Addition Of Flagellin Research Articles

A8 REPEATED SUBMERGENCE OF AIR-LIQUID INTERFACE COLONOID CULTURES IMPAIRS INFLAMMATORY AND REGENERATIVE RESPONSES

Abstract Background Damage in the intestinal epithelium is repaired via de-differentiation of mature intestinal epithelial cells to a stem-like state. Indeed, literature has primarily focused on acute forms of intestinal damage, but there is a lack of models to study how intestinal stem cells function after chronic injury, such as in inflammatory bowel disease (IBD). A previous report found that growth of mouse intestinal organoids in air-liquid interface (ALI) follows by submergence caused differentiation and reversible injury, but this has not been demonstrated in human cells or with repeated cycles of injury. Understanding how chronic damage alters human intestinal stem cell fate and function is imperative to developing novel therapies that repair the epithelium in people with IBD Aims To develop a robust in vitro model to differentiate and damage human intestinal epithelial cells, with or without the addition of bacterial flagellin to mimic pathogen exposure. Methods Human colonoid monolayers were seeded on Transwell inserts for 10 days until fully confluent and then differentiated by removing the apical media to create ALI growth conditions for 7 days. To induce damage, media was added to the apical side of the Transwell, with or without the addition of flagellin in the basolateral compartment. Following submergence induced damage, the apical media was removed and collected for chemokine analysis, and the cells were grown back in ALI for 3 days to recover them from injury. This cycle was repeated 5 times to induce chronic damage. Cells were collected for qPCR analysis, immunofluorescence imaging, RNA sequencing and DNA methylation analysis Results Repeated rounds of damage impaired the ability of intestinal epithelial cells (IECs) to respond to TLR stimulation (a decrease in basolateral IL-8 with each round), likely due to a decrease in TLR signaling pathways, as demonstrated by GSEA and qPCR. Chronic submergence damage led to an increase in differentiation of cells expressing MUC2, SLC26a3 and CHGA, and a decrease in stemness as shown by qPCR for BMI1, HOPX, and LGR5. After several rounds of damage, colonoid monolayers were unable to regrow as monolayers after passaging, likely due to a decrease in YAP signaling. We also identified mRNA expression and DNA methylation changes in genes associated with IBD and colon cancer. Conclusions We have developed a novel chronic damage model of recurrent IEC injury, which possibly mimics pathologies seen in people with inflammatory bowel disease. This model can be used to understand how chronic damage alters the ability of IECs to respond to pathogens and regenerate to repair and protect the epithelium from further damage. Funding Agencies CCC

Vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge

Current influenza vaccines should be improved by the addition of universal influenza vaccine antigens in order to protect against multiple virus strains. We used our self-assembling protein nanoparticles (SAPNs) to display the two conserved influenza antigens M2e and Helix C in their native oligomerization states. To further improve the immunogenicity of the SAPNs, we designed and incorporated the TLR5 agonist flagellin into the SAPNs to generate self-adjuvanted SAPNs. We demonstrate that addition of flagellin does not affect the ability of SAPNs to self-assemble and that they are able to stimulate TLR5 in a dose-dependent manner. Chickens vaccinated with the self-adjuvanted SAPNs induce significantly higher levels of antibodies than those with unadjuvanted SAPNs and show higher cross-neutralizing activity compared to a commercial inactivated virus vaccine. Upon immunization with self-adjuvanted SAPNs, mice were completely protected against a lethal challenge. Thus, we have generated a self-adjuvanted SAPN with a great potential as a universal influenza vaccine.

Flagellin-stimulated Cl− secretion and innate immune responses in airway epithelia: role for p38

Activation of an innate immune response in airway epithelia by the human pathogen Pseudomonas aeruginosa requires bacterial expression of flagellin. Addition of flagellin (10(-7) M) to airway epithelial cell monolayers (Calu-3, airway serous cell-like) increased Cl(-) secretion (I(Cl)) beginning after 3-10 min, reaching a plateau after 20-45 min at DeltaI(Cl) = 15-50 microA/cm(2). Similar, although 10-fold smaller, responses were observed in well-differentiated bronchial epithelial cultures. Flagellin stimulated I(Cl) in the presence of maximally stimulating doses of the purinergic agonist ATP, but had no effects following forskolin. IL-1beta (produced by both epithelia and neutrophils during infections) stimulated I(Cl) similar to flagellin. Flagellin-, IL-1beta-, ATP-, and forskolin-stimulated I(Cl) were inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) blockers GlyH101, CFTRinh172, and glibenclamide. Neither flagellin nor IL-1beta altered transepithelial fluxes of membrane-impermeant dextran (10 kDa) or lucifer yellow (mol wt = 457), but both activated p38, NF-kappaB, and IL-8 secretion. Blockers of p38 (SB-202190 and SB-203580) reduced flagellin- and IL-1beta-stimulated I(Cl) by 33-50% but had smaller effects on IL-8 and NF-kappaB. It is concluded that: 1) flagellin and IL-1beta activated p38, NF-kappaB, IL-8, and CFTR-dependent anion secretion without altering tight junction permeability; 2) p38 played a role in regulating I(Cl) and IL-8 but not NF-kappaB; and 3) p38 was more important in flagellin- than IL-1beta-stimulated responses. During P. aeruginosa infections, flagellin and IL-1beta are expected to increase CFTR-dependent ion and fluid flow into and bacterial clearance from the airways. In cystic fibrosis, the secretory response would be absent, but activation of p38, NF-kappaB, and IL-8 would persist.

Gram-negative Flagellin-induced Self-tolerance Is Associated with a Block in Interleukin-1 Receptor-associated Kinase Release from Toll-like Receptor 5

Flagellin from a number of Gram-negative bacteria induces cytokine and nitric oxide production by inflammatory cell types. In view of the evidence that flagellin responsiveness is subject to modulation, we explored the possibilities that a prior exposure to flagellin might result in a state of reduced flagellin responsiveness or tolerance and that lipopolysaccharide (LPS) and flagellin may induce a state of cross-tolerance to each other. Our results demonstrate that a prior exposure to flagellin results in a subsequent state of flagellin tolerance in human monocytes, THP1 cells, Jurkat cells, and COS-1 cells. Tolerance occurs within 2 h after addition of flagellin and does not require protein synthesis. Flagellin did not induce tolerance to LPS in monocytes and THP1 cells; however, LPS treatment of monocytes and THP1 cells resulted in a state of flagellin cross-tolerance. Flagellin-induced self-tolerance is not the result of a decrease in the steady-state level of toll-like receptor 5 (TLR5) or interleukin-1 receptor associated kinase (IRAK), but it is associated with a block in the release of IRAK from the TLR5 complex in flagellin-tolerant cells. Release is essential for IRAK activity because the TLR5-associated IRAK lacks kinase activity. LPS-induced cross-tolerance to flagellin is also associated with a block in IRAK release from TLR5. These results provide evidence for a novel mechanism of TLR signaling control.

On flagellar formation in Caulobacter crescentus: novel flagellin synthesis in stub-forming non-motile mutants of C. crescentus.

Defective flagellar organella, stubs, and flagellin synthesis by L-[35S]methionine labeling of two non-motile mutants of C. crescentus CB15 were studied. CB15 wild type synthesized two flagellin species, flagellin A of molecular weight 26,000 and flagellin B of molecular weight 28,000. A DNA phage phi0CbK-resistant non-motile mutant, CB15 pdr-816 [Fukuda et al. (1981) J. Bacteriol. 145, 559-572] did not synthesize flagellin B but synthesized large amounts of flagellin A and a novel flagellin of smaller molecular weight 22,000 (22K flagellin), and formed stubs composed of both flagellin A and 22K flagellin. The other mutant, CB15 fla-801 synthesized only 22K flagellin and formed very short stubs composed of 22K flagellin. Motile revertants were isolated from these mutants. Full revertants were isolated from CB15 fla-801 at a frequency of ca. 10(-7). However, only partial revertants swimming slowly were obtained from CB15 pdr-816 at a similar frequency. Such partial revertants synthesized only flagellin A and formed short flagella in spite of the fact that flagellin A was still synthesized at a higher level than in the wild type. Given the previous finding that flagellin B first polymerizes onto the hook followed by addition of flagellin A onto flagellin B filament [Koyasu et al. (1981) J. Mol. Biol. 153, 471-475], flagellin B seems very important in the formation of normal flagella in C. crescentus. A small amount of 22K flagellin was detected even in wild type cell. Neither conversion nor alteration was observed in flagellin A or B or 22K flagellin by short-pulse and pulse-chase experiments. It was thus suggested that 22K flagellin was not a degradation product formed from other flagellins but was of different origin from flagellins A and B. C. crescentus flagellins consist of several subspecies of different isoelectric points. 22K flagellin consisted of at least three subspecies, in two-dimensional gel, with more basic isoelectric points than other flagellins. Heterogeneity in isoelectric point of polymerized and nascent flagellins was similar, indicating that each subspecies of different isoelectric point can polymerize into filament, and that conversion between subspecies is not needed to form flagellar filament.