Human corneal epithelial cells respond to ocular-pathogenic, but not to nonpathogenic-flagellin

In this study, we investigated the expression of TLR5 in human corneal epithelial cells (CEC), and the functional outcome of TLR5 triggering by flagellins of pathogenic- and nonpathogenic bacteria. Flagellins derived from Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescense or Bacillus subtilis were used. The TLR5 protein and TLR5 specific mRNA expression was evident on human CEC. In human corneal epithelium tissues, TLR5 protein was detected at the basal and wing cells of the tissues. Ocular pathogenic bacteria, namely P. aeruginosa and S. marcescense, derived flagellin induced the significantly increased level of gene activation and IL-6 and IL-8 production. In contrast, ocular nonpathogenic S. typhimurium- and B. subtilis-derived flagellin induced neither the gene activation nor the increased production of IL-6 and IL-8 in human CEC. Human CEC would respond only to flagellin derived of ocular pathogenic bacteria, but not to those derived of ocular nonpathogenic bacteria, to generate pro-inflammatory cytokines.     

Pseudomonas evades immune recognition of flagellin in both mammals and plants

The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.     

Thymic stromal lymphopoietin induces tight junction protein claudin-7 via NF-κB in dendritic cells

Epithelial-derived thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that triggers dendritic cell (DC)-mediated Th2-type inflammatory responses. The activated DCs can penetrate the epithelium to directly take up antigen without compromising the barrier function. Although it is reported that DCs express tight junction molecules and can establish tight junction-like structures with adjacent epithelial cells to preserve the epithelial barrier, the regulation of expression of tight junction molecules in DCs remains unknown. In the present study, to investigate the mechanical regulation of expression of tight junction molecules in DCs, XS52 DCs that was a long-term DC line established from the epidermis of a newborn BALB/c mouse, were treated with TSLP or toll-like receptor (TLR) ligands. In XS52 cells, tight junction molecules claudin-1, -3, -4, -6, -7, -8, and occludin were detected. mRNA expression of TSLP receptor and all these tight junction molecules was significantly increased in activated XS52 cells after treatment with TSLP. In addition, expression of claudin-7 protein was increased in dose- and time-dependent manner. In XS52 cells, which express TLR2, TLR3, TLR4, and TLR7, but not TLR9, expression of claudin-7 protein was also increased after treatment with ligands of TLR2, TLR4 or TLR7/8, Pam₃Cys-Ser-(Lys)₄, LPS, or CL097. The NF-κB inhibitor IMD-0354 prevented upregulation of claudin-7 after treatment with TSLP or TLR ligands. These findings indicate that TSLP induces expression of tight junction protein claudin-7 in DCs via NF-κB as well as via TLRs and may control tight junctions of DCs to preserve the epithelial barrier during allergic inflammation.     

KU812 cells provide a novel in vitro model of the human IL-33/ST2L axis: Functional responses and identification of signaling pathways

Activation of interleukin-1 family receptor ST2L by its ligand interleukin-33 (IL-33) is an important component in inflammatory responses. Peripheral blood basophils, recognized as major effector cells in allergic inflammation that play a role in both innate and adaptive immunity, are activated by IL-33 through ST2L. However, studies are challenging due to the paucity of this cell population, representing less than 1% of peripheral blood leukocytes. We identified a basophil-like chronic myelogenous leukemia cell line, KU812, that constitutively expresses ST2L and demonstrates functional responses to IL-33 stimulation. IL-33 induced production of multiple inflammatory mediators in KU812 cells that were blocked by anti-ST2L and anti-IL-33 antibodies. The interaction of IL-33 and ST2L activated NF-κB, JNK, and p38 MAPK, but not ERK1/2 signaling pathways. Studies using pharmacological inhibitors to IKK-2 and MAP kinases revealed that one of the functional responses, IL-33-induced IL-13 production, was regulated through NF-κB, but not JNK or p38 MAPK signaling. The requirement of NF-κB was confirmed by IKK-2 knockdown using shRNA. KU812 represents the first human cell line-based in vitro model of the IL-33/ST2L axis and provides a valuable tool to aid in understanding the mechanism and significance of IL-33 and ST2L interaction and function.     

CMRF-35-like molecule 3 preferentially promotes TLR9-triggered proinflammatory cytokine production in macrophages by enhancing TNF receptor-associated factor 6 ubiquitination

TLRs are critical innate immune sensors in the induction of proinflammatory cytokines to eliminate invading pathogens. However, the mechanisms for the full activation of TLR-triggered innate immune response need to be fully understood. The murine CMRF-35-like molecule (CLM)-3 is a representative of CLM family belonging to the Ig superfamily. Considering that CLM-3 is selectively expressed in macrophages and the roles of CLM members in innate immune response remain unclear, in this study we investigated the role of CLM-3 in the regulation of TLR-triggered innate response. We found that CLM-3 was an endosome/lysosome-localized molecule, and was downregulated in macrophages by stimulation with TLR9 ligand, but not TLR4 and TLR3 ligands. Interestingly, CLM-3 selectively promoted production of TNF-α and IL-6 in macrophages triggered by TLR9, but not TLR4 or TLR3. CLM-3 enhanced activation of MAPKs and NF-κB pathways in TLR9-triggered macrophages. Furthermore, CLM-3-transgenic mice were generated, and CLM-3 expression was confirmed by mAb against CLM-3 that we prepared. Accordingly, the macrophages derived from CLM-3-transgenic mice were more sensitive to TLR9 ligand stimulation, with more pronounced production of TNF-α, IL-6, and increased activation of MAPKs and NF-κB pathways. Moreover, ubiquitination of TNFR-associated factor 6, a crucial signaling transducer of TLR-triggered MAPKs and NF-κB activation, was found to be significantly promoted by CLM-3 in macrophages. Collectively, the endosome/lysosome-localized CLM-3 can promote full activation of TLR9-triggered innate responses by enhancing TNFR-associated factor 6 ubiquitination and subsequently activating MAPKs and NF-κB.     

Cigarette smoke alters IL-33 expression and release in airway epithelial cells

Airway epithelium is a regulator of innate immune responses to a variety of insults including cigarette smoke. Cigarette smoke alters the expression and the activation of Toll Like Receptor 4 (TLR4), an innate immunity receptor. IL-33, an alarmin, increases innate immunity Th2 responses. The aims of this study were to explore whether mini-bronchoalveolar lavage (mini-BAL) or sera from smokers have altered concentrations of IL-33 and whether cigarette smoke extracts (CSE) alter both intracellular expression (mRNA and protein) and release of IL-33 in bronchial epithelial cells. The role of TLR4 in the expression of IL-33 was also explored.Mini-BALs, but not sera, from smokers show reduced concentrations of IL-33. The expression of IL-33 was increased also in bronchial epithelium from smokers. 20% CSE reduced IL-33 release but increased the mRNA for IL-33 by real time PCR and the intracellular expression of IL-33 in bronchial epithelial cells as confirmed by flow cytometry, immunocytochemistry and western blot analysis. The effect of CSE on IL-33 expression was also observed in primary bronchial epithelial cells. IL-33 expression was mainly concentrated within the cytoplasm of the cells. LPS, an agonist of TLR4, reduced IL-33 expression, and an inhibitor of TLR4 increased the intracellular expression of IL-33. In conclusion, the release of IL-33 is tightly controlled and, in smokers, an altered activation of TLR4 may lead to an increased intracellular expression of IL-33 with a limited IL-33 release.     

Interleukin-32 induced thymic stromal lymphopoietin plays a critical role in the inflammatory response in human corneal epithelium

Interleukin (IL)-32, a novel cytokine, participates in a variety of inflammatory disorders. Thymic stromal lymphopoietin (TSLP) plays important roles in mucosal epithelial cells, especially in allergy-induced inflammation, through the TSLP-TSLPR (thymic stromal lymphopoietin receptor) signalling pathway. However, the association of IL-32 with TSLP on the ocular surface remains unclear. The present work aimed to assess the functional association of IL-32 with TSLP in the control of pro-inflammatory cytokine levels in the corneal epithelium. Human corneal tissue specimens and human corneal epithelial cells (HCECs) were administered different concentrations of IL-32 in the presence or absence of various inhibitors to assess TSLP levels and localization, as well as the molecular pathways that control pro-inflammatory cytokine production. TSLP mRNA levels were determined by real time RT- PCR, while protein levels were quantitated by ELISA and immunohistochemical staining. TSLP protein expression was examined in donor corneal epithelium samples. IL-32 significantly upregulated TSLP and pro-inflammatory cytokines (TNFα and IL-6) in HCECs at the gene and protein levels. The production of pro-inflammatory molecules by IL-32 was increased by recombinant TSLP. Interestingly, both NF-κB (quinazoline) and caspase-1 (VX-765) inhibitors suppressed the IL-32-related upregulation of pro-inflammatory cytokines (TNFα and IL-6). These findings demonstrate that IL-32 and IL-32-induced-TSLP are critical cytokines that participate in inflammatory responses through the caspase-1 and NF-κB signalling pathways in the corneal epithelium, suggesting new molecular targets for inflammatory diseases of the ocular surface. The effects of IL-32 on cell proliferation and apoptosis were investigated by MTT assays and RT-PCR,respectively. The results demonstrated that IL-32 inhibits cells apoptosis in HCECs.     

The ex vivo response of human intestinal mucosa to enteropathogenic Escherichia coli infection

In vitro organ culture (IVOC) represents a gold standard model to study enteropathogenic E. coli (EPEC) infection of human intestinal mucosa. However, the optimal examination of the bacterial–host cell interaction requires a directional epithelial exposure, without serosal or cut surface stimulation. A polarized IVOC system (pIVOC) was developed in order to overcome such limitations: apical EPEC infection produced negligible bacterial leakage via biopsy edges, resulted in enhanced colonization compared with standard IVOC, and showed evidence of bacterial detachment, as in natural rabbit EPEC infections. Examination of mucosal innate immune responses in pIVOC showed both interleukin (IL)-8 mRNA and protein levels were significantly increased after apical EPEC infection. Increased IL-8 levels mainly depended on flagellin expression as fliC -negative EPEC did not elicit a significant IL-8 response despite increased mucosal colonization compared with wild-type EPEC. In addition, apical application of purified flagella significantly increased IL-8 protein levels over non-infected controls. Immunofluorescence staining of EPEC-infected small intestinal biopsies revealed apical and basolateral distribution of Toll-like receptor (TLR) 5 on epithelium, suggesting that EPEC can trigger mucosal IL-8 responses by apical flagellin/TLR5 interaction ex vivo and does not require access to the basolateral membrane as postulated in cell culture models.     

Opisthorchis viverrini excretory/secretory products induce toll-like receptor 4 upregulation and production of interleukin 6 and 8 in cholangiocyte

Biliary tract infection with the Group I carcinogenic liver fluke Opisthorchis viverrini is associated with severe inflammation leading to cholangiocarcinoma--a major biliary cancer in Southeast Asia. However, mechanism(s) by which the liver fluke induces host mucosal immune/inflammatory responses is unclear. In the present study we address whether a normal immortalized human cholangiocyte cell line (H69 cells) recognizes and responds to O. viverrini excretory/secretory products (OVES). Expression of multiple TLRs, activation of NF-κB, and expression of pro-inflammatory cytokines were monitored in the presence and absence of OVES. Our results showed that OVES induced increased cholangiocyte TLR4 mRNA expression, induced IκB-α degradation in a MyD88-dependent manner, and activated NF-κB nuclear translocation. Moreover, OVES induced expression and secretion of the strong chemoattractant chemokine interleukin 8 (IL-8) and pro-inflammatory cytokine IL-6. These results demonstrate that secreted/excreted products of O. viverrini are recognized by human cholangiocytes and initiate innate mucosal immunity/inflammatory cascades, a primary event in the pathogenesis of opisthorchiasis and cholangiocarcinoma.     

CpG oligonucleotides induce an immune response of odontoblasts through the TLR9, MyD88 and NF-κB pathways

Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-α expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-κB or IκBα markedly inhibited CpG ODN-induced IL-6 and TNF-α expression. Moreover, the CpG ODN-mediated increase of κB-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9, MyD88 and IκBα, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-κB are involved in this process.     

Interleukin-10 activates Toll-like receptor 4 and requires MyD88 for cardiomyocyte survival

Toll-like receptors (TLRs) are important in a variety of inflammatory diseases including acute cardiac disorders. TLR4 innate signaling regulates the synthesis of anti-inflammatory cytokine, interleukin-10 (IL-10) upon TLR4 agonists’ re-stimulation. Anti-apoptotic action of IL-10 in cardiac dysfunction is generally accepted but its protective mechanism through TLR4 is not yet understood. We studied the effect of IL-10 in the activation of TLR4 downstream signals leading to cardiomyocytes survival. IL-10 caused a significant increase in the expression of CD14, MyD88 and TLR4. TLR4 activation led to the translocation of the interferon regulatory factor 3 (IRF3) into the nucleus. Phosphorylation of IRF3 enhanced mRNA synthesis for IL-1β but not TNF-α and was elevated even after removal of IL-10 stimulation. Furthermore, degradation of inhibitory kappa B (IκB) kinase (Ikk) suggested that IκBβ was the main activating kinase for IRF3-regulated NF-κB activation and phosphorylation of p65. Phosphorylated NF-κB p65 was translocated into the nucleus. Concomitantly, an increase in Bcl-xL activity inhibited Bax and the proteolytic activity of caspase 3 as well as a decrease in PARP cleavage. An inhibition of MyD88, modulated the above listed responses to IL-10 as there was a decrease in TLR4 and IRF3 and an increase in TNF-α mRNA. This was associated with a decrease in NF-κB p65, Bcl-xL mRNA and protein levels as well as there was an activation of Bax and PARP cleavage independent of caspase 3 activation. These data in cardiomyocytes suggest that IL-10 induced anti-apoptotic signaling involves upregulation of TLR4 through MyD88 activation.     

TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation caused by proteinuria

Proteinuria is an important risk factor for chronic kidney diseases (CKD). Several studies have suggested that proteinuria initiates tubulointerstitial inflammation, while the mechanisms have not been fully understood. In this study, we hypothesized whether the activation of the TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation induced by proteinuria. We observed expression of TLR2, MyD88, NF-κB, as well as TNF-α and IL-6 detected by immunohistostaining, Western blotting and real-time PCR in albumin-overloaded (AO) nephropathy rats. In vitro, we observed these markers in HK-2 cells stimulated by albumin. We used TLR2 siRNA or the NF-κB inhibitor BAY 11-7082 to observe the influence of TNF-α and IL-6 expression caused by albumin overload. Finally, we studied these markers in non-IgA mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria. It was demonstrated that expression of TLR2, MyD88 and NF-κB were significantly increased in AO rats and in non-IgA MsPGN patients with high levels of proteinuria, and TNF-α and IL-6 expressions were increased after NF-κB activation. Furthermore, TNF-α and IL-6 expression was positively correlated with the level of proteinuria. Albumin-overload induced TNF-α and IL-6 secretions by the TLR2–MyD88–NF-κB pathway activation, which could be attenuated by the TLR2 siRNA or BAY 11-7082 in HK-2 cells. In summary, we demonstrated that proteinuria may exhibit an endogenous danger-associated molecular pattern (DAMP) that induces tubulointerstitial inflammation via the TLR2–MyD88–NF-κB pathway activation.     

Flagellin administration protects respiratory tract from Burkholderia cepacia infection

Burkholderia cepacia is an important pathogen that often causes pneumonia in immunocompromised individuals. Here, it was demonstrated that the TLR5 agonist flagellin could locally activate innate immunity. This was characterized by rapid expressions of IL-1beta, TNF-alpha, and iNOS mRNA and a delay in the expression of IL-10 mRNA. A significant elevation in the IL-1beta, TNF-alpha, and nitric oxide levels was also noted. In the respiratory tract, flagellin induced neutrophil infiltration into the airways, which was observed by histopathological examination and confirmed by the neutrophil count and level of myeloperoxidase activity. This was concomitant with a high activity of alveolar macrophages that engulfed and killed B. cepacia in vitro. The flagellin mucosal treatment improved the B. cepacia clearance in the mouse lung. Thus, the present findings illustrate the profound stimulatory effect of flagellin on the lung mucosal innate immunity, a response that needs to be exploited therapeutically to prevent the development of respiratory tract infection by B. cepacia.     

Synergism of Toll-like receptor-induced interleukin-12p70 secretion by monocyte-derived dendritic cells is mediated through p38 MAPK and lowers the threshold of T-helper cell type 1 responses

Toll-like receptors (TLRs) recognise specific molecular signatures of pathogens and trigger antimicrobial defence responses. Thereby, two independent signalling pathways can be distinguished: The inflammatory signalling pathway acting via the adapter molecule MyD88, leading to the activation of nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPK) such as SAPK/JNK and p38 MAPK and the interferon (IFN) dependent pathway that signals via TRIF and results in the production of IFN-α/β. Several evolutionarily conserved molecular patterns are expressed by pathogens, leading to the question if concerted targeting of different TLRs may induce exaggerated immune responses by signalling via both TLR pathways. Here we report that monocyte-derived dendritic cells (MoDCs) combine and integrate signals received via the IFN-dependent pathway by engagement of TLR3 (poly I:C) and activation of TRIF with the MyD88-dependent pathway by ligation of TLR2 (PGN), TLR2/TLR6 (zymosan) and TLR5 (flagellin). The generally low IL-12p70 inducers resulted in combination of both pathways in cytokine levels similar to LPS, which acts via TLR4 and induces recruitment of MyD88/Tirap and TRIF/TRAM adapter proteins. The combination of TLR3 (poly I:C) or TLR4 (LPS) engagement with TLR8 (R848) ligation induced synergistic effects on cytokine production with a boost especially in IL-12p70 secretion. SB203580, a specific p38 MAPK inhibitor, completely blocked TLR ligand mediated IL-12p70 secretion, whereby specific inhibitors for SAPK/JNK (SP600125) and NF-κB (PDTC) only repressed partially the IL-12p70 secretion. Enhanced phosphorylation in poly I:C and R848 activated MoDCs revealed the critical contribution of p38 MAPK in synergistically induced IL-12p70 induction. Further investigation of primary and recall CD8⁺ T cell responses to the MUC_12-20 M1.2 peptide LLLLTVLTV and the influenza A virus matrix_58-66 peptide GILGFVFTL proved that synergistically activated MoDCs were superior compared with LPS or R848 alone. The results indicate that dendritic cells process, combine and integrate signals delivered by pathogens to launch effective adaptive immune responses.     

Development of a mucosal complex vaccine against oral Salmonella infection in mice

We examined the immunogenicity of a Salmonella enterica complex vaccine (CV), consisting of flagellin and polysome purified from serotype Typhimurium LT2. CV plus cholera toxin (CT), in three oral doses given at 7-day intervals, conferred complete protection on C57BL/6 mice against lethal oral infection with a wild-type strain. It elicited mucosal IgA > IgG2a > IgG1 and systemic IgG2a > IgG1 > IgA antibodies to flagellin and polysome, and delayed footpad response (DFR) to both antigens. In Peyer's patches (PPs) and lamina propria (LP), IgA was produced under a Th1-dominant environment; CD4+T cells from produced interleukin (IL)-2, interferon (IFN)-gamma, and IL-10 by stimulation with salmonella extract. On the same protocol, flagellin plus CT induced flagellin-specific mucosal and systemic IgA and IgG1 antibodies, CD4+T cells producing IL-10 and IFN-gamma in PPs and LP, and only minimal levels of flagellin-specific DFR. Polysome plus CT induced polysome-specific mucosal and systemic IgG2a in addition to IgG1 and IgA antibodies, CD4+T cells producing IFN-gamma and IL-2 in PPs and LP, and polysome-specific DFR. These two vaccines, however, conferred at most 50-60% survival rates. Our results suggest that polysomes in CV provide effective adjuvant activity for the induction of both mucosal and systemic Th1-biased responses toward flagellin.     

Myoloid-Related Protein 8, an Endogenous Ligand of Toll-Like Receptor 4, Is Involved in Epileptogenesis of Mesial Temporal Lobe Epilepsy Via Activation of the Nuclear Factor-κB Pathway in Astrocytes

The role of Toll-like receptor 4 (TLR4) in the activation of innate immunity has been extensively studied in the past several years. Here, we are the first to report that myeloid-related protein 8 (MRP8), an endogenous TLR4 ligand, is involved in the epileptogenesis of mesial temporal lobe epilepsy (MTLE). We find that the expression of MRP8, TLR4, and interleukin 1-β (IL-1β) was upregulated in a MTLE model during both acute and chronic disease stages. We next investigated the possible roles played by astrocytes, which have been shown to be the major source of IL-1β during epilepsy. Stimulation via MRP8 led to the induction of IL-1β in astrocytes in vitro, accompanied by the activation of Nuclear Factor-κB, while knockdown of TLR4 or inhibition of NF-κB in astrocytes prevented this IL-1β induction. Thus, MRP8 may potentiate the perpetuation of MTLE by activating the NF-κB pathway in astrocytes, and could be a new target for anticonvulsant therapies.     

Luminal-applied flagellin is internalized by polarized intestinal epithelial cells and elicits immune responses via the TLR5 dependent mechanism

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn's lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.