Changes in the in utero environment result in generational transfer of maladapted physiology in the context of conditions such as stress, obesity, and anxiety. Given the significant contribution of noncommunicable diseases – which are characterised by chronic inflammation – to population mortality, the potential for chronic maternal inflammation mediating foetal programming is a growing concern. The extent of generational transfer in terms of immune functionality and leukocyte glucocorticoid sensitivity was investigated over two generations of offspring (F1 and F2) in a model of chronic LPS-induced maternal inflammation in C57/BL/6 mice. Maternal inflammation resulted in glucocorticoid hypersensitivity (increased glucocorticoid receptor expression levels) in the majority of leukocyte subpopulations in both F1 and F2 offspring. Furthermore, splenocytes stimulated with LPS in vitro exhibited exacerbated inflammatory cytokine responses, which were even more prominent in F2 than F1; this effect could be ascribed to NLRP3 inflammasome hyperactivity in F1 but not F2. Current data illustrates that parental chronic inflammation may mediate the inflammatory profile in offspring, potentially propagating a maladapted proinflammatory phenotype in subsequent generations.
Ethical clearance was obtained from the Stellenbosch University Animal Research Ethics Committee (SU-ACUM14-00004). C57/BL/6 mice were housed under temperature-controlled conditions under a 12-hour dark-light cycle, with ad libitum access to standard rodent chow. After one-week acclimatization, 6-week-old dams (generation F0) were naturally mated with age-matched males. The breeding and propagation of the mice is illustrated in Figure 1. Females were placed with males overnight and removed the following morning. Successful mating was confirmed by the presence of a vaginal plug. The plug-positive dams were moved to separate cages and randomised to receive either LPS (from Escherichia coli; Sigma, USA; serotype 0127: B8) at 10 μg/kg bodyweight, prepared in 0.9% saline solution, or 0.9% saline solution (control) at a final volume of 50 μl. Breeding schematic for (a) saline and (b) LPS-exposed groups. The n represents the number of mice for each subsequent generation and gender group. The LPS or saline treatment was administered via intraperitoneal injection and repeated every seven days for the duration of gestation. The dose was based on the body mass prior to the first injection (200 ng LPS per 20 g body weight) and was kept constant throughout the duration of the gestation. A total of 3 injections were administered to each dam over the duration of gestation, which was either 19 or 20 days for all animals. For the duration of the intervention protocol, females were monitored daily for any signs of morbidity, such as lethargy, weight loss, or vaginal bleeding—none was observed. F0 was terminated 4 weeks after weaning of offspring (i.e., at 14 weeks of age and 5 weeks after the last LPS injection). The first generation of offspring (F1), resulting from the F0 mating, was weaned at 3 weeks of age. Offspring siblings were grouped together, but sexes were separated into two cages until 8 weeks of age; after which, 4 males and 4 females per treatment group were terminated for further experimental analysis. The remaining animals were bred with wild-type animals for the second generation of offspring (F2). For F2 generation, the F1 mice were mated with wild-type C57/BL/6 mice and the same procedure was followed for weaning but with no further intervention. Data on breeding, offspring litter size, and gestational duration did not seem to be different across generations or as result of LPS exposure (Supplementary ). As for F1, F2 mice were killed by cervical dislocation at 8 weeks of age. Whole blood was collected by cardiac puncture and transferred to K2EDTA microtubes. An aliquot was assigned for full blood and differential leukocyte counts on the CELL-DYN 3700CS haemocytometer (Abbott Diagnostics), while the remaining blood sample was used to collect plasma for assessment of corticosterone concentrations. Corticosterone concentrations were determined by quantitative ELISA (Demeditec Corticosterone rat/mouse ELISA, Demeditec Diagnostics, Germany), as per manufacturer’s instructions. The concentrations were calculated in Microsoft Excel using a 6-point standard curve with a logistic regression algorithm. The detection range of the kit was 6.1-2250 ng/ml. The kit has an intra-assay variation of 8.9% and interassay variation of 7.2%. Mouse spleens were dissected under sterile conditions and collected into ice-cold complete RPMI 1640 medium (supplemented with 10% foetal bovine serum, 1% penicillin-streptomycin, and 1% gentamicin). Both LPS-treated dams and LPS-affected offspring displayed macroscopically visible larger spleen sizes in comparison to saline-treated dams or saline-affected offspring, respectively. Exact organ mass was however not determined due to the requirement for sterility in culturing splenocytes. A single-cell suspension of murine splenocytes was generated by mechanical dissociation, by passing dissected tissue through a sterile 70 μm cell strainer (BD Biosciences, USA). The cell strainer was rinsed with complete RPMI 1640 medium to remove any attached cells. Red blood cells were lysed with 1x ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM NA2EDTA in ddH2O) for 5 minutes at room temperature, and splenocytes were washed with 1x Dulbecco’s phosphate-buffered saline (DPBS, Gibco, USA). The cells were pelleted at 300×g for 5 minutes at room temperature, the supernatant was aspirated, and the pellet was resuspended in complete RPMI 1640. The cells were counted and adjusted to 1 × 107/ml viable cells and used immediately for cell counting assays or frozen and stored in liquid nitrogen for subsequent batch analysis of the inflammasome and splenocyte functional capacity. All reagents were prepared as per manufacturer’s instructions prior to use. For permeabilisation of samples, the BD Cytofix/Cytoperm kit was used. The staining buffer was prepared as 1x DPBS with 5% bovine serum albumin (Invitrogen, USA) and 1% NaN3 and stored at 4°C until use. The antibodies were titrated to determine optimal dilution for experiments. The antibodies and dyes and their respective dilutions are as follows: CD16/32 Fc block (BD Biosciences); Zombie Aqua Fixable Viability dye (BioLegend); NK1.1 BV421, clone PK136 (BioLegend); TCRβ FITC, clone H57-597 (BD Biosciences); F4/80 PE-CF594, clone T45-2342 (BD Biosciences); CD11b PerCP-Cy5.5, clone M1/70 (BD Biosciences); NR3C1 Ax647, clone BugR2 (Novus Biologicals); and Ly6G APC-Cy7, clone 1A8 (BD Biosciences). Briefly, 1 × 106 splenocytes were incubated with Zombie Aqua dye in DPBS for 30 minutes at room temperature. After incubation, the cells were washed twice with DPBS and the supernatant was aspirated. The cells were then incubated with CD16/32 mouse Fc block antibody for 5 minutes in staining buffer, where after a master mix of the appropriate cell surface marker, antibodies are added. The cells were mixed thoroughly and incubated for 30 minutes at 4°C. The cells were then washed twice with staining buffer and permeabilised for 20 minutes at 4°C. After incubation, the cells were washed in 1x perm buffer and pelleted at 600×g for 5 minutes. Splenocytes were then resuspended with the appropriate dilution of the intracellular NR3C1 antibody. The samples were incubated at 4°C for 30 minutes in the dark. After incubation, the cells were washed twice with 1x perm buffer and, as a last step, resuspended in 300 μl staining buffer after centrifugation. The samples were stored at 4°C for a maximum of 6 hours before acquisition using a flow cytometer. Splenocytes were thawed at 37°C and washed twice (300×g, 5 minutes) with prewarmed complete RPMI 1640 media. Cells were seeded at a density 2 × 106/ml in 10 cm bacteriological plates in 20 ml complete RPMI 1640 media supplemented with 10% L929 media and incubated at 37°C at 5% CO2. On day 3, the plate was washed with prewarmed DPBS, to remove unattached cells, and the media was replaced. On day 6, the cells were harvested using 5 ml Accutase and resuspended at a concentration of 2 × 105 cells per well in poly-HEMA-coated 48-well plates in 490 μl RPMI 1640. Splenocytes were incubated in RPMI 1640 medium with either LPS (100 ng/ml) (two wells per sample) or RPMI 1640 only (one well per sample) for 6 hours at 37°C. For each sample, nigericin (10 μM, Sigma-Aldrich, USA) was added to one LPS well for the last 30 minutes of incubation. Following incubation, the cells were transferred into 1.5 ml microcentrifuge tubes and centrifuged at 300×g for 5 minutes to pellet cells; after which, they were fixed with 4% paraformaldehyde. Antibodies used for labelling were titrated to determine optimal dilution for experiments. The antibodies and dyes and their respective dilutions used for the study are as follows: mouse Fc block (BD Biosciences); CD11b BV421, clone M1/70 (BD Biosciences); F4/80 PE, clone T45-2342 (BD Biosciences); pro-IL-1β PE-Cy7, clone NJTEN3 (eBiosciences); and ASC/TMS1 Ax647 (Novus Biologicals). The cells were permeabilised with BD CytoFix/Cytoperm buffer for 20 minutes at 4°C and subsequently washed twice. Prior to staining, CD16/32 mouse Fc block was added to the samples for 5 minutes at 4°C to block nonspecific binding. Thereafter, a master mix of the appropriate antibodies for intracellular and extracellular markers was added and the samples were incubated for 30 minutes at 4°C in the dark. After incubation, the cells were resuspended in 1x BD perm buffer and centrifuged at 600×g for 5 minutes, at room temperature. After washing, the supernatant was discarded and the cells were resuspended in staining buffer before acquisition on the flow cytometer. Acquisition was performed on the BD FACSAria IIu flow cytometer (BD Biosciences), with BD FACSDiva™ version 8.1 software for data acquisition and analysis. Application settings in the BD FACSDiva software were used to standardize experimental data. As an experimental control, lot-specific 8-peak bead control was included as daily standardization validation to ensure that all settings were valid and reproducible on any flow cytometer employed for this purpose. All data files were exported as FCS 3.1 files and further analysed in FlowJo™ v10.4.2. The samples were resuspended by vortexing for 5 seconds prior to data acquisition. For the assessment of the glucocorticoid receptor expression level on specific leukocyte subpopulations, a minimum of 200 000 and a maximum of 500 000 live, gated, and singlet events were collected for each sample. The gating strategies are defined in Figure 2. Splenocytes were identified using FSC vs. SSC; thereafter, dead cells were excluded. Doublet discrimination was performed by applying a gate around the linear population in the SSC-H vs. SSC-A plot. Cells of interest were then identified from the single-cell population as follows: T-lymphocytes (TCRβ+ NK1.1-), NKT lymphocytes (TCRβ+ NK1.1+) NK cells (TCRβ- CD11b+ NK1.1+), neutrophils (TCRβ- CD11b+ Ly6G+), monocytes (TCRβ- CD11b+ F4/80-), and macrophages (TCRβ- CD11b+ F4/80+). Relative glucocorticoid receptor (NR3C1) expression for each cell population was quantified as relative median fluorescence intensity (MFI). Bulk gating was used to apply these gate coordinates to each generation, and all the gates were inspected and adjusted manually for each sample, if needed. All data for the experimental design was exported to Microsoft Excel. Representative images illustrating the gating strategy for basal glucocorticoid receptor expression on splenocytes (a) and assessment of inflammasome function (b). For the inflammasome assay, a minimum of 5000 CD11b+F4/80+ macrophages were collected per sample. All samples were run on application settings, and compensation was performed every run. The gating strategies are defined in Figure 2. Macrophages were gated on the FSC vs. SSC dot plot, and doublets were excluded using FSC-H vs. FSC-A. Macrophages were further identified by CD11b+F4/80+ expression, and within this population, pro-IL-1β expression was quantified as relative median fluorescent intensity (MFI). Inflammasome adaptor protein Apoptosis-Associated Speck-Like Protein Containing CARD (ASC) speck formation was assessed by plotting ASC-A vs. ASC-H. The ASC speck-containing cells were gated for quantification in the doublet gate as defined by accepted methodology [33]. As a brief introduction, the inflammasome complex is a multiprotein protein structure, responsible for the tightly controlled secretion of both IL-1β and IL-18 that recognises pathogens via Toll-like receptor binding in combination with NOD-like receptor binding. Inflammasome assembly, and thereby the release of biologically active IL-1β, is a two-step process: firstly, by the production of inactive pro-IL-1β, stimulated by TLR ligand binding, and secondly, the formation of the inflammasome complex (ASC formation) which cleaves inactive pro-IL-1β into active IL-1β (Jha, Brickey, Pan, & Ting, 2017; Strowig, Henao-Mejia, Elinav, & Flavell, 2012). The NLRP3 is the best-studied inflammasome complex and has been implicated in obesity, heart disease, neuroinflammation, and other systemic inflammatory dysregulation (Jha et al., 2017; Menu, Vince, Vince, & Menu, 2011; Strowig et al., 2012). Functional capacity of splenocytes, in terms of their basal and LPS-induced cytokine secretion profile, was determined for samples from all three generations of mice. Isolated splenocytes were resuspended in RPMI 1640 at a cell concentration of 1 × 106 cell/ml and plated in 24-well plates at 1 ml per well. The splenocytes were treated with either LPS (from Escherichia coli; Sigma, USA; serotype 0127: B8) at 1 μg/ml in RPMI 1640 (LPS-induced/stimulated) or complete RPMI 1640 only (basal/unstimulated) and incubated for 18 hours at 37°C, 5% CO2. After stimulation, culture supernatants were collected and stored at -80°C for batch analysis. The MAP Mouse Cytokine/Chemokine Magnetic Bead panel kit (Millipore, USA) was employed to assess the cytokine profile (IL-1β, IL-6, IL-10, TNF-α, and IFN-γ) in stimulated and unstimulated supernatant samples, using the Bio-Plex 200 system (Bio-Rad, USA) equipped with the Bio-Plex Manager™ software. Cytokine concentrations were automatically calculated based on a 6-point standard curve (in duplicate) fitted with a five-parameter logistic regression algorithm. The lowest limit for the detection of IL-1β, IL-6, TNF-α, IL-10, and IFN-γ was 1.1 pg/ml, 2.3 pg/ml, 2.0 pg/ml, and 1.1 pg/ml, respectively. The highest limit of detection was 10 000 pg/ml for all cytokine kits used. For flow cytometric data, percentage of cells for each leukocyte population identified and median fluorescent intensity (MFI) were used in statistical analyses. All data was exported to Microsoft Excel from respective analysis programs and consolidated. Data was analysed in Statistica version 13.2 (StatSoft Software, USA), and graphs were generated in GraphPad Prism 7.04 (GraphPad Software Incorporated, USA). After confirming normalcy of data distribution, one-way analysis of variance (ANOVA) was performed for the F0 LPS and saline comparison and a two-way analysis of variance (ANOVA) was employed for the F1 and F2 comparison. Fisher’s LSD post hoc tests were employed to analyse the statistical significance of differences between control and LPS-affected groups within the same generation. Data is presented as means and standard errors of the mean (SEM), and p < 0.05 or less was regarded as significant.
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