Fig 1: 8-AG reduces the expression of pro-inflammation genes, reduces the levels of pro-inflammatory cytokines, and affects the purine metabolome in F344 rat retinae.Retina from young, water-treated aged, and 8-AG-treated aged rats were harvested, retinal lysates were used for qPCR, multiplex cytokine profiling using a flow-cytometer, and purine metabolome quantification using UPLC-MS/MS. a–f Transcript levels in the retinae for the following genes normalized with Gapdh level. N = 3. a Mt2a, b Adgre1, c Itgal, d Cd68, e Itgb2, and f Rt1-da. g–l Retinal cytokine/chemokine levels normalized by net retina weight and calibrated by standard curves. N = 6. g IL-33, h GM-CSF, i IL-28, j IL-1-alpha, k MCP-1, and l IL12p-70 (the 70 kDa complex of IL-12). m A brief illustration reminding the salvage and degradation of purine nucleosides. The tissue-damaging purines are in red background; 8-aminoguanine (8-AG) shown in light blue background, is a PNPase inhibitor. n–z Concentrations of retinal purine metabolites from young (black circle), water-treated aged (magenta square), and 8-AG-treated aged rats (blue triangle), normalized with the total protein in micrograms (ng of metabolite/µg of total protein). N = 4. n 8-AG; o Guanosine; p Adenosine; q Inosine; r 8-Aminoguanosine; s Guanine; t Adenine; u Hypoxanthine; v Xanthine; w 3’5’-cGMP; x 5’-GMP; y 3’5’-cAMP; z 5’-AMP. aa Dot plot of transcripts involved in the purine salvage and degradation pathway from young (black) and aged (magenta) rat retinae, using RNA-seq data. b Dot plot of transcripts involved in the purine salvage and degradation pathway from aged (magenta) and 8-AG-treated aged (blue) rat retinae, using the RNA-seq data. N = 3. Data and error bars are means±SDs. P values were calculated using One-way ANOVA for all the plots. ns stands for no significance.
Fig 2: Sleep deprivation is associated with an increase in LBP and proinflammatory cytokines levels in circulation, colon, and reproductive organs in female and male rats.(A) Comparison of LBP, IL-1β, IL-6, and TNF-α protein levels in the circulatory system between control and sleep-deprived groups in female (superior section) and male (inferior section) rats. (B) Comparison of mRNA levels of IL-1β, IL-6, and TNF-α in the colon between control and sleep-deprived groups in female (left section) and male (right section). (C) Comparison of TNF-α, IL-1β, and IL-6 mRNA levels in reproductive organs between control and sleep-deprived groups in female (left section) and male (right section) rats. Data are presented as mean ± SEM (n = 6 in each group), *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 3: FimHDG-Ferritin mRNA vaccination induces FimH-specific cellular immune responses in the spleen of UPEC-naïve animals. FimH-specific T cell responses were measured in the splenocytes of FimHDG-Ferritin mRNA-vaccinated animals in dose-response immunogenicity studies in (A, B) female BALB/c AnNRj mice (n=12/dose schedule) and (C) female Wistar rats (n=12/dose schedule). UPEC-naïve animals received either 2 or 3 IM administrations of FimHDG-Ferritin mRNA vaccine on Days 0, 28 (all) and 56 (3-dose only) at doses of 0.04, 0.2, 1 or 5 μg (mice) and 0.08, 0.4, 2, 10 or 20 μg (rats). Control animals received 0.9% NaCl (n=6/dose schedule). Isolated splenocytes were re-stimulated with a 15-mer peptide library spanning the FimH full length protein. FimH-specific (A) CD8+ and (B) CD4+ and (C) IFNγ-secreting T cell responses were analysed in (A, B) mice samples by ICS and flow cytometry (gating analysis shown in Supplementary Figure 6) and (C) rat samples by ELISpot, two weeks post second administration (Day 42, orange bars, 2-dose schedule only) or third administration (Day 70, blue bars). Due to limited sample availability, sample sizes for specific groups were reduced as follows: (C) Day 42: 0.4 µg (n=11), 10 µg (n=11). Each dot represents an individual animal, bars depict the geometric mean, error bars depict the geometric SD. (A, B) Dotted lines represent the respective LLOQ. The statistical difference between 2- and 3-dose schedules across all dose levels was analysed using a two-way ANCOVA (***P < 0.001; ns: not significant). ANCOVA, Analysis of Covariance; CD, cluster of differentiation; ELISpot, enzyme linked immunospot; ICS, intracellular cytokine staining; IM, intramuscular; LLOQ, lower limit of quantification; mRNA, messenger RNA; SD, standard deviation TNF, tumour necrosis factor; UPEC, uropathogenic E. coli..
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