c rodentium strain dbs100  (ATCC)

Journal: bioRxiv

Article Title: Myeloid PINK1 represses mtDNA release and immune signaling that impacts neuronal pathology in patient-derived idiopathic PD models

doi: 10.64898/2026.01.07.694713

Figure Lengend Snippet: A) Schematic diagram illustrating gastrointestinal infection with C. rodentium via gavage of mice where tdTomato (Ai9) is controlled under the dopamine transporter promoter (referred to as DAT-Cre). Both wild type (WT) and PINK1 KO (PiKO) mice were assessed including uninfected controls. Immunofluorescence was performed for enteric neuronal characterization at 2-w.p.i.; single-cell RNAseq was conducted on lamina propria cells for immunophenotyping at 1-w.p.i. (previously published by Recinto et al . ) B, C) Representative immunofluorescence staining with cross-section view of colonic tissue from DAT-Cre mice. Anti-tubulin beta 3 class III (TUBB3) antibody was used to detect enteric neurons in green (B). Dopaminergic neurons immunoreactive for tdTomato are labelled in red and cells expressing G-protein-regulated inward-rectifier potassium channel 2 (GIRK2) are shown in green (C). Nuclei are stained with Hoechst (blue). Myenteric ganglia are outlined by white dotted box. The inset is a magnified view of enteric neurons and arrow heads indicate immunoreactive cells. Scale bars: 50 μm and 20 μm (for the inset). The density of neurons normalized to ganglionic area (in mm 2 ) is quantified across all groups. Two-way ANOVA followed by Tukey’s multiple comparison test was applied. Mean ± SEM, n=3-4 mice per group with 5 ganglia per mouse evaluated. D) Re-analysis of single-cell RNAseq of mouse intestinal immune compartment following C. rodentium infection taken from Recinto et al . . Lollipop plot displays proportions of significant differentially expressed genes (DEG) per major immune cell type previously annotated between infected WT and PiKO mice normalized to total DEGs detected in all immune cells. Wilcoxon Rank Sum test using Bonferroni correction was applied. Enriched Gene Ontological (GO) terms in the monocytes/macrophages (Mϕ) population were assessed. Amongst the top 10%, key GO terms were highlighted in the dot plot. The number of DEG underlying a specified GO term is denoted by the size of the dots while the color scale represents the adj. p-value. The False Discovery Rate (FDR) test was applied. E) Heatmap depicting the likelihood of a given ligand from neighboring immune cells could influence the expression of the DEG in infected PiKO monocyte/Mϕ population, termed as regulatory potential. Regulatory potential is determined computationally based on the pearson correlation coefficient between a ligand’s target predictions and the observed transcriptional response. Arrowhead indicates the highest ranked prioritized ligand, likely contributing to transcriptional alterations in infected PiKO monocyte/Mϕ population. F, G) Bone marrow-derived Mϕ (BMDM) were cultured from WT and PiKO mice and stimulated for 24 h with lipopolysaccharide (LPS) and mouse recombinant interleukin-1beta (IL-1β). Cell surface expression of major histocompatibility complex 2 (MHC2) and Toll-like receptor 2 (TLR2) were assessed by flow cytometry (F) as well as cell supernatant for secreted amounts of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) by ELISA (G). Shown are relative mean fluorescence intensities (MFI) of cell surface activation markers and relative concentrations of pro-inflammatory cytokines (in pg/mL) across all groups and normalized to the average level in non-stimulated (NS) WT cells. Two-way ANOVA followed by Tukey’s multiple comparison test was applied. Mean ± SEM, n=3-5 mice per group. H) Graphical representation of a functional in vitro assay to examine the impact of PiKO macrophages on enteric neuronal survival. Primary enteric neurons (1’) and BMDM (1’’) were cultured separately from WT only or both WT and PiKO mice, respectively. Following differentiation of BMDM, cells were stimulated with LPS and mouse recombinant IL-1β for 24h to induce activation (2). Seven days after plating, enteric neurons were treated with conditioned medium (CM) from BMDM for 48h (3). I) Representative immunofluorescence staining of mouse primary enteric neurons using a pan-neuronal marker TUBB3. Scale bars: 100 μm. J) The proportion of TUBB3 + and TUBB3 + DAT-tdTOM + neurons across all groups normalized to the cells receiving BMDM basal medium in the presence of stimuli (to account for possible effects from base medium) was quantified using an automated software for high-throughput imaging (MetaXpress). Staurosporine was used as positive control. Two-way ANOVA followed by Tukey’s multiple comparison test was applied. Mean ± SEM, n=3 independent experiment with each experiment consisted of pooled CM from 3 mice and comprised of triplicates each with 45 sites analyzed.

Article Snippet: As described in Recinto et al. [ ], mice were infected with chloramphenicol-resistant C. rodentium (4 × 10 9 CFU, ATCC 51459) by oral gavage.

Techniques: Infection, Immunofluorescence, Staining, Expressing, Comparison, Derivative Assay, Cell Culture, Recombinant, Immunopeptidomics, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Fluorescence, Activation Assay, Functional Assay, In Vitro, Marker, Software, High Throughput Screening Assay, Imaging, Positive Control