Germ-free and conventional male Sprague Dawley rats were obtained from Taconic Biosciences (Germantown, NY). These mice were provided autoclaved LabDiets (Cat #5001) and water ad libitum, and were maintained in glove box isolators (Park Bioservices, Groveland, MA) and IsoCages (Techniplast Inc., Buguggiate, Italy). Germ-free mice on C57BL/6 background from Taconic Biosciences (Rensselaer, NY) were bred and housed in the germ-free animal facility at Georgia State University. These mice were maintained on specific pathogen-free conditions and housed 4–5 per cage with Enrich-n’Pure paper bedding material, and provided ad libitum standard rodent chow diet from LabDiets (Cat #5001) and autoclaved water. The aforementioned genetically altered mice were bred with WT mice to generate their respective littermates.
#Crossout text Activator
These results suggest that flagellin (a protein and the principal component of bacterial flagella) is a dominant innate immune activator of gut microbiota, particularly in scenarios involving breach of the intestine.Ĭonventional WT, Myd88KO, Tlr4KO, Tlr5KO and Il10KO mice on C57BL/6 background, which were originally obtained from Jackson Laboratories, were housed and bred in the Department of Laboratory Animal Resources, University of Toledo College of Medicine and Life Sciences, Toledo, OH. However, such activation did not require TLR4 rather, such immune response was gut microbiota-dependent and driven by a heat-stable, protease-sensitive ligand whose signaling required the flagellin receptor, TLR5. Herein, we report that murine cecal content and fecal extracts indeed had high potential to activate innate immunity as reflected by induction of pro-inflammatory cytokine expression. To this end, we administered mice with gastrointestinal extract contents via intraperitoneal injection to emulate scenarios where intestinal barrier was bypassed. The goal of this study was to better define the potential of the microbe-derived luminal contents to activate innate immunity when given broad access to extraintestinal host tissues. These protective mechanisms could be undermined by acute mucosal breaches and/or persistently compromised epithelial permeability ( alias leaky gut), resulting in MAMPs translocating from the gut to other organs/tissues resulting in PRR activation and, consequently, inflammation. Several mechanisms are in place to prevent or subdue activation of PRR by MAMPs, which include limiting the expression of PRR on the basolateral side of IEC thus separating them from their ligands in the gut lumen 2, and secreting soluble forms of TLR that bind and sequester their ligands, thus preventing the ligands from inducing membrane-bound TLR signaling 3–5. These microbe-associated molecular patterns (MAMPs) include ligands for pathogen recognition receptors (PRR) such as toll-like receptors (TLR) and nod-like receptors, which are in active dialog with intestinal epithelial cells (IEC) and mucosal immune cells in the lamina propria. It has been estimated that there are 10 11 bacteria per gram of stool in the healthy colon 1, thereby putting the colonic mucosa in very close proximity to the highly dense concentration of various microbial products. The mammalian intestines are residence for numerous microorganisms collectively known as the ‘gut microbiota’. Taken together, this work serves to underscore flagellin as a major, heat-stable and bioactive MAMP in the intestinal content that confers this milieu strong potential to trigger innate immune responses. Accordingly, pre-treating intestinal extracts with proteinase, thereby degrading flagellin, was sufficient to block their ability to activate innate immune responses. Such immune responses were abrogated in the absence of either myeloid differentiation factor 88 (MyD88) or Toll-like receptor (TLR) 5, but not TLR4, suggesting that the stimuli was flagellin ( i.e., protein subunit of flagella that drives bacterial motility). Here, we demonstrated that intestinal contents from conventional, but not germ-free, mice and rats triggered robust innate immune responses in vitro and in vivo. We undertook this study to assess the predominant microbe-associated molecular patterns (MAMPs) present therein and the receptors) that mediate the innate immune responses to them. Intestinal contents comprise the largest repository of immunogenic ligands of microbial origin.
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