baseline measurement at 9 weeks across the female age groups. In contrast to this, levels significantly above those of females across all ages assayed were present in males, already at 13 weeks of age (Fig. S7).
Fig. 5Anti-dsDNA antibody levels in BXSB/MpJ, C57BL/6 J, and DBA/2JRj dams.
(A) Anti-dsDNA IgG2A in BXSB/MpJ (n = 4), C57BL/6 J (n = 5), and DBA/2JRj (n = 5) controls.
Anti-dsDNA IgG2A was hardly detectable in all groups.
(B) Anti-dsDNA IgG2C in BXSB/MpJ (n = 4), C57BL/6 J (n = 5), and DBA/2JRj (n = 8) controls. Between all groups, levels of anti-dsDNA IgG2C were diminutive and comparable to the DBA/2JRj controls.
Statistical significance was tested by one-way ANOVA with Tukey's test for multiple comparisons. No significant differences were detected.
3.6 No difference in microglial activation between embryo transfers of BXSB/MpJ and C57BL/6 J
Although the absence of autoantibodies in the BXSB/MpJ mothers and the embryo transfer offspring from these indicated the lack of a robust autoimmune phenotype, the possibility remained that more subtle components of the autoimmune phenotype could still be at play. For example, elevated levels of inflammatory cytokines have been shown to exacerbate permeability of the blood-brain barrier and promote leukocyte recruitment, thereby allowing for immune cell-mediated activation of microglia and subsequent inflammation in the CNS (
To determine whether subtle autoimmune influences could affect the CNS of offspring independently of genetic factors, we analysed the microglia in the CNS of embryo transfer offspring and controls.
Brains were sectioned coronally and to ensure a comprehensive and representative analysis throughout the CNS we stained a full 1:6 series of 40 μm coronal sections (i.e., one section every 240 μm). The tissue was immunostained for class II major histocompatibility complex molecules (MHCII), the expression and upregulation of which is associated with microglial activation (
Regulation and function of class II major histocompatibility complex, CD40, and B7 expression in macrophages and microglia: implications in neurological diseases.
), and subsequently counterstained with cresyl violet. MHCII-positive (MHCII+) microglia were identified and counted across sections. Both ramified and hyper-ramified microglia were detected in all groups, although no phagocytic microglia, with amoeboid morphology, were observed (Fig. 6A-C ). Additionally, the total number of MHCII+ microglia did not significantly differ between groups (Fig. 6D).
Fig. 6MHCII-positive microglia found BXSB/MpJ and C57BL/6ET offspring and DBA/2JRj controls.
Both ramified (white arrows) and hyper-ramified (red arrows) were observed in (A) BXSB/MpJ ET offspring, (B) C57BL/6 J ET offspring and (C) DBA/2JRj controls.
(D) The total number of MHCII-positive microglia did not differ significantly between C57BL/6 J (n = 7) and BXSB/MpJ (n = 11) embryo transfer offspring or DBA/2JRj controls (n = 6).
Statistical tests were performed using one-way ANOVA with Holm-Sidak's test for multiple comparisons. No significant differences were detected.
ET = embryo transfer, n = sample size.
Scale bars indicate 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
In addition to investigating the presence of MHCII+ cells, the microglia morphology was studied using the general microglia-marker, the ionized calcium-binding adaptor molecule 1 (Iba1) in an immunofluorescent stain (Fig. 7).
Fig. 7Quantification of microglial morphology in DBA/2JRj embryos transferred to either an autoimmune BXSB/MpJ or a normal C57BL/6 J mother.
(D),(E) Summary of (D) microglial endpoints (number of processes) and (E) process length in ET offspring from BXSB/MpJ and C57BL/6 J mothers, and offspring from DBA/2JRj controls (n = 6 for all groups).
Statistical tests performed using two-way ANOVA with Holm-Sidak's test for multiple comparisons. No significant differences detected.
This included CNS sections from embryo transfers of BXSB/MpJ and C57BL/6 J mothers, and conventionally reared DBA/2JRj controls. From each animal, two sections representing the striatum and hippocampus were selected and stained for Iba1, along with DAPI, and background signal was subtracted during image processing. We did not observe morphological differences in microglia from BXSB/MpJ and C57BL/6 J embryo transfer offspring, and age-matched DBA/2JRj controls (Fig. 7A-C). Finally, using an already established protocol from Young & Morrison (
), the number of processes (endpoints) and the process lengths were quantified and assessed.
However, we did not detect any meaningful change in the morphology between groups (Fig. 7D and E).
In accordance with this, no significant differences were observed in the expression of microglial activation marker CD68 between the embryo transfer groups and controls (Fig. 8).
Fig. 8Microglial CD68 expression in DBA/2JRj embryos transferred to either an autoimmune BXSB/MpJ or a normal C57BL/6 J mother.
(A) Representative images (40× magnification) of CD68-expressing microglia (arrowheads) in the hippocampal cortex of an embryo transfer offspring from a C57BL/6 J mother. Scale bars indicate 20 μm.
(B) Mean fluorescence intensity of the microglial activation marker CD68 in striatal and hippocampal cortex of offspring from BXSB/MpJ embryo transfers, C57BL/6 J embryo transfers, and DBA/2JRj controls (n = 6 for all groups).
Statistical tests performed using two-way ANOVA with Holm-Sidak's test for multiple comparisons. No significant differences detected.
Image contrast were enhanced for representative purposes.
Taken together, this did not indicate an activated microglial phenotype in the embryo transfer offspring from BXSB/MpJ or C57BL/6 J mothers.
4. Discussion
Our experiments demonstrate that maternal immunoglobulins are transferred to the foetus during pregnancy, independent of gender and background strain, because we could detect maternally derived immunoglobulin in all the embryo transfer offspring. Interestingly, both male and female offspring from autoimmune BXSB/MpJ mothers had higher levels of IgG2C, compared with their counterparts from healthy C57BL/6 J mothers. This indicates a specific increase in transfer of maternal immunoglobulin from the BXSB/MpJ mothers, potentially as a consequence of maternal immune activation. However, although a maternofoetal transfer of IgG2C was detected in offspring from both BXSB/MpJ and C57BL/6 J mothers, there was no significant indication of anti-dsDNA antibodies in the offspring at 6 weeks of age. One likely explanation could be that the catabolic rate of anti-dsDNA, and autoantibodies in general, is much higher than that of other immunoglobulins, as the former would likely form immune complexes and be rapidly cleared in vivo, whereas the latter would likely not be exposed to their cognate antigens.
Hence the possibility remained that such autoantibodies, potentially in conjunction with maternal cytokines and other factors, could have exerted an immune activating effect in the offspring during the prenatal or immediate postnatal period. To investigate this, we analysed cellular markers of immune activation across secondary lymphoid tissues, including germinal center B cell frequencies and plasmablast/cell frequencies. There were slight, and counterbalancing skews in B, CD4 T and CD8 T cell levels between embryo transfers and DBA/2JRj controls. Although these did in several instances reach statistical significance, the differences were marginal and of unclear biological significance. No differences were found in germinal center B cell levels across tissues. However, we did observe an increased frequency of plasmablasts/cells in embryo transfer offspring of BXSB/MpJ mothers compared to those of C57BL/6 J mothers, although they were comparable to those of DBA/2JRj controls. This could indicate an effect of the maternal environment on the generation or longevity of plasmablasts/cells. One potential explanation is that this plasmablast/cell population trails an earlier germinal center response, which had itself already subsided at the time of analysis 6 weeks postnatally. Another possibility is that the plasmablast/cells originated from extrafollicular responses, either earlier or still ongoing. Finally, the increased frequency could be caused by immune signals increasing the longevity of plasmablasts/cells, as has previously been seen for ambient commensal immune stimulation via TLR5 (
). Notwithstanding the reason for the increase, the relatively small magnitude of the increase in plasmablast/cell levels would, however, suggest that this was a very mild, and potentially subsiding, phenotype.
Nonetheless, it does track with our observation of endogenously produced anti-dsDNA of the IgG2A allotype in the BXSB/MpJ embryo transfer group, specifically, although this was seen in a subset of the offspring only.
Subtle immune activation could manifest in perturbations of CNS homeostasis, as previously noted (
). Accordingly, we also investigated whether there was any observable CNS phenotype in the offspring, which could be connected to their maternal origin. Based on their role as immune sentinels of the CNS, microglia are particularly responsive to inflammatory signals and perturbations of normal homeostasis (
For this reason, we specifically asked whether there was any difference in microglia frequency or activation status across series of representative coronal sections, covering all areas of the cerebrum with an immunohistochemical staining for MHCII. Both ramified and hyper-ramified microglia were detected in all groups, as determined based on morphology and MHCII expression. No phagocytic microglia with amoeboid morphology were observed, indicating homeostatic conditions across groups. Accordingly, we also did not observe any differences in frequency of MHCII+ microglia. These findings were further corroborated by immunofluorescence microscopy using Iba1 and DAPI staining and analysing for microglia endpoints and process lengths, as well as CD68 expression, using computational methods. Taken together, our findings indicated no apparent impact of the maternal immune environment on the immune status of the brain in the offspring.
Following our initial experiments, we became aware of earlier findings from Denenberg and colleagues (
) suggesting that embryo transfer offspring born to autoimmune BXSB/MpJ mothers displayed both significant levels of serum anti-dsDNA antibodies and learning impairment when faced with behavioural tests. To try to understand this discrepancy with our findings, we investigated the phenotype of females of the BXSB/MpJ background in our colony. Due to the genetic makeup of the BXSB/MpJ strain, we expected a clearly detectable disease phenotype in both the BXSB/MpJ males and females, of high and low severity, respectively (
). BXSB/MpJ males did indeed present with much higher anti-dsDNA antibody titers compared to BXSB/MpJ females, however, BXSB/MpJ females did not display anti-dsDNA antibody levels above those of female DBA/2JRj controls. The autoimmune phenotypic variation observed when compared to Denenberg's work is not clear. We considered the possibility that age could be a factor, as it is well-known that autoimmune manifestations worsen over time, as observed for e.g., SLE patients (
). However, the females used as embryo recipients in our study at 15–29 weeks of age, were not significantly different from the age range of those used by Denenberg and colleagues, 10–20 weeks range with a mean of 15 weeks (
). Moreover, offspring were analysed at similar postnatal age, 6–7 weeks in our study and 5–12 weeks with a mean of 8 weeks in that of Denenberg and colleagues (
). Finally, the BXSB/MpJ female controls we analysed for presence of anti-dsDNA autoantibodies were 16 weeks, closely resembling the maternal age used by Denenberg and colleagues. Additionally, we measured levels of IgG2C anti-dsDNA in BXSB/MpJ females across different ages, at 9, 21, 34, 46, and 59 weeks, and in 13 weeks old BXSB/MpJ males as controls. Although there was a trend towards an increase with age, we did not observe a statistically significant difference from the baseline measurement at 9 weeks across the female age groups. In contrast to this, levels significantly above those of females across all ages assayed were present in males, already at 13 weeks of age. Finally, our experimental approaches to assay anti-dsDNA were similar: Denenberg and colleagues employed an ELISA-based assay, we employed a TRIFMA; both are solid-phase assays, but TRIFMA is generally considered more sensitive than ELISA (
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As an improvement over the studies of Denenberg and colleagues, we additionally employed assays that allowed us to discriminate maternally derived vs. endogenously produced antibody allotypes, as well as total and isotype-specific antibody levels.
), it was mentioned that other assays were also carried out on the plasma and would be the subject of a later report; however, we were unable to identify any such follow-up study presenting these additional investigations.
Our BXSB/MpJ founders were ordered from Jackson Laboratories for the described experiments and only maintained for a relatively short time before conducting the experiments. The fact that we observed a robust phenotype in the males confirmed the overall phenotype of the strain. However, differences in cage enrichment, diet, and the microbial environment could possibly affect the physiology of the mice, with a more severe impact on the mild phenotype displayed by BXSB/MpJ females (
Animal husbandry practices have developed significantly over time, and our animal facility has a long-standing SPF status with the use of an individually ventilated cage system, which together could decrease overall microbial load and immune tonicity. Furthermore, genetic distance in generations of inbred mice from the original breeding colony have been shown to correlate with an increase of spontaneous mutations causing genetic drift (
). Considering that the experiments of Denenberg and colleagues were carried out close to three decades ago, this could be a significant factor.
Combined, these results indicate that the attenuated disease phenotype displayed by female BXSB/MpJ mice does not appear sufficient for creating a maternal immune activation to promote maternal immune activation-associated adverse effects in the offspring. Ongoing studies are aimed at interrogating such phenomena in more severe models of maternal inflammation.
Declaration of Competing Interest
The authors declare that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Acknowledgements
The authors wish to thank Charlotte Christie Petersen and Anja Bille Bohn for help with flow cytometry, Anna Lorentzen and Nina Glöckner for assistance with imaging, and Gitte Ulbjerg Toft for expert technical assistance with histology.
Selected figures presented in this publication and in the associated graphical abstract were created with BioRender.com.
This work was supported by the Lundbeck Foundation through a Lundbeckfonden Fellowship to Søren E. Degn (grant ID: R238-2016-2954). Cecilia Fahlquist-Hagert was supported by a postdoctoral fellowhip from Lundbeckfonden (grant ID: R303-2018-3415).
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