Genomic and Transcriptomic Diversification of Flagellin Genes Provides Insight into Environmental Adaptation and Phylogeographic Characteristics in Aeromonas hydrophila.

Aeromonas hydrophila is an opportunistic motile pathogen with a broad host range, infecting both terrestrial and aquatic animals. Environmental and geographical conditions exert selective pressure on both geno- and phenotypes of pathogens. Flagellin, directly exposed to external environments and containing important immunogenic epitopes, may display significant variability in response to external conditions. In this study, we conducted a comparative analysis of ~ 150 A. hydrophila genomes, leading to the identification of six subunits of the flagellin gene (fla-1 to fla-4, flaA, and flaB). Individual strains harbored different composition of flagellin subunits and copies. The composition of subunits showed distinct patterns depending on environmental sources. Strains from aquatic environments were mainly comprised of fla-1 to fla-4 subunits, while terrestrial strains predominated in groups harboring flaA and flaB subunits. Each flagellin showed varying levels of expression, with flaA and flaB demonstrating significantly higher expression compared to others. One of the chemotaxis pathways that control flagellin movement through a two-component system was significantly upregulated in flaA(+ 1)/flaB(+ 1) group, whereas flaA and flaB showed different transcriptomic expressions. The genes positively correlated with flaA expression were relevant to biofilm formation and bacterial chemotaxis, but flaB showed a negative correlation with the genes in ABC transporters and quorum sensing pathway. However, the expression patterns of fla-2 to fla-4 were identical. This suggests various types of flagellin subunits may have different biological functions. The composition and expression levels of flagellin subunits could provide valuable insights into the adaptation of A. hydrophila and the differences among strains in response to various external environments.

Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-terminal cleavage of flg22 epitope from bacterial flagellin.

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure.

A potential virulence factor: Brucella flagellin FliK does not affect the main biological properties but inhibits the inflammatory response in RAW264.7 cells.

The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.

Dendritic cell targeting peptide plus Salmonella FliCd flagellin fused outer membrane protein H (OmpH) demonstrated increased efficacy against infections caused by different Pasteurella multocida serogroups in mouse models.

As the major outer membrane protein (OMP) presents in the Pasteurella multocida envelope, OmpH was frequently expressed for laboratory assessments of its immunogenicity against P. multocida infections, but the results are not good. In this study, we modified OmpH with dendritic cell targeting peptide (Depeps) and/or Salmonella FliCd flagellin, and expressed three types of recombinant proteins with the MBP tag (rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, rFliC-OmpH-MBP). Assessments in mouse models revealed that vaccination with rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, or rFliC-OmpH-MBP induced significant higher level of antibodies as well as IFN-γ and IL-4 in murine sera than vaccination with rOmpH-MBP (P < 0.5). Vaccination with the three modified proteins also provided increased protection (rDepeps-FliC-OmpH-MBP, 70 %; rDepeps-OmpH-MBP, 50 %; rFliC-OmpH-MBP, 60 %) against P. multocida serotype D compared to vaccination with rOmpH-MBP (30 %). In mice vaccinated with different types of modified OmpHs, a significantly decreased bacterial strains were recovered from bloods, lungs, and spleens compared to rOmpH-MBP-vaccinated mice (P < 0.5). Notably, our assessments also demonstrated that vaccination with rDepeps-FliC-OmpH-MBP provided good protection against infections caused by a heterogeneous group of P. multocida serotypes (A, B, D). Our above findings indicate that modification with DCpep and Salmonella flagellin could be used as a promising strategy to improve vaccine effectiveness.

Structure and biological activity in vitro of Flagellin and its mutants from Escherichia coli Nissle 1917.

Bacterial flagellin is a potent immunomodulatory agent. Previously, we successfully obtained flagellin from Escherichia coli Nissle 1917 (FliCEcN) and constructed two mutants with varying degrees of deletion in its highly variable regions (HVRs). We found that there was a difference in immune stimulation levels between the two mutants, with the mutant lacking the D2-D3 domain pair of FliCEcN having a better adjuvant effect. Therefore, this study further analyzed the structural characteristics of the aforementioned FliCEcN and its two mutants and measured their levels of Caco-2 cell stimulation to explore the impact of different domains in the HVRs of FliCEcN on its structure and immune efficacy. This study utilized AlphaFold2, SERS (Surface-enhanced Raman spectroscopy), and CD (circular dichroism) techniques to analyze the structural characteristics of FliCEcN and its mutants, FliCΔ174-506 and FliCΔ274-406, and tested their immune effects by stimulating Caco-2 cells in vitro. The results indicate that the D2 and D3 domains of FliCEcN have more complex interactions compared to the D1-D2 domain pair., and these domains also play a role in molecular docking with TLR5 (Toll-like receptor 5). Furthermore, FliCΔ274-406 has more missing side chain and characteristic amino acid peaks than FliCΔ174-506. The FliCEcN group was found to stimulate higher levels of IL-10 (interleukin 10) secretion, while the FliCΔ174-506 and FliCΔ274-406 groups had higher levels of IL-6 (interleukin 6) and TNF-α (tumor necrosis factor-α) secretion. In summary, the deletion of different domains in the HVRs of FliCEcN affects its structural characteristics, its interaction with TLR5, and the secretion of immune factors by Caco-2 cells.

Nasal vaccination of triple-RBD scaffold protein with flagellin elicits long-term protection against SARS-CoV-2 variants including JN.1.

Developing a mucosal vaccine against SARS-CoV-2 is critical for combatting the epidemic. Here, we investigated long-term immune responses and protection against SARS-CoV-2 for the intranasal vaccination of a triple receptor-binding domain (RBD) scaffold protein (3R-NC) adjuvanted with a flagellin protein (KFD) (3R-NC + KFDi.n). In mice, the vaccination elicited RBD-specific broad-neutralizing antibody responses in both serum and mucosal sites sustained at high level over a year. This long-lasting humoral immunity was correlated with the presence of long-lived RBD-specific IgG- and IgA-producing plasma cells, alongside the Th17 and Tfh17-biased T-cell responses driven by the KFD adjuvant. Based upon these preclinical findings, an open labeled clinical trial was conducted in individuals who had been primed with the inactivated SARS-CoV-2 (IAV) vaccine. With a favorable safety profile, the 3R-NC + KFDi.n boost elicited enduring broad-neutralizing IgG in plasma and IgA in salivary secretions. To meet the challenge of frequently emerged variants, we further designed an updated triple-RBD scaffold protein with mutated RBD combinations, which can induce adaptable antibody responses to neutralize the newly emerging variants, including JN.1. Our findings highlight the potential of the KFD-adjuvanted triple-RBD scaffold protein is a promising prototype for the development of a mucosal vaccine against SARS-CoV-2 infection.

Protective Activity of Inactivated Rabies Vaccine Using Flagellin-Based Adjuvant.

Rabies is a zoonotic disease with high lethality. Most human deaths are associated with the bites received from dogs and cats. Vaccination is the most effective method of preventing rabies disease in both animals and humans. In this study, the ability of an adjuvant based on recombinant Salmonella typhimurium flagellin to increase protective activity of the inactivated rabies vaccine in mice was evaluated. A series of inactivated dry culture vaccine for dogs and cats "Rabikan" (strain Shchelkovo-51) with addition of an adjuvant at various dilutions were used. The control preparation was a similar series of inactivated dry culture vaccine without an adjuvant. Protective activity of the vaccine preparations was evaluated by the NIH potency test, which is the most widely used and internationally recommended method for testing effectiveness of the inactivated rabies vaccines. The value of specific activity of the tested rabies vaccine when co-administered with the adjuvant was significantly higher (48.69 IU/ml) than that of the vaccine without the adjuvant (3.75 IU/ml). Thus, recombinant flagellin could be considered as an effective adjuvant in the composition of future vaccine preparations against rabies virus.

Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation.

Glycosyltransferases play a fundamental role in the biosynthesis of glycoproteins and glycotherapeutics. In this study, we investigated protein glycosyltransferase FlgGT1, belonging to the GT2 family. The GT2 family includes cysteine S-glycosyltransferases involved in antimicrobial peptide biosyntheses, sharing conserved catalytic domains while exhibiting diverse C-terminal domains. Our in vitro studies revealed that FlgGT1 recognizes structural motifs rather than specific amino acid sequences when glycosylating the flagellin protein Hag. Notably, FlgGT1 is selective for serine or threonine O-glycosylation over cysteine S-glycosylation. Molecular dynamics simulations provided insights into the structural basis of FlgGT1's ability to accommodate various sugar nucleotides as donor substrates. Mutagenesis experiments on FlgGT1 demonstrated that truncating the relatively large C-terminal domain resulted in a loss of flagellin glycosylation activity. Our classification based on sequence similarity network analysis and AlphaFold2 structural predictions suggests that the acquisition of the C-terminal domain is a key evolutionary adaptation conferring distinct substrate specificities on glycosyltransferases within the GT2 family.

Preventive nasal administration of flagellin restores antimicrobial effect of gentamicin and protects against a multidrug-resistant strain of Pseudomonas aeruginosa.

The increasing prevalence of multidrug-resistant Pseudomonas aeruginosa (PA) is a significant concern for chronic respiratory disease exacerbations. Host-directed drugs, such as flagellin, an agonist of toll-like receptor 5 (TLR5), have emerged as a promising solution. In this study, we evaluated the prophylactic intranasal administration of flagellin against a multidrug-resistant strain of PA (PAMDR) in mice and assessed the possible synergy with the antibiotic gentamicin (GNT). The results indicated that flagellin treatment before infection decreased bacterial load in the lungs, likely due to an increase in neutrophil recruitment, and reduced signs of inflammation, including proinflammatory cytokines. The combination of flagellin and GNT showed a synergistic effect, decreasing even more the bacterial load and increasing mice survival rates, in comparison to mice pre-treated only with flagellin. These findings suggest that preventive nasal administration of flagellin could restore the effect of GNT against MDR strains of PA, paving the way for the use of flagellin in vulnerable patients with chronic respiratory diseases.

Immunoprotection of FliBc chimeric fiber2 fusion proteins targeting dendritic cells against Fowl adenovirus serotype 4 infection.

Hepatitis-hydropericardium syndrome (HHS) is a highly fatal disease in chickens caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4), which has severe economic consequences. The fiber2 protein exhibits excellent potential as a candidate for a subunit vaccination against FAdV-4. Despite having a high safety profile, subunit vaccines have low immunogenicity due to their lack of infectivity, which leads to low levels of immune response. As a vaccine adjuvant, Salmonella flagellin possesses the potential to augment the immunological response to vaccinations. Additionally, a crucial strategy for enhancing vaccine efficacy is efficient presentation of immune antigens to dendritic cells (DC) for targeted vaccination. In this study, we designed FAdV-4-fiber2 protein, and a recombinant protein called FliBc-fiber2-SP which based on FAdV-4-fiber2 protein, was generated using the gene sequence FliBc, which retains only the conserved sequence at the amino and carboxyl termini of the flagellin B subunit, and a short peptide SPHLHTSSPWER (SP), which targets chicken bone marrow-derived DC. They were separately administered via intramuscular injection to 14-day-old specific pathogen-free (SPF) chickens, and their immunogenicity was compared. At 21 d postvaccination (dpv), it was found that the FliBc-fiber2-SP recombinant protein elicited significantly higher levels of IgG antibodies and conferred a vaccine protection rate of up to 100% compared to its counterpart fiber2 protein. These results suggest that the DC-targeted peptide fusion strategy for flagellin chimeric antigen construction can effectively enhance the immune protective efficacy of antigen proteins.

The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.

Different roles of Ca2+ and chitohexose in peanut (Arachis Hypogaea) photosynthetic responses to PAMP-immunity.

Background: During active infections, plants prevent further spread of pathogenic microorganisms by inducing the rapid programmed death of cells around the infection point. This phenomenon is called the hypersensitive response and is a common feature of plant immune responses. Plants recognize conserved structures of pathogenic microorganisms, called pathogen-associated molecular patterns (PAMPs), e.g., flagellin 22 (flg22) and chitohexose, which bind to receptors on plant cells to induce various immune-response pathways. Although abiotic stresses are known to alter photosynthesis, the different effects of flg22 and chitohexose, which are involved into PAMP-induced signaling, on photosynthesis needs further study. Methods: In the present study, we assessed the role of PAMPs in peanut (Arachis hypogaea) photosynthesis, particularly, the interaction between PAMPs and Ca2+ signal transduction pathway. Results: Both flg22 and chitohexose significantly promoted the expression of the pathogenesis-related genes PR-4 and PR-10, as did Ca2+. We found that Ca2+ is involved in downregulating the photosystem II (PSII) reaction center activity induced by the flg22 immune response, but the role of chitohexose is not obvious. Additionally, Ca2+ significantly reduced the non-photochemical energy dissipation in the flg22- and chitohexose-induced immune response. Conclusion: These results indicated that flg22 and chitohexose can trigger peanut immune pathways through the Ca2+ signaling pathway, but they differ in their regulation of the activity of the PSII reaction center.

Spatial IMA1 regulation restricts root iron acquisition on MAMP perception.

Iron is critical during host-microorganism interactions1-4. Restriction of available iron by the host during infection is an important defence strategy, described as nutritional immunity5. However, this poses a conundrum for externally facing, absorptive tissues such as the gut epithelium or the plant root epidermis that generate environments that favour iron bioavailability. For example, plant roots acquire iron mostly from the soil and, when iron deficient, increase iron availability through mechanisms that include rhizosphere acidification and secretion of iron chelators6-9. Yet, the elevated iron bioavailability would also be beneficial for the growth of bacteria that threaten plant health. Here we report that microorganism-associated molecular patterns such as flagellin lead to suppression of root iron acquisition through a localized degradation of the systemic iron-deficiency signalling peptide Iron Man 1 (IMA1) in Arabidopsis thaliana. This response is also elicited when bacteria enter root tissues, but not when they dwell on the outer root surface. IMA1 itself has a role in modulating immunity in root and shoot, affecting the levels of root colonization and the resistance to a bacterial foliar pathogen. Our findings reveal an adaptive molecular mechanism of nutritional immunity that affects iron bioavailability and uptake, as well as immune responses.

Development of antibody to virulence factor flagellin and its evaluation in screening Ralstonia pseudosolanacearum.

The bacterial wilt disease caused by Ralstonia pseudosolanacearum presents a notable economic risk to a variety of crucial crops worldwide. During preliminary isolation of this phytopathogen, several colonies of other saprophytic bacteria may be mistaken with it. So, the present study aims to address this issue by proposing the application of immunogenic proteins, particularly flagellin (FliC), to enable a rapid and early identification of bacterial wilt. In this study, a novel approach is unveiled for the early detection of R. pseudosolanacearum. The study exploits the immunogenic attributes of flagellin (FliC), by generating polyclonal antibodies against recombinant FliC within model organisms-rabbits and mice. The efficacy of these antibodies is meticulously assessed through discerning techniques, including DAS-ELISA and Western blot analyses, which elucidate their remarkable specificity in identifying various R. pseudosolanacearum strains. Furthermore, the introduction of antibody-coated latex agglutinating reagents offers an additional layer of confirmation, substantiating the feasibility of establishing a laboratory-based toolkit for swift screening and unambiguous identification of the bacterial wilt pathogen. This study presents a significant stride toward enhancing early diagnostic capabilities, potentially revolutionizing agricultural practices by safeguarding crop yield and quality through proactive pathogen detection and mitigation strategies.

Spermidine constitutes a key determinant of motility and attachment of Salmonella Typhimurium through a novel regulatory mechanism.

Spermidine is a poly-cationic molecule belonging to the family of polyamines and is ubiquitously present in all organisms. Salmonella synthesizes, and harbours specialized transporters to import spermidine. A group of polyamines have been shown to assist in Salmonella Typhimurium's virulence and regulation of Salmonella pathogenicity Inslad 1 (SPI-1) genes and stress resistance; however, the mechanism remains elusive. The virulence trait of Salmonella depends on its ability to employ multiple surface structures to attach and adhere to the surface of the target cells before invasion and colonization of the host niche. Our study discovers the mechanism by which spermidine assists in the early stages of Salmonella pathogenesis. For the first time, we report that Salmonella Typhimurium regulates spermidine transport and biosynthesis processes in a mutually inclusive manner. Using a mouse model, we show that spermidine is critical for invasion into the murine Peyer's patches, which further validated our in vitro cell line observation. We show that spermidine controls the mRNA expression of fimbrial (fimA) and non-fimbrial adhesins (siiE, pagN) in Salmonella and thereby assists in attachment to host cell surfaces. Spermidine also regulated the motility through the expression of flagellin genes by enhancing the translation of sigma-28, which features an unusual start codon and a poor Shine-Dalgarno sequence. Besides regulating the formation of the adhesive structures, spermidine tunes the expression of the two-component system BarA/SirA to regulate SPI-1 encoded genes. Thus, our study unravels a novel regulatory mechanism by which spermidine exerts critical functions during Salmonella Typhimurium pathogenesis.

Mucosal TLR5 activation controls healthspan and longevity.

Addressing age-related immunological defects through therapeutic interventions is essential for healthy aging, as the immune system plays a crucial role in controlling infections, malignancies, and in supporting tissue homeostasis and repair. In our study, we show that stimulating toll-like receptor 5 (TLR5) via mucosal delivery of a flagellin-containing fusion protein effectively extends the lifespan and enhances the healthspan of mice of both sexes. This enhancement in healthspan is evidenced by diminished hair loss and ocular lens opacity, increased bone mineral density, improved stem cell activity, delayed thymic involution, heightened cognitive capacity, and the prevention of pulmonary lung fibrosis. Additionally, this fusion protein boosts intestinal mucosal integrity by augmenting the surface expression of TLR5 in a certain subset of dendritic cells and increasing interleukin-22 (IL-22) secretion. In this work, we present observations that underscore the benefits of TLR5-dependent stimulation in the mucosal compartment, suggesting a viable strategy for enhancing longevity and healthspan.

The role of TRIF protein in regulating the proliferation and antigen presentation ability of myeloid dendritic cells through the ERK1/2 signaling pathway in chronic low-grade inflammation of intestinal mucosa mediated by flagellin-TLR5 complex signal.

Objective: The objective is to explore whether the flagellin-TLR5 complex signal can enhance the antigen presentation ability of myeloid DCs through the TRIF-ERK1/2 pathway, and the correlation between this pathway and intestinal mucosal inflammation response. Methods: Mouse bone marrow-derived DC line DC2.4 was divided into four groups: control group (BC) was DC2.4 cells cultured normally; flagellin single signal stimulation group (DC2.4+CBLB502) was DC2.4 cells stimulated with flagellin derivative CBLB502 during culture; TLR5-flagellin complex signal stimulation group (ov-TLR5-DC2.4+CBLB502) was flagellin derivative CBLB502 stimulated ov-TLR5-DC2.4 cells with TLR5 gene overexpression; TRIF signal interference group (ov-TLR5-DC2.4+CBLB502+Pepinh-TRIFTFA) was ov-TLR5-DC2.4 cells with TLR5 gene overexpression stimulated with flagellin derivative CBLB502 and intervened with TRIF-specific inhibitor Pepinh-TRIFTFA. WB was used to detect the expression of TRIF and p-ERK1/2 proteins in each group of cells; CCK8 was used to detect cell proliferation in each group; flow cytometry was used to detect the expression of surface molecules MHCI, MHCII, CD80, 86 in each group of cells; ELISA was used to detect the levels of IL-12 and IL-4 cytokines in each group. Results: Compared with the BC group, DC2.4+CBLB502 group, and ov-TLR5-DC2.4+CBLB502+Pepinh-TRIFTFA group, the expression of TRIF protein and p-ERK1/2 protein in ov-TLR5-DC2.4+CBLB502 group was significantly upregulated (TRIF: p = 0.02,  = 0.007,  = 0.048) (ERK1: p < 0.001, =0.0003,  = 0.0004; ERK2:p = 0.0003,  = 0.0012,  = 0.0022). The cell proliferation activity in ov-TLR5-DC2.4+CBLB502 group was enhanced compared with the other groups (p = 0.0001, p < 0.0001, p = 0.0015); at the same time, the expression of surface molecules MHCI, MHCII, CD80, 86 on DCs was upregulated (p < 0.05); and the secretion of IL-12 and IL-4 cytokines was increased, with significant differences (IL-12: p < 0.0001, p < 0.0001, p = 0.0005; IL-4: p =  < 0.0001, p =  < 0.0001, p = 0.0001). However, the ov-TLR5-DC2.4+CBLB502+Pepinh-TRIFTFA group, which was treated with TRIF signal interference, showed a decrease in intracellular TRIF protein and p-ERK1/2 protein, as well as a decrease in cell proliferation ability and surface stimulation molecules, and a decrease in the secretion of IL-12 and IL-4 cytokines (p < 0.05). Conclusion: After stimulation of flagellin protein-TLR5 complex signal, TRIF protein and p-ERK1/2 protein expression in myeloid dendritic cells were significantly up-regulated, accompanied by increased proliferation activity and maturity of DCs, enhanced antigen presentation function, increased secretion of pro-inflammatory cytokines IL-12 and IL-4. This process can be inhibited by the specific inhibitor of TRIF signal, suggesting that the TLR5-TRIF-ERK1/2 pathway may play an important role in abnormal immune response and mucosal chronic inflammation infiltration mediated by flagellin protein in DCs, which can provide a basis for our subsequent animal experiments.

Notch2 with retinoic acid license IL-23 expression by intestinal EpCAM+ DCIR2+ cDC2s in mice.

Despite the importance of IL-23 in mucosal host defense and disease pathogenesis, the mechanisms regulating the development of IL-23-producing mononuclear phagocytes remain poorly understood. Here, we employed an Il23aVenus reporter strain to investigate the developmental identity and functional regulation of IL-23-producing cells. We showed that flagellin stimulation or Citrobacter rodentium infection led to robust induction of IL-23-producing EpCAM+ DCIR2+ CD103- cDC2s, termed cDCIL23, which was confined to gut-associated lymphoid tissues, including the mesenteric lymph nodes, cryptopatches, and isolated lymphoid follicles. Furthermore, we demonstrated that Notch2 signaling was crucial for the development of EpCAM+ DCIR2+ cDC2s, and the combination of Notch2 signaling with retinoic acid signaling controlled their terminal differentiation into cDCIL23, supporting a two-step model for the development of gut cDCIL23. Our findings provide fundamental insights into the developmental pathways and cellular dynamics of IL-23-producing cDC2s at steady state and during pathogen infection.