The role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in immune responses mediated by T-helper 2 (T(H)2) lymphocytes is unknown. Here we characterize the development of allergic airway disease in TRAIL-deficient (Tnfsf10(-/-)) mice and in mice exposed to short interfering RNA targeting TRAIL. We show that TRAIL is abundantly expressed in the airway epithelium of allergic mice and that inhibition of signaling impairs production of the chemokine CCL20 and homing of myeloid dendritic cells and T cells expressing CCR6 and CD4 to the airways. Attenuated homing limits T(H)2 cytokine release, inflammation, airway hyperreactivity and expression of the transcriptional activator STAT6. Activation of STAT6 by interleukin-13 restores airway hyperreactivity in Tnfsf10(-/-) mice. Recombinant TRAIL induces pathognomic features of asthma and stimulates the production of CCL20 in primary human bronchial epithelium cells. TRAIL is also increased in sputum of asthmatics. The function of TRAIL in the airway epithelium identifies this molecule as a target for the treatment of asthma.

Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response. By using HEK 293 reporter cells for pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and α-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling revealed a core innate immune response partly shared by LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greater magnitude than those induced by SaS. Microarray data analysis suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. The higher upregulation of chemokines (Cxcl10, Ccl2, Ccl5 and Ccl20) that target mononuclear leucocytes by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. The MEC responses to the two stimuli were different, as LPS was associated with NF-κB and Fas signaling pathways, whereas SaS was associated with AP-1 and IL-17A signaling pathways. It is noteworthy that at the protein level secretion of TNF-α and IL-1β was not induced by either stimulus. These results suggest that the response of MEC to diffusible stimuli from E. coli and S. aureus contributes to the onset of the response with differential leucocyte recruitment and distinct inflammatory and innate immune reactions of the mammary gland to infection.

Antimicrobial factors are efficient defense components of the innate immunity, playing a crucial role in the intestinal homeostasis and protection against pathogens. In this study, we report that upon infection of polarized human intestinal cells in vitro, virulent Shigella flexneri suppress transcription of several genes encoding antimicrobial cationic peptides, particularly the human beta-defensin hBD-3, which we show to be especially active against S. flexneri. This is an example of targeted survival strategy. We also identify the MxiE bacterial regulator, which controls a regulon encompassing a set of virulence plasmid-encoded effectors injected into host cells and regulating innate signaling, as being responsible for this dedicated regulatory process. In vivo, in a model of human intestinal xenotransplant, we confirm at the transcriptional and translational level, the presence of a dedicated MxiE-dependent system allowing S. flexneri to suppress expression of antimicrobial cationic peptides and promoting its deeper progression toward intestinal crypts. We demonstrate that this system is also able to down-regulate additional innate immunity genes, such as the chemokine CCL20 gene, leading to compromised recruitment of dendritic cells to the lamina propria of infected tissues. Thus, S. flexneri has developed a dedicated strategy to weaken the innate immunity to manage its survival and colonization ability in the intestine.

Mouse bone marrow-derived macrophages (BMDM) grown in M-CSF (CSF-1) have been used widely in studies of macrophage biology and the response to TLR agonists. We investigated whether similar cells could be derived from the domestic pig using human rCSF-1 and whether porcine macrophages might represent a better model of human macrophage biology. Cultivation of pig bone marrow cells for 5-7 d in presence of human rCSF-1 generated a pure population of BMDM that expressed the usual macrophage markers (CD14, CD16, and CD172a), were potent phagocytic cells, and produced TNF in response to LPS. Pig BMDM could be generated from bone marrow cells that had been stored frozen and thawed so that multiple experiments can be performed on samples from a single animal. Gene expression in pig BMDM from outbred animals responding to LPS was profiled using Affymetrix microarrays. The temporal cascade of inducible and repressible genes more closely resembled the known responses of human than mouse macrophages, sharing with humans the regulation of genes involved in tryptophan metabolism (IDO, KYN), lymphoattractant chemokines (CCL20, CXCL9, CXCL11, CXCL13), and the vitamin D3-converting enzyme, Cyp27B1. Conversely, in common with published studies of human macrophages, pig BMDM did not strongly induce genes involved in arginine metabolism, nor did they produce NO. These results establish pig BMDM as an alternative tractable model for the study of macrophage transcriptional control.

Macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19 are members of the CC chemokine subfamily which exert their effects through specific receptors, CCR6 and CCR7, respectively. Previously, we have reported that human neutrophils have the capacity to produce a number of chemokines, including IL-8/CXCL8, GROalpha/CXCL1, IP-10/CXCL10, and MIG/CXCL9. Herein, we show that neutrophils also have the ability to express and release MIP-3alpha/CCL20 and MIP-3beta/CCL19 when cultured with either LPS or TNF-alpha. We also report that MIP-3alpha/CCL20 and MIP-3beta/CCL19 production by LPS-stimulated neutrophils is negatively modulated by IL-10. Remarkably, we found that supernatants harvested from stimulated neutrophils not only induced chemotaxis of both immature and mature dendritic cells (DC), but also triggered rapid integrin-dependent adhesion of CCR6- and CCR7-expressing lymphocytes to purified VCAM-1 and ICAM-1, respectively. Importantly, both chemotaxis and rapid integrin-dependent adhesion were dramatically suppressed by anti-MIP-3alpha/CCL20 and anti-MIP-3beta/CCL19 neutralizing antibodies, indicating that MIP-3alpha/CCL20 and MIP-3beta/CCL19 present in the supernatants were both biologically active. As these chemokines are primarily chemotactic for DC and specific lymphocyte subsets, the ability of neutrophils to produce MIP-3alpha/CCL20 and MIP-3beta/CCL19 might be significant in orchestrating the recruitment of these cell types to the inflamed sites and therefore in contributing to the regulation of the immune response.

Interleukin (IL) 1alpha produced by human endothelial cells (ECs), in response to tumor necrosis factor (TNF) or to co-culture with allogeneic T cells in a TNF-dependent manner, can augment the release of cytokines from alloreactive memory T cells in vitro. In a human-mouse chimeric model of artery allograft rejection, ECs lining the transplanted human arteries express IL-1alpha, and blocking IL-1 reduces the extent of human T cell infiltration into the artery intima and selectively inhibits IL-17 production by infiltrating T cells. In human skin grafts implanted on immunodeficient mice, administration of IL-17 is sufficient to induce mild inflammation. In cultured cells, IL-17 acts preferentially on vascular smooth muscle cells rather than ECs to enhance production of proinflammatory mediators, including IL-6, CXCL8, and CCL20. Neutralization of IL-17 does not reduce T cell infiltration into allogeneic human artery grafts, but markedly reduces IL-6, CXCL8, and CCL20 expression and selectively inhibits CCR6(+) T cell accumulation in rejecting arteries. We conclude that graft-derived IL-1 can promote T cell intimal recruitment and IL-17 production during human artery allograft rejection, and suggest that targeting IL-1 in the perioperative transplant period may modulate host alloreactivity.

BACKGROUND

Airway dendritic cells (DCs) are crucial for allergen-induced sensitization and inflammation in allergic asthma. After allergen challenge, an increased number of DCs is observed in airway epithelium from patients with allergy.

OBJECTIVE

Because Der p 1, a cysteine protease allergen from Dermatophagoides pteronyssinus , induces chemokine production by bronchial epithelial cells (BECs), the purpose of this investigation was to evaluate the capacity of BEC exposed to Der p 1 to recruit DCs.

METHODS

Chemotactic activity of BEAS-2B, a bronchial epithelial cell line, and BECs from nonatopic controls and patients with allergic asthma was evaluated on the migration of precursors, immature and mature monocyte-derived DCs (MDDCs), and CD34 + -derived Langerhans cells (LCs).

RESULTS

C-C chemokine ligand (CCL)-2, CCL5, and C-X-C chemokine ligand 10 production by BEAS-2B and BEC was increased after Der p 1 exposure, whereas the proenzyme proDer p 1 devoid of enzymatic activity had no effect. Der p 1 stimulation of BEAS-2B and BEC from both groups increased significantly the recruitment of MDDC precursors, depending on CCL2, CCL5, and C-X-C chemokine ligand 10 production. In a reconstituted polarized epithelium, apical application of Der p 1 enhanced MDDC precursor migration into the epithelial layer. Moreover, Der p 1 stimulation of BEC from patients with asthma but not from controls increased the migration of LC precursors, mainly dependent on CCL20 secretion. No migration of immature and mature DCs was observed.

CONCLUSIONS

These data confirmed that BECs participate in the homeostasis of the DC network present within the bronchial epithelium through the secretion of chemokines. In allergic asthma, upregulation of CCL20 production induced LC recruitment, the role of which remains to be determined.

BACKGROUND

T(H)17 cells play a critical role in the pathogenesis of several autoimmune and allergic diseases. Intravenous immunoglobulin (IVIg), a therapeutic preparation of polyclonal IgG that is increasingly used in the treatment of diverse autoimmune and allergic diseases, might target T(H)17 cells to exert therapeutic effects.

OBJECTIVE

We sought to examine whether IVIg interferes with the development and function of human T(H)17 cells.

METHODS

T(H)17 cells were differentiated from naive human CD4(+) T cells in the presence of TGF-β and IL-21. T(H)17 cells were amplified by stimulating memory CD4(+) T cells in the presence of IL-1β and IL-6. The effect of IVIg was examined on the differentiation and amplification of T(H)17 cells, expression of the T(H)17 lineage-specific transcription factor retinoic acid-related orphan receptor C, secretion of T(H)17 effector cytokines, and phosphorylation of signal transducer and activator of transcription 3, a transcription factor that plays an important role in T(H)17 cell development and function.

RESULTS

IVIg inhibits the differentiation and amplification of human T(H)17 cells, as well as the production of their effector cytokines IL-17A, IL-17F, IL-21, and CCL20. The inhibitory effects of IVIg on T(H)17 cells are F(ab')(2) dependent and involve interference with the expression of retinoic acid-related orphan receptor C and activation of signal transducer and activator of transcription 3. Also, IVIg significantly enhanced forkhead box protein 3-positive regulatory T cells among the memory CD4(+) T cells.

CONCLUSIONS

These results reveal a novel mechanism of action of IVIg in achieving a therapeutic effect in autoimmune and allergic diseases, in which T(H)17 cells play a key modulatory role in sustaining the chronic inflammatory response. Our results also suggest a reciprocal regulation of T(H)17 and regulatory T-cell populations by IVIg.

To reach the site of antigen deposition at epithelial surfaces, dendritic cells (DC) have to traverse the endothelial barrier, progress through the tissue (i.e., dermis) and cross the dermo-epithelial junction (basal membrane). In the present study, we demonstrate that (1) circulating blood DC and monocytes express high levels of CCR2 and primarily respond to monocyte chemotactic protein (MCP) and not to macrophage inflammatory protein (MIP)-3alpha/CCL20; (2) while the CD34(+) hematopoietic progenitor cells (HPC)-derived CD1a(+) precursors committed to Langerhans cell differentiation primarily respond to MIP-3alpha/CCL20, the HPC-derived CD14(+) precursors respond to both MCP and MIP-3alpha/CCL20; (3) in concordance with the sequential expression of CCR2 and CCR6, the HPC-derived CD14(+) precursors initially acquire the ability to migrate in response to MCP-4/CCL13 and subsequently in response to MIP-3alpha/CCL20; and (4) in vivo, in inflamed epithelium, MCP-4/CCL13 and MIP-3alpha/CCL20 form complementary gradients, with MCP-4/CCL13 expressed in basal epithelial cells at the contact of blood vessels, while MIP-3alpha/CCL20 expression is restricted to epithelial cells bordering the external milieu. These observations suggest that the recruitment of DC to the site of infection is controlled by the sequential action of different chemokines: (i) CCR2(+) circulating DC or DC precursors are mobilized into the tissue via the expression of MCP by cells lining blood vessels, and (ii) these cells traffic from the tissue to the site of pathogen invasion via the production of MIP-3alpha/CL20 by epithelial cells and the up-regulation of CCR6 in response to the tissue environment.

OBJECTIVE

To clarify the involvement of dendritic cells (DCs), chemokines, and proinflammatory Th1 cytokines in the pathogenesis of the chronic muscle diseases dermatomyositis (DM) and polymyositis (PM).

METHODS

We characterized by immunohistochemistry the DC subsets and their interaction with cells producing chemokines and the Th1 cytokines interleukin-17 (IL-17) and interferon-gamma (IFNgamma). Immature and mature DCs were defined by the expression of CD1a and DC-LAMP/CD83, respectively.

RESULTS

Immature DCs were mainly detected in lymphocytic infiltrates in DM and PM muscle tissue samples. Mature DCs were detected in perivascular infiltrates and surrounded muscle fibers. IL-17-positive and IFNgamma-positive cells were also observed in perivascular infiltrates in both cases. We then focused on the expression of the CCL20/CCR6 chemokine/receptor complex, which controls immature DC migration, and on the expression of the CCL19/CCR7 and CCL21/CCR7 chemokine/receptor complexes, which control mature DC migration. CCL20 and CCR6 colocalized in lymphocytic infiltrates in DM and PM samples. CCL21 was rarely observed in DM samples and never observed in PM samples. CCL19- and CCR7-expressing cells were absent in both tissues.

CONCLUSIONS

The close association between CCL20/CCR6 and immature DCs suggests the contribution of CCL20 to CCR6+ immature DC homing. Detection of mature DCs in DM and PM muscle tissue samples despite the lack of CCL19 and CCR7 is evidence for a local maturation of DCs in inflammatory muscle tissue without lymphoid organ organization.

Recent reports of tumor regression following delivery of autologous tumor antigen-pulsed DCs suggest that defective antigen presentation may play a key role in tumor escape. Here we show in two different murine tumor models, CT26 (colon adenocarcinoma) and B16 (melanoma), that the number and activation state of intratumoral DCs are critical factors in the host response to tumors. We used CCL20/macrophage inflammatory protein-3alpha (MIP-3alpha) chemokine to increase the number of tumoral DCs and intratumoral injections of CG-rich motifs (CpGs) to activate such cells. Expression of CCL20 in the tumor site attracted large numbers of circulating DCs into the tumor mass and, in the case of CT26 tumors, led to complete tumor regression. Intratumoral CpG injections, in addition to CCL20, were required to induce therapeutic immunity against B16 tumors. In this model CpG overcame tumor-mediated inhibition of DC activation and enabled tumoral DCs to cross-present tumor antigens to naive CD8 T cells. CpG activation of tumoral DCs alone was not sufficient to induce tumor regression in either tumor model, nor was systemic delivery of the DC growth factor, Flt3 ligand, which dramatically increased the number of circulating DCs but not the number of tumoral DCs. These results indicate that the number of tumoral DCs as well as the tumor milieu determines the ability of tumor-bearing hosts to mount an effective antitumor immune response. Our results also suggest that DCs can be manipulated in vivo without delivery of defined tumor antigens to induce a specific T cell-mediated antitumor response and provide the basis for the use of chemokines in DC-targeted clinical strategies.

OBJECTIVE

Caspase recruitment domain 9 (CARD9) is an adaptor protein that integrates signals downstream of pattern recognition receptors. CARD9 has been associated with autoinflammatory disorders, and loss-of-function mutations have been associated with chronic mucocutaneous candidiasis, but the role of CARD9 in intestinal inflammation is unknown. We characterized the role of Card9 in mucosal immune responses to intestinal epithelial injury and infection.

METHODS

We induced intestinal inflammation in Card9-null mice by administration of dextran sulfate sodium (DSS) or Citrobacter rodentium. We analyzed body weight, assessed inflammation by histology, and measured levels of cytokines and chemokines using quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Cell populations were compared between wild-type and Card9-null mice by flow cytometry analysis.

RESULTS

Colon tissues and mesenteric lymph nodes of Card9-null mice had reduced levels of interleukin (IL)-6, interferon-γ, and T-helper (Th)17 cytokines after administration of DSS, compared with wild-type mice. IL-17A and IL-22 expression were reduced in the recovery phase after DSS administration, coincident with decreased expression of antimicrobial peptides and the chemokine (C-C motif) ligand 20 (Ccl20). Although Card9-null mice had more intestinal fungi based on 18S analysis, their Th17 responses remained defective even when an antifungal agent was administered throughout DSS exposure. Moreover, Card9-null mice had impaired immune responses to C rodentium, characterized by decreased levels of colonic IL-6, IL-17A, IL-22, and regenerating islet-derived 3 gamma (RegIIIγ), as well as fewer IL-22-producing innate lymphoid cells (ILCs) in colon lamina propria.

CONCLUSIONS

The adaptor protein CARD9 coordinates Th17- and innate lymphoid cell-mediated intestinal immune responses after epithelial injury in mice.

BACKGROUND

The cause of corticosteroid-resistant (CR) asthma is unknown.

OBJECTIVE

We sought to perform gene microarray analyses by using bronchoalveolar lavage (BAL) cells from well-characterized subjects with CR asthma and subject with corticosteroid-sensitive (CS) asthma to elucidate the differential expression of genes that contribute to the development of corticosteroid resistance.

METHODS

The patients were characterized as having CR or CS asthma based on FEV(1) percent predicted improvement after a 1-week course of oral prednisone. Expression of selected gene targets was verified by means of real-time PCR and ELISA.

RESULTS

Microarray analyses demonstrated significantly higher levels (>3-fold increase, P < .05) of transcripts for TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, CXCL1, CXCL2, CXCL3, CXCL8 (IL-8), CCL3, CCL4, and CCL20 in BAL cells of subjects with CR asthma. These findings, confirmed by means of RT-PCR in additional BAL samples, were consistent with classical macrophage activation by bacterial products. In contrast, markers of alternatively activated macrophages, arginase I and CCL24, were decreased. Genes associated with activation of the LPS signaling pathway (early growth response 1, dual-specificity phosphatase 2, molecule possessing ankyrin repeats induced by LPS, and TNF-alpha-induced protein 3) were significantly increased in BAL samples from subjects with CR asthma (P < .05). These patients had significantly higher amounts (1444.0 +/- 457.3 pg/mg total protein) of LPS in BAL fluid than seen in subjects with CS asthma (270.5 +/- 216.0 pg, P < .05), as detected by using the LAL assay and confirmed by means of gas chromatographic/mass spectrometric analysis. Prolonged exposure to LPS induced functional steroid resistance to dexamethasone in normal human monocytes, as demonstrated by persistently increased IL-6 levels in the presence of dexamethasone.

CONCLUSIONS

Classical macrophage activation and induction of LPS signaling pathways along with high endotoxin levels detected in BAL fluid from subjects with CR asthma suggest that LPS exposure might contribute to CR asthma.

T cells recruited to the kidney contribute to tissue damage in crescentic and proliferative glomerulonephritides. Chemokines and their receptors regulate T cell trafficking, but the expression profile and functional importance of chemokine receptors for renal CD4+ T cell subsets are incompletely understood. In this study, we observed that renal FoxP3+CD4+ regulatory T cells (Tregs) and IL-17-producing CD4+ T (Th17) cells express the chemokine receptor CCR6, whereas IFNgamma-producing Th1 cells are CCR6-. Induction of experimental glomerulonephritis (nephrotoxic nephritis) in mice resulted in upregulation of the only CCR6 ligand, CCL20, followed by T cell recruitment, renal tissue injury, albuminuria, and loss of renal function. CCR6 deficiency aggravated renal injury and increased mortality (from uremia) among nephritic mice. Compared with wild-type (WT) mice, CCR6 deficiency reduced infiltration of Tregs and Th17 cells but did not affect recruitment of Th1 cells in the setting of glomerulonephritis. Adoptive transfer of WT but not CCR6-deficient Tregs attenuated morphologic and functional renal injury in nephritic mice. Furthermore, reconstitution with WT Tregs protected CCR6-/- mice from aggravated nephritis. Taken together, these data suggest that CCR6 mediates renal recruitment of both Tregs and Th17 cells and that the reduction of anti-inflammatory Tregs in the presence of a fully functional Th1 response aggravates experimental glomerulonephritis.

OBJECTIVE

IL-2ralpha (CD25)(-/-) mice develop autoimmunity and lymphoproliferative disorders, including SS-like disease. The objective of this study was to evaluate the severity of corneal epithelial disease and T-cell cytokine profile in the ocular surface tissues of CD25KO mice.

METHODS

CD25KO mice were evaluated at 8, 12 and 16 weeks of age. Corneal epithelial smoothness and corneal permeability were measured. Phenotype of infiltrating lymphocytes was evaluated by immunohistochemistry. Th-1, -2 and -17 associated factors were measured by real-time PCR in cornea and conjunctiva and by Luminex immunobead assay in tears.

RESULTS

Compared with 8-week-old wild-type (WT) mice, CD25KO mice of the same age had significantly greater corneal irregularity and a significant increase in the number of CD4(+) and CD8(+) T cells infiltrating the conjunctiva. CD25KO mice had significantly higher levels of IL-6, TGF-beta1, IL-23R, IL-17A, IL-17F, IL-21, CCL20, IL-10, GATA-3 and IFN-gamma mRNA transcripts in their cornea and conjunctiva than WT mice at 8 weeks. IL-17A and IL-17F mRNA transcripts peaked at 12 weeks, whereas IFN-gamma spiked at 16 weeks in CD25KO mice. Increased expression of IL-17A and IL-17F at 12 weeks in CD25KO mice was accompanied by a worsening of corneal surface parameters and an increase of CD4(+) T cell infiltrating the cornea.

CONCLUSIONS

Disruption of IL-2 signalling in CD25KO mice results in age-dependent SS-like autoimmune lacrimal-keratoconjunctivitis. A mix of Th-1 and Th-17 cytokines was detected. The peak severity of corneal epithelial disease corresponded to the peak of IL-17 expression.

Streptococcus pneumoniae is a major cause of pneumonia in infants and the elderly. Innate defenses are essential to the control of pneumococcal infections, and deficient responses can trigger disease in susceptible individuals. Here we showed that flagellin can locally activate innate immunity and thereby increase the resistance to acute pneumonia. Flagellin mucosal treatment improved S. pneumoniae clearance in the lungs and promoted increased survival of infection. In addition, lung architecture was fully restored after the treatment of infected mice, indicating that flagellin allows the reestablishment of steady-state conditions. Using a flagellin mutant that is unable to signal through Toll-like receptor 5 (TLR5), we established that TLR5 signaling is essential for protection. In the respiratory tract, flagellin induced neutrophil infiltration into airways and upregulated the expression of genes coding for interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), CXCL1, CXCL2, and CCL20. Using depleting antibodies, we demonstrated that neutrophils are major effectors of protection. Further, we found that B- and T-cell-deficient SCID mice clear S. pneumoniae challenge to the same extent as immunocompetent animals, suggesting that these cell populations are not required for flagellin-induced protection. In conclusion, this study emphasizes that mucosal stimulation of innate immunity by a TLR not naturally engaged by S. pneumoniae can increase the potential to cure pneumococcal pneumonia.

Chemokine ligand 20 (CCL20) and human beta-defensins (HBDs) share structural and functional properties, including antiparallel beta-pleated sheet core structures, charge distribution, and signaling to adaptive immune cells via the highly selective CCR6 receptor. Because of their similarities, we hypothesized that in addition to its known adaptive immune signaling functions, CCL20 has antimicrobial properties and participates in pulmonary innate immunity. We found that primary cultures of human airway epithelial and cultured fetal lung explants expressed CCL20 mRNA. Expression of CCL20 transcripts were significantly induced by interleukin (IL)-1beta and tumor necrosis factor-alpha, and inhibited by dexamethasone. Primary cultures of airway epithelia secreted CCL20 both apically and basolaterally, and CCL20 abundance was increased over 30-fold with IL-1beta stimulation, achieving an estimated concentration of 167 ng/ml in airway surface liquid. CCL20 abundance in bronchoalveolar lavage fluid from patients with cystic fibrosis was nearly 90-fold higher compared with bronchoalveolar lavage fluid from healthy volunteers. Interestingly, CCL20 exhibited salt-sensitive antimicrobial activity, mainly against Gram-negative bacteria in low mug/ml concentrations. Additionally, apical washings from IL-1beta-stimulated primary cultures of human airway epithelia had significantly more antimicrobial activity than unstimulated controls. CCL20 rapidly permeabilized bacterial membranes with a time course intermediate to HBD-2 and HBD-3. Thus, CCL20 is a bi-functional peptide with both innate and adaptive immune properties that is regulated by inflammatory mediators, expressed by airway epithelia, and increased in cystic fibrosis airway secretions.

Isolated lymphoid follicles (ILFs) are organized lymphoid structures that facilitate the efficient interaction of antigen, antigen-presenting cells, and lymphocytes to generate controlled adaptive immune responses within the intestine. Because CC chemokine receptor 6 (CCR6) deficiency affects the generation of mucosal immune responses, we evaluated a potential role for CCR6 in the development of ILFs. We observed that CCR6 and its ligand CCL20 are highly expressed within ILFs and that B lymphocytes are the largest CCR6-expressing population within ILFs. ILF development was profoundly arrested in the absence of CCR6. Concordant with a block in ILF development at a stage corresponding to the influx of B lymphocytes, we observed that CCR6-deficient mice had a diminished population of intestinal B lymphocytes. Bone marrow reconstitution studies demonstrated that ILF development is dependent on CCR6-sufficient B lymphocytes, and adoptive transfers demonstrated that CCR6(-/-) B lymphocytes were inefficient at localizing to intestinal lymphoid structures. Paralleling these findings, we observed that CCR6-deficient mice had a reduced proportion of Peyer's patch B lymphocytes and an associated re-duction in the number and size of Peyer's patch follicular domes. These observations define an essential role for CCR6 expression by B lymphocytes in localizing to intestinal lymphoid structures and in ILF development.

The expression of CCL20 (MIP-3alpha), which chemoattracts leukocytes to sites of inflammation, has been shown in intestinal epithelial cells (IEC). Aim of this study was to analyze the role of the CCL20 receptor CCR6 in IEC and colorectal cancer (CRC) cells. Expression of CCR6 and CCL20 was analyzed by RT-PCR and immunohistochemistry. Signaling was investigated by Western blotting, proliferation by MTS assays and chemotactic cell migration by wounding assays. The effect of CCL20 on Fas-induced apoptosis was determined by flow cytometry. CCR6 and its ligand CCL20 are expressed in IEC. Moreover, CRC and CRC metastases express CCR6, which is upregulated during IEC differentiation. Stimulation of IEC with CCL20 and proinflammatory stimuli (TNF-alpha, IL-1beta, LPS) significantly upregulates CCL20 mRNA expression. CCL20 expression was significantly increased in inflamed colonic lesions in Crohn's disease and correlated significantly with the IL-8 mRNA expression in these lesions (r = 0.71) but was downregulated in CRC metastases. CCL20 activated Akt, ERK-1/2, and SAPK/JNK MAP kinases and increased IL-8 protein expression. The CCL20 mediated activation of these pathways resulted in a 2.6-fold increase of cell migration (P = 0.001) and in a significant increase of cell proliferation (P < 0.05) but did not influence Fas-induced apoptosis. In conclusion, IEC and CRC express CCL20 and its receptor CCR6. CCL20 expression is increased in intestinal inflammation, while CCR6 is upregulated during cell differentiation. CCR6 mediated signals result in increased IEC migration and proliferation suggesting an important role in intestinal homeostasis and intestinal inflammation by mediating chemotaxis of IEC but also in mediating migration of CRC cells.

Endometrial cancer is the third most common gynecologic malignancy and the ninth most common malignancy for females overall in Hong Kong. Approximately 80% or more of these cancers are endometrioid endometrial adenocarcinomas. The aim of this study was to reveal genes contributing to the development of endometrioid endometrial cancer, which may impact diagnosis, prognosis and treatment of the disease. Whole-genome gene expression analysis was completed for a set of 55 microdissected sporadic endometrioid endometrial adenocarcinomas and 29 microdissected normal endometrium specimens using the Affymetrix Human U133 Plus 2.0 oligonucleotide microarray. Selected genes of interest were validated by quantitative real-time-polymerase chain reaction (qRT-PCR). Pathway analysis was performed to reveal gene interactions involved in endometrial tumorigenesis. Unsupervised hierarchical clustering displayed a distinct separation between the endometrioid adenocarcinomas and normal endometrium samples. Supervised analysis identified 117 highly differentially regulated genes >>or=4.0-fold change), which distinguished the endometrial cancer specimens from normal endometrium. Twelve novel genes including DKK4, ZIC1, KIF1A, SAA2, LOC16378, ALPP2, CCL20, CXCL5, BST2, OLFM1, KLRC1 and MBC45780 were deregulated in the endometrial cancer, and further validated in an independent set of 56 cancer and 29 normal samples using qRT-PCR. In addition, 10 genes were differentially regulated in late-stage cancer, as compared to early-stage disease, and may be involved in tumor progression. Pathway analysis of the expression data from this tumor revealed an interconnected network consisting of 21 aberrantly regulated genes involved in angiogenesis, cell proliferation and chromosomal instability. The results of this study highlight the molecular features of endometrioid endometrial cancer and provide insight into the events underlying the development and progression of endometrioid endometrial cancer.

Neutrophils, historically known for their involvement in acute inflammation, are also targets for infection by many different DNA and RNA viruses. However, the mechanisms by which they recognize and respond to viral components are poorly understood. Polyinosinic:polycytidylic acid (poly(I:C)) is a synthetic mimetic of viral dsRNA that is known to interact either with endosomal TLR3 (not expressed by human neutrophils) or with cytoplasmic RNA helicases such as melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). In this study, we report that intracellularly administered poly(I:C) stimulates human neutrophils to specifically express elevated mRNA levels encoding type I IFNs, immunoregulatory cytokines, and chemokines, such as TNF-alpha, IL-12p40, CXCL10, CXCL8, CCL4, and CCL20, as well as classical IFN-responsive genes (IRG), including IFIT1 (IFN-induced protein with tetratricopeptide repeats 1)/IFN-stimulated gene (ISG)56, G1P2/ISG15, PKR (dsRNA-dependent protein kinase), and IFN-regulatory factor (IRF)7. Investigations into the mechanisms whereby transfected poly(I:C) promotes gene expression in neutrophils uncovered a crucial involvement of the MAPK-, PKR-, NF-kappaB-, and TANK (TNF receptor-associated NF-kappaB kinase)-binding kinase (TBK1)/IRF3-signaling transduction pathways, as illustrated by the use of specific pharmacological inhibitors. Consistent with the requirement of the cytoplasmic dsRNA pathway for antiviral signaling, human neutrophils were found to constitutively express significant levels of both MDA5 and RIG-I, but not TLR3. Accordingly, neutrophils isolated from MDA5-deficient mice had a partial impairment in the production of IFN-beta and TNF-alpha upon infection with encephalomyocarditis virus. Taken together, our data demonstrate that neutrophils are able to activate antiviral responses via helicase recognition, thus acting at the frontline of immunity against viruses.

OBJECTIVE

CCL20/MIP3alpha is a chemokine for immature dendritic cells as well as an antibacterial against gram-positive and gram-negative bacteria. The role of CCL20/MIP3alpha as an antiviral is unknown. In this study, we have examined the production of CCL20/MIP3alpha by epithelial cells from the upper female reproductive tract as well as its activity as an antiviral molecule.

METHODS

Primary uterine and Fallopian tube epithelial cells were treated with Poly(I:C) and CCL20/MIP3alpha mRNA and protein was measured by Realtime RT-PCR and ELISA assays. Anti-HIV activity was determined using an indicator cell line TZM-bl and quantified by using a luminometer.

RESULTS

Primary uterine and Fallopian tube epithelial cells produce CCL20/MIP3alpha constitutively and the production is enhanced following stimulation with viral double-stranded RNA mimic Poly(I:C). Recombinant CCL20/MIP3alpha was able to inhibit both T-cell-tropic X4/IIIB and macrophage-tropic R5/BaL HIV-1 when virus was directly incubated with CCL20/MIP3alpha but not when CCL20/MIP3alpha was added to cells either prior to infection or post-infection. This suggests that the mechanism of inhibition is likely to be a direct interaction between HIV-1 and CCL20/MIP3alpha.

CONCLUSIONS

This study demonstrates that CCL20/MIP3alpha is an important endogenous anti-HIV-1 microbicide of the female reproductive tract.

The IL-1 family of cytokines, which now includes 11 members, is well known to participate in inflammation. Although the most recently recognized IL-1 family cytokines (IL-1F5-11) have been shown to be expressed in airway epithelial cells, the regulation of their expression and function in the epithelium has not been extensively studied. We investigated the regulation of IL-1F5-11 in primary normal human bronchial epithelial cells. Messenger (m)RNAs for IL-1F6 and IL-1F9, but not IL-1F5, IL-1F8 or IL-1F10, were significantly up-regulated by TNF, IL-1β, IL-17 and the Toll-like receptor (TLR)3 ligand double-stranded (ds)RNA. mRNAs for IL-1F7 and IL-1F11 (IL-33) were weakly up-regulated by some of the cytokines tested. Notably, mRNAs for IL-1F6 and IL-1F9 were synergistically enhanced by the combination of TNF/IL-17 or dsRNA/IL-17. IL-1F9 protein was detected in the supernatant following stimulation with dsRNA or a combination of dsRNA and IL-17. IL-1F6 protein was detected in the cell lysate but was not detected in the supernatant. We screened for the receptor for IL-1F9 and found that lung fibroblasts expressed this receptor. We found that IL-1F9 activated mitogen-activated protein kinases and the transcription factor NF-κB in primary normal human lung fibroblasts. IL-1F9 also stimulated the expression of the neutrophil chemokines IL-8 and CXCL3 and the Th17 chemokine CCL20 in lung fibroblasts. These results suggest that epithelial activation by TLR3 (e.g., by respiratory viral infection) and exposure to cytokines from Th17 cells (IL-17) and inflammatory cells (TNF) may amplify neutrophilic inflammation in the airway via induction of IL-1F9 and activation of fibroblasts.

In LPS-stimulated human neutrophils, engagement of the adenosine A2A receptor selectively prevented the expression and release of TNF-alpha, MIP-1alpha/CCL3, MIP-1beta/CCL4, MIP-2alpha/CXCL2, and MIP-3alpha/CCL20. In mice lacking the A2A receptor, granulocytes that migrated into the air pouch 4 h after LPS injection expressed higher mRNA levels of TNF-alpha, MIP-1alpha, and MIP-1beta than PMNs from wild-type mice. In mononuclear cells present in the air pouch 72 h after LPS injection, expression of IL-1beta, TNF-alpha, IL-6, and MCP-2/CCL6 was higher in A2AR knockout mice. In addition to highlighting neutrophils as an early and pivotal target for mediating adenosine anti-inflammatory activities, these results identify TNF-alpha and the MIP chemokine family as gene products whose expression is pivotally affected by activation of A2AR in LPS-activated PMNs. Modulation by A2AR in the production of inflammatory signals by PMNs may thus influence the evolution of an inflammatory response by reducing the activation status of inflammatory cells.