High Mobility Group Box 1 and TLR4 Signaling Pathway in Gnotobiotic Piglets Colonized/Infected with L. amylovorus, L. mucosae, E. coli Nissle 1917 and S. Typhimurium

Abstract

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can be actively secreted by immune cells after different immune stimuli or passively released from cells undergoing necrosis. HMGB1 amplifies inflammation, and its hypersecretion contributes to multiple organ dysfunction syndrome and death. We tested possible immunomodulatory effect of commensal Lactobacillus amylovorus (LA), Lactobacillus mucosae (LM) or probiotic Escherichia coli Nissle 1917 (EcN) in infection of gnotobiotic piglets with Salmonella Typhimurium (ST). Transcription of HMGB1 and Toll-like receptors (TLR) 2, 4, and 9 and receptor for advanced glycation end products (RAGE), TLR4-related molecules (MD-2, CD14, and LBP), and adaptor proteins (MyD88 and TRIF) in the ileum and colon were measured by RT-qPCR. Expression of TLR4 and its related molecules were highly upregulated in the ST-infected intestine, which was suppressed by EcN, but not LA nor LM. In contrast, HMGB1 expression was unaffected by ST infection or commensal/probiotic administration. HMGB1 protein levels in the intestine measured by ELISA were increased in ST-infected piglets, but they were decreased by previous colonization with E. coli Nissle 1917 only. We conclude that the stability of HMGB1 mRNA expression in all piglet groups could show its importance for DNA transcription and physiological cell functions. The presence of HMGB1 protein in the intestinal lumen probably indicates cellular damage.

Toll-Like Receptor 4 Signaling in the Ileum and Colon of Gnotobiotic Piglets Infected with Salmonella Typhimurium or Its Isogenic ∆rfa Mutants

“…However, these mutants resulted from spontaneous mutations with non-defined defects. A similar protective effect showed association of the gnotobiotic piglets with serum-sensitive probiotic E. coli Nissle 1917 with R chemotype of LPS [ 63 ]. In our experiments, we used isogenic ∆ rfa S .…”

“…The TLR2 mRNA can be also upregulated indirectly, e.g., by a nuclear protein HMGB1 (high mobility group box1) that is an intrinsic ligand of TLR2 [78]. It was released from damaged enterocytes of the wild-type and ∆rfaL S. Typhimurium-infected gnotobiotic piglets [63,64].…”

“…It was released from damaged enterocytes of the wild-type and ∆ rfaL S . Typhimurium-infected gnotobiotic piglets [ 63 , 64 ].…”

High Mobility Group Box 1 in Pig Amniotic Membrane Experimentally Infected with E. coli O55

“…Two µL cDNA template was added to 18 µL of the FastStart Universal Probe Master (Roche Diagnostic, Manheim, Germany) containing 100 nM LNA probe (Roche Diagnostic), and 500 nM each of the forward and reverse primers (Generi-Biotech, Hradec Kralove, Czechia) to quantify specific sequences in the cDNA templates. The used LNA probe-based Real-Time PCR, and primers for HMGB1, RAGE, TLR2, TLR4, MyD88, and TRIF were listed elsewhere [33]. PCR was performed by the iQ5 Real-Time PCR cycler (Bio-Rad, Hercules, CA, USA) at 95 • C for 10 min (1×); 95 • C for 15 s; and 60 • C for 60 s (45×).…”

“…Amniotic membranes were embedded in Tissue-Tek (Sakura, Tokyo, Japan), snapfrozen in isopentane cooled in liquid nitrogen vapor and stored at −70 • C. Five µm acetone-fixed cryosections were cut on a cryostat CM1860 UV (Leica Microsystems, Wetzlar, Germany), put on SuperFrost/Plus slides (Thermo Fisher Scientific, Darmstadt, Germany), and were kept at −40 • C until labeling. Later, the sections were processed as described elsewhere [33]. Briefly, they were blocked with 10% normal rabbit serum (Life Technologies, Carlsbad, CA, USA) for 1 h at RT, incubated with anti-HMGB1 rabbit polyclonal antibodies (Novus Biologicals, Centennial, CO, USA) for 16 h at 4 • C, and labeled with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibodies (Life Technologies) for 2 h at RT After embedding in ProLong Gold Antifade Reagent (Life Technologies), the sections were evaluated under an Olympus BX 40 microscope with an Olympus Camedia C-2000 digital camera (Olympus, Tokyo, Japan).…”