Fig 2: Escitalopram mitigated D-galactose/ovariectomy-induced alterations in the hippocampal protein expression of (a) p-PI3K, (b) p-Akt, (c) p-GSK-3ß, (d) p-Raf-1, (e) p-MEK1/2, and (f) p-ERK1/2 in rats. The cropped blots for these proteins were presented relative to that of ß-actin, and the uncropped images are available in the Supplementary File. Each bar with a vertical line represents mean ± SD (n= 5). * vs SO group, # vs D-gal/OVX group, using one-way ANOVA followed by Tukey’s multiple comparisons test; p < 0.05. SO sham operation, D-gal D-galactose, OVX ovariectomy, Esc escitalopram, PI3K phosphoinositide 3-kinase, Akt protein kinase B, GSK-3ß glycogen synthase kinase-3ß, ERK extracellular signal-regulated kinase.

Fig 3: Analysis of PGE2 biosynthesis and MEK function in THP-1 macrophages in response to treatment with ?yopB Y. enterocolitica, YopJC172A Y. pseudotuberculosis, or the chemical MEK inhibitor PD184161. PMA-differentiated THP-1 macrophages were pretreated with or without 10 µM PD184161 (MEK1/2 inhibitor) 1 h before infection. Macrophages were uninfected (Ctrl) or were infected with wild-type or ?yopB Y. enterocolitica (Ye) (A) or wild-type or YopJC172A Y. pseudotuberculosis (Yptb) (B and C) for 2 h at an MOI of 50:1. The PGE2 concentration from infected cell supernatants was measured by a commercial monoclonal ELISA recognizing PGE2 (A and B). Western blotting was used to quantify MEK1/2 and phosphorylated MEK/2 (C and D), where the fold change was calculated using densitometry analysis by normalizing to phosphorylated MEK 1/2 to MEK1/2 levels (D). The ß-actin was used as a loading control. Two-way ANOVA and Tukey’s post hoc test were used to calculate significance for Western blot densitometry (n = 3). P values are indicated (*, P = 0.05; **, P = 0.01; ***, P = 0.001; ****, P = 0.0001). The images shown are representative of three independent experiments.

Fig 4: Model of PGE2 and IL-1ß biosynthesis in macrophages during Gram-negative bacterial infection. (Step 1) LPS ligand binding to TLR4 induces rapid phosphorylation of host MAPKs, such as MEK1/2, with adaptor protein assistance, including MyD88. (Step 2) MEK1/2 becomes activated and phosphorylates ERK1/2, leading to the activation and migration of transcription factors to the nucleus to induce proinflammatory gene transcription, including upregulation of COX-2 transcripts. (Step 3) Activated ERK1/2 phosphorylates and activates cPLA2 at Ser 515/505 residue, resulting in the liberation of AA from membrane phospholipids and AA’s conversion into PGH2/PGE2 by COX-2. (Step 4) PGE2 is secreted and binds in an autocrine fashion to specific EP2/4 receptors to activate IL-1ß transcription by altering cAMP levels or PI3K-induced migration of transcription factors NF-?B and AP-1. In the presence of Gram-negative bacteria, pro-IL-1ß is cleaved to mature IL-1ß by caspase-1 due to inflammasome activation. (Step 5) IL-1ß forms a positive-feedback loop with PGE2 by binding to IL-1R and increasing COX-2 transcription. Y. pseudotuberculosis and S. flexneri modulate inflammasome activation and PGE2 biosynthesis by manipulating host MAPK/ERK-dependent signaling such as T3SS factors YopJ (Y. pseudotuberculosis) or OspB/OspF (S. flexneri).