Rationale: Uncontrolled inflammatory innate response and impaired adaptive immune response are associated with clinical severity in patients with Coronavirus disease 2019 (COVID-19).

Objectives: To compare the immunopathology of COVID-19 acute respiratory distress syndrome (ARDS) to that of non-COVID-19 ARDS, and to identify biomarkers associated with mortality in patients with COVID-19 ARDS.

Methods: Prospective observational monocenter study. Immunocompetent patients diagnosed with RT-PCR-confirmed SARS-CoV-2 infection and ARDS admitted between March 8 and March 30, 2020 were included and compared with patients with non-COVID-19 ARDS. The primary clinical endpoint of the study was mortality at day 28. Flow cytometry analyses and serum cytokines measurements were performed at days 1-2 and 4-6 of ICU admission.

Measurements and main results: As compared with patients with non-COVID-19 ARDS (n=36), those with COVID-19 (n=38) were not significantly different regarding age, gender, and SOFA and SAPS II scores but exhibited a higher day-28 mortality (34% vs 11%, p=0.030). COVID-19 patients showed profound and sustained T CD4+ (p=0.002), CD8+ (p<0.0001) and B (p<0.0001) lymphopenia, higher HLA-DR expression on monocytes (p<0.001) and higher serum concentrations of EGF, GM-CSF, IL-10, CCL2/MCP-1, CCL3/MIP-1a, CXCL10/IP-10, CCL5/RANTES, and CCL20/MIP-3a. After adjusting on age and SOFA, serum CXCL10/IP-10 (p=0.047) and GM-CSF (p=0.050) were higher and naso-pharyngeal RT-PCR cycle threshold values lower (p=0.010) in COVID-19 patients who were dead at day-28.

Conclusions: Profound global lymphopenia and a "chemokine signature" were observed in COVID-19 ARDS. Increased serum concentrations of CXCL10/IP-10 and GM-CSF, together with higher naso-pharyngeal SARS-CoV-2 viral load, were associated with day-28 mortality. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: SARS-CoV-2, COVID-19, ARDS, chemokines, cytokines.

CCR6 is a chemokine receptor that is expressed at the cell surface of Th17 cells, an IL-17- and IL-22-secreting population of CD4(+) T cells with antipathogenic, as well as inflammatory, properties. In the current study, we have determined the involvement of CCR6 in human Th17 lymphocyte migration toward inflamed tissue by analyzing the capacity of its ligands to induce arrest of these cells onto inflamed endothelium in vitro under flow conditions. We show that polarized, in situ-differentiated, skin-derived Th17 clones activated via the TCR-CD3 complex produce CCL20 in addition to IL-17 and IL-22. The latter cytokines induce, in a synergic fashion, the production of human β-defensin (hBD)-2, but neither hBD-1 nor hBD-3, by epidermal keratinocytes. Both CCL20 and hBD-2 are capable of inducing the arrest of Th17 cells, but not Th1 or Th2 cells, on HUVEC in an CD54-dependent manner that is CCR6 specific and independent from the expression of CXCR4, reported to be an alternative receptor for hBD-2. In addition, Ag-specific activation induces a transient loss of CCR6 expression, both at the transcriptional and protein level, which occurs with slow kinetics and is not due to endogenous CCL20-mediated internalization of CCR6. Together, these results indicate that Ag-specific activation will initially contribute to CCR6-mediated Th17 cell trafficking toward and sequestration in inflamed tissue, but that it eventually results in a transitory state of nonresponsiveness to further stimulation of these cells with CCR6 ligands, thus permitting their subsequent migration out of the inflamed site.

The human steroid receptor RNA activator (SRA) gene encodes both noncoding RNAs (ncRNAs) and protein-generating isoforms. In reporter assays, SRA ncRNA enhances nuclear receptor and myogenic differentiation 1 (MyoD)-mediated transcription but also participates in specific corepressor complexes, serving as a distinct scaffold. That SRA RNA levels might affect some biological functions, such as proliferation, apoptosis, steroidogenesis, and myogenesis, has been reported. However, the breadth of endogenous target genes that might be regulated by SRA RNAs remains largely unknown. To address this, we depleted SRA RNA in two human cancer cell lines with small interfering RNAs and then assayed for changes in gene expression by microarray analyses. The majority of significantly changed genes were reduced upon SRA knockdown, implicating SRA RNAs as endogenous coactivators. Unexpectedly, only a small subset of direct estrogen receptor-alpha target genes was affected in estradiol-treated MCF-7 cells. Eight bona fide SRA downstream target genes were identified (SLC2A3, SLC2A12, CCL20, TGFB2, DIO2, TMEM65, TBL1X, and TMPRSS2), representing entirely novel SRA targets, except for TMPRSS2. These data suggest unanticipated roles for SRA in glucose uptake, cellular signaling, T(3) hormone generation, and invasion/metastasis. SRA depletion in MDA-MB-231 cells reduced invasiveness and expression of some genes critical for this process. Consistent with the knockdown data, overexpressed SRA ncRNA coactivates certain target promoters and may enhance the activity of some coregulatory proteins. This study is a valuable resource because it represents the first genome-wide analysis of a mammalian RNA coregulator.

Beta-defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, members of the beta-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses, which are in part mediated by the CC-chemokine receptor CCR6. Here we report the expression of recombinant mBD4 and its human orthologue hBD2 fused to the constant domain of human IgG(1) to obtain correct folding and to increase stability and solubility using the Drosophila S2 expression system. Purified recombinant mBD4:Ig and hBD2:Ig fusion proteins retained potent antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, these beta-defensin fusion proteins showed specific binding to CCR6-expressing cells as revealed by flow cytometry. Interestingly, although hBD2:Ig bound to both human and mouse CCR6-expressing cells, mBD4:Ig did only bind to mCCR6-expressing cells but not to hCCR6-expressing cells. Both beta-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CC-chemokine receptor CCR6. The chemokine ligand CCL20 competed with the beta-defensin fusion proteins for specific binding to CCR6 as analyzed by fluorescence-activated cell sorter analysis. Both beta-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CCR6 receptor, but mBD4:Ig did not induce chemotactic activity of cells expressing human CCR6. This result supports our finding that mBD4 does not interact with human CCR6-expressing cells. Further evidence for specific interaction of the beta-defensin fusion proteins with CCR6-expressing cells is demonstrated by the observation that CCL20 and beta-defensin fusion proteins desensitize each other in inducing chemotactic activity. In addition both mBD4:Ig and hBD2:Ig demonstrated CCR6-independent chemotaxis of freshly isolated mouse resident peritoneal cells and human peripheral blood mononuclear cells, indicating the interaction with another chemotaxis-inducing receptor. Thus, the beta-defensin fusion proteins used in this study retained their biological activity and are a feasible tool to identify and analyze specific beta-defensin receptor interactions.

OBJECTIVE

To assess the expression of Toll-like receptor 3 (TLR-3) and TLR-7 in muscle tissue from patients with polymyositis (PM) and dermatomyositis (DM) and to investigate the function and regulation of TLR-3 in cultured muscle cells.

METHODS

The expression of TLR-3, TLR-7, HLA class I, and CD56, a marker of immature myoblast precursors, was analyzed using immunohistochemistry. TLR-3 regulation and signaling were assessed in myoblasts and in differentiated myotubes with the TLR-3 agonist poly(I-C), necrotic myoblasts, and Th1 and Th17 cytokines, in the presence or absence of neutralizing anti-TLR-3 antibody. Levels of TLR-3 messenger RNA (mRNA) were quantified by reverse transcription-polymerase chain reaction. Levels of interleukin-6 (IL-6), CCL20, and IL-8 were determined by enzyme-linked immunosorbent assay.

RESULTS

TLR-3 and TLR-7 were expressed in PM/DM tissues, but not in noninflammatory muscle tissues, and were primarily detected in inflammatory infiltrates, although a few muscle cells were also positive. These TLR-3- and TLR-7-positive fibers expressed high levels of CD56 and HLA class I antigens. A synergy between poly(I-C) and IL-17 was observed for the production of IL-6 and CCL20. Similarly, stimulation with necrotic myoblasts increased IL-6 production, and stimulation with necrotic myoblasts in combination with IL-17 further increased the induction of IL-6. TLR-3 blockade decreased the inducing effect of necrotic myoblasts and IL-17 on IL-6 production. Stimulation with interferon-gamma (IFNgamma) increased TLR-3 mRNA levels, but IL-17 down-regulated the inducing effect of IFNgamma.

CONCLUSIONS

Our findings indicate that TLR-3 and TLR-7 are expressed in inflammatory myopathic tissues, particularly in immature myoblast precursors. Necrotic muscle cells activate cytokine production, in part, through the TLR-3 pathway, with a differential regulatory effect of Th1 and Th17 cytokines.

Proinflammatory cytokines play important roles in insulin resistance. Here we report that mice with a T-cell-specific conditional knockout of HGK (T-HGK cKO) develop systemic inflammation and insulin resistance. This condition is ameliorated by either IL-6 or IL-17 neutralization. HGK directly phosphorylates TRAF2, leading to its lysosomal degradation and subsequent inhibition of IL-6 production. IL-6-overproducing HGK-deficient T cells accumulate in adipose tissue and further differentiate into IL-6/IL-17 double-positive cells. Moreover, CCL20 neutralization or CCR6 deficiency reduces the Th17 population or insulin resistance in T-HGK cKO mice. In addition, leptin receptor deficiency in T cells inhibits Th17 differentiation and improves the insulin sensitivity in T-HGK cKO mice, which suggests that leptin cooperates with IL-6 to promote Th17 differentiation. Thus, HGK deficiency induces TRAF2/IL-6 upregulation, leading to IL-6/leptin-induced Th17 differentiation in adipose tissue and subsequent insulin resistance. These findings provide insight into the reciprocal regulation between the immune system and the metabolism.

Th17 cells have emerged as a key factor in the pathogenesis of uveitis as well as other autoimmune disorders. They secrete a number of cytokines including IL-17A, IL-17F and IL-22 and until now the effects of these cytokines on resident cells of the eye were not yet clear. The purpose of this study was to investigate the effects of Interleukin (IL)-17A, IL-17F and IL-22 on the production of inflammatory mediators and barrier function of retinal pigment epithelium cells. We showed that ARPE-19 cells, a spontaneously arisen cell line of retinal pigment epithelium (RPE), constitutively expressed IL-17RC and IL-22R, but not IL-17RA. IL-17A significantly enhanced the production of CXCL8, CCL2, CCL20 and IL-6 by these cells. IL-17F had a similar effect on the production of CXCL8, CCL2 and IL-6 by ARPE-19 cells, but did not influence the expression of CCL20. Both IL-17A and IL-17F significantly decreased the transepithelial electrical resistance (TER) of the ARPE-19 monolayer and increased the diffusion rate of fluorescein isothiocyanate (FITC)-dextran. They also disrupted the distribution of the junction proteins zonula occludens (ZO)-1 and occludin at the interface of adjacent cells. IL-22 did not have a detectable effect on the production of the tested inflammatory mediators by ARPE-19 cells, TER of the ARPE-19 monolayer, the diffusion rate of FITC-dextran or the distribution of ZO-1 and occludin. This study demonstrates that IL-17A and IL-17F, but not IL-22, significantly promoted ARPE-19 cells to secrete inflammatory mediators and compromised the ARPE-19 monolayer barrier function in association with a disrupted distribution of ZO-1 and occludin. These results suggest that both IL-17A and IL-17F may play a role in posterior segment inflammation of the eye.

Natural killer (NK) cell-based adoptive immunotherapy is an attractive adjuvant treatment option for patients with acute myeloid leukemia. Recently, we reported a clinical-grade, cytokine-based culture method for the generation of NK cells from umbilical cord blood (UCB) CD34⁺ hematopoietic progenitor cells with high yield, purity and in vitro functionality. The present study was designed to evaluate the in vivo anti-leukemic potential of UCB-NK cells generated with our GMP-compliant culture system in terms of biodistribution, survival and cytolytic activity following adoptive transfer in immunodeficient NOD/SCID/IL2Rg(null) mice. Using single photon emission computed tomography, we first demonstrated active migration of UCB-NK cells to bone marrow, spleen and liver within 24 h after infusion. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Thereafter, we showed that low dose IL-15 mediates efficient survival, expansion and maturation of UCB-NK cells in vivo. Most importantly, we demonstrate that a single UCB-NK cell infusion combined with supportive IL-15 administration efficiently inhibited growth of human leukemia cells implanted in the femur of mice, resulting in significant prolongation of mice survival. These preclinical studies strongly support the therapeutic potential of ex vivo-generated UCB-NK cells in the treatment of myeloid leukemia after immunosuppressive chemotherapy.

The inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and ulcerative colitis (UC) are illness characterized by a chronic clinical course of relapse and remission associated with self-destructive inflammation of the gastrointestinal tract. In both UC and CD, leukocyte infiltration into the intestine is fundamental event in disease development and progression where the chemokines and their receptors are orchestrating the tissue-specific and the cell type-selective trafficking of leukocytes. In this review, we will discuss the homeostatic and inflammatory roles of the chemokines and their receptors with their potentials and promise as molecular targets for therapeutic interventions in human IBD, focusing on the recently identified role of the CX3CL1-CX3CR1 axis, as well as the CCL20-CCR6, CCL25-CCR9, and CXCL10-CXCR3 pathways.

Lactobacillus johnsonii (Ljo) N6.2 has been shown to mitigate the development of type 1 diabetes when administered to diabetes-prone rats. The specific mechanisms underlying this observed response remain under investigation. The objective of this study was to assess the effect of Ljo N6.2 on mucosal inflammatory response using differentiated Caco-2 monolayers. The mRNA expression levels of CCL20, CXCL8, and CXCL10 chemokines were determined by qRT-PCR. Ljo at 10(11) CFU/L induced a strong response in all chemokines examined. To assess the specific host-signaling pathways involved, we performed RT-PCR amplification of Toll-like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors. TLR7 and TLR9 expression levels were induced 4.2- and 9-fold, respectively, whereas other TLR and nucleotide-binding oligomerization domain receptors were not modified. A similar effect was observed in Caco-2 monolayers treated with Ljo cell-free extract or purified nucleic acids (NA). Increased levels of IFN type 1 and IFN regulators Stat1 and IRF7 followed the upregulation of TLR9. Activation of TLR9 was also evidenced by increased Frizzled 5 expression in Ljo-treated Caco-2 cells and an increase in the number of Paneth cells in Ljo-fed, diabetes-prone rats. These results are in agreement with the polarizing-tolerizing mechanism recently described in which the apical stimulation of TLR9 in intestinal epithelial cells leads to a higher state of immunologic alertness. Furthermore, these results suggest that live probiotics could be, in the future, replaced with select cellular components.

The mechanisms responsible for the evolution of steatosis towards NASH (non-alcoholic steatohepatitis) and fibrosis are not completely defined. In the present study we evaluated the role of CD4(+) T-helper (Th) cells in this process. We analysed the infiltration of different subsets of CD4(+) Th cells in C57BL/6 mice fed on a MCD (methionine choline-deficient) diet, which is a model reproducing all phases of human NASH progression. There was an increase in Th17 cells at the beginning of NASH development and at the NASH-fibrosis transition, whereas levels of Th22 cells peaked between the first and the second expansion of Th17 cells. An increase in the production of IL (interleukin)-6, TNFα (tumour necrosis factor α), TGFβ (transforming growth factor β) and CCL20 (CC chemokine ligand 20) accompanied the changes in Th17/Th22 cells. Livers of IL-17(-/-) mice were protected from NASH development and characterized by an extensive infiltration of Th22 cells. In vitro, IL-17 exacerbated the JNK (c-Jun N-terminal kinase)-dependent mouse hepatocyte lipotoxicity induced by palmitate. IL-22 prevented lipotoxicity through PI3K (phosphoinositide 3-kinase)-mediated inhibition of JNK, but did not play a protective role in the presence of IL-17, which up-regulated the PI3K/Akt inhibitor PTEN (phosphatase and tensin homologue deleted on chromosome 10). Consistently, livers of IL-17(-/-) mice fed on the MCD diet displayed decreased activation of JNK, reduced expression of PTEN and increased phosphorylation of Akt compared with livers of wild-type mice. Hepatic infiltration of Th17 cells is critical for NASH initiation and development of fibrosis in mice, and reflects an infiltration of Th22 cells. Th22 cells are protective in NASH, but only in the absence of IL-17. These data strongly support the potentiality of clinical applications of IL-17 inhibitors that can prevent NASH by both abolishing the lipotoxic action of IL-17 and allowing IL-22-mediated protection.

BACKGROUND

The importance of IL-17A, IL-17F, and IL-25 in allergic rhinitis (AR), as well as their possible role in regulation on thymic stromal lymphopoietin (TSLP) production in nasal epithelial cells, is not well understood.

OBJECTIVE

To determine the possible regulation of IL-17A, IL-17F, and IL-25 on TSLP production in the initiation of allergic responses.

METHODS

The levels of IL-17A, IL-17F, IL-25, and TSLP in nasal lavages of patients with AR were measured using an enzyme-linked immunosorbent assay (ELISA) and compared with that in normal controls. Then, primary human nasal epithelial cells (HNECs) were stimulated with dsRNA (0-75 microg/ml), as well as IL-17A (100 ng/ml), IL-17F (100 ng/ml), and IL-25(100 ng/ml). The mRNA expression of IL-17A, IL-17F, IL-25, TSLP, as well as the chemokines CCL20, IL-8, and eotaxin was analyzed using quantitative real-time PCR, and their protein levels in the supernatants of cultured HNECs were determined by ELISA.

RESULTS

Both TSLP and IL-17 cytokines are significantly elevated in patients with AR. dsRNA was found to increase the production of IL-17F, IL-25, TSLP, CCL20, and IL-8 in HNECs. Furthermore, IL-25 significantly enhanced dsRNA-induced TSLP production in primary HNECs and was dominant to the inhibitory effect of IL-17A on TSLP regulation.

CONCLUSIONS

Our study provides the first evidence that both IL-17F and IL-25 can be induced by dsRNA in HNECs. Despite of the opposing effects of IL-17A and IL-25 on TSLP regulation in HNECs, IL-25 was dominant to IL-17A, providing a plausible explanation for the simultaneous upregulation of IL-17 cytokines and TSLP in patients with AR.

Lactoferrin (LF) is an important protein component of the innate immune system that is broadly distributed within the body fluids. LF is endowed with multiple biological activities. Talactoferrin (TLF), a recombinant human LF, is in clinical development as an anticancer agent and is entering Phase III clinical trials. Here, we show that TLF induces the maturation of human dendritic cells (DCs) derived from monocytes. TLF, at physiologically relevant concentrations (100 microg/ml) up-regulates the expression of human leukocyte antigen (HLA) class II, CD83, CD80, and CD86 costimulatory molecule and CXCR4 and CCR7 chemokine receptors, acting primarily through the p38 MAPK signaling pathway. DCs matured by TLF displayed an enhanced release of IL-8 and CXCL10, as well as a significantly reduced production of IL-6, IL-10, and CCL20. They also display a reduced ability to take up antigen and increased capacity to trigger proliferation and release IFN-gamma in the presence of allogeneic human T cells. TLF-matured DCs are able to prime naive T cells to respond to KLH antigen and display a significantly increased capacity to present Flu-MA(58-66) peptide to HLA-A2-matched T cells. These data suggest that a key immunomodulatory function that may be mediated by TLF is to link the innate with adaptive immunity through DC maturation.

CCR6 is the only known receptor for the chemokine macrophage-inflammatory protein (MIP)-3alpha/CC chemokine ligand (CCL)20. We have shown previously that CCR6 is expressed on peripheral blood B cells, but CCR6 activity on these cells is low in in vitro assays. We report that MIP-3alpha/CCL20-induced calcium flux and chemotaxis can be enhanced significantly on peripheral blood and tonsillar B cells after activation by cross-linking surface Ag receptors. Of particular interest is the fact that the enhanced activity on B cells was not associated with an increase in CCR6 expression as assessed by levels of receptor mRNA, surface staining, or MIP-3alpha/CCL20 binding sites, or by a change in the affinity of the receptor for ligand. These data convincingly demonstrate that responses to a chemokine can be regulated solely by changes in the downstream pathways for signal transduction resulting from Ag receptor activation, and establish CCR6 as an efficacious receptor on human B cells.

Chemokines are important mediators of the immune response to pathogens, but can also promote chronic inflammatory states. Chemokine receptor 6 (CCR6) is found on immature DC and effector/memory T cells, and binds a single ligand, CCL20, with high affinity. Here, we investigated the role of CCL20 and CCR6 in a pulmonary viral infection caused by RSV, a ubiquitous virus that can cause severe pulmonary complications. Neutralization of CCL20 during RSV infection significantly reduced lung pathology and favored a Th1 effector response. CCR6-deficient animals recapitulated this phenotype, and additionally showed enhanced viral clearance when compared with WT mice. No differences were observed in migration of T cells to the lungs of CCR6(-/-) animals; however, a significant reduction was observed in numbers of conventional DC (cDC), but not plasmacytoid DC, in CCR6(-/-) mice. A pathogenic phenotype could be reconstituted in CCR6(-/-) mice by supplying cDC into the airway, indicating that mere number of cDC dictates the adverse response. Our data suggest that blockade of the CCL20/CCR6 pathway provides an environment whereby the attenuated recruitment of cDC alters the balance of innate immune cells and mediates the efficient antiviral response to RSV.

Inhalation of antigenic matter stimulates rapid recruitment of dendritic cells (DCs) into the lung. Recent studies propose that the chemokine CCL20 (macrophage inflammatory protein-3alpha) may play an important role in DC recruitment. We previously showed that primary human alveolar type II epithelial (ATII) cells are a rich source of chemokines and so hypothesized that the ATII cell produces CCL20 and might therefore be a key regulator of DC recruitment into the lung. Here, we show that primary human ATII cells, but not human alveolar macrophages, produce CCL20 both constitutively (403.5 +/- 85.4 pg/ml; 24 h) and in response to endotoxin (lipopolysaccharide) exposure (1,525.0 +/- 169.4 pg/ml; 1 mug/ml lipopolysaccharide; 24 h) in a time- and dose-dependent manner. In addition, we show that peripheral blood monocyte-derived CD1a+ DCs migrate in response to conditioned media from ATII cells but not those from alveolar macrophages; DC migration was significantly correlated with the amount of CCL20 (r(2)>> 0.9; P < 0.05) detected in the media but not with any other chemokine measured. We therefore conclude that the alveolar epithelium is an important source of CCL20 in the lung and that the ATII cell may play a critical role in controlling the movement of DCs through the lung both under normal and inflammatory conditions.

OBJECTIVE

To analyze the ability of nine different potentially probiotic bacteria to induce maturation and cytokine production in human monocyte-derived dendritic cells (moDCs).

METHODS

Cytokine production and maturation of moDCs in response to bacterial stimulation was analyzed with enzyme-linked immunosorbent assay (ELISA) and flow cytometric analysis (FACS), respectively. The kinetics of mRNA expression of cytokine genes was determined by Northern blotting. The involvement of different signaling pathways in cytokine gene expression was studied using specific pharmacological signaling inhibitors.

RESULTS

All studied bacteria induced the maturation of moDCs in a dose-dependent manner. More detailed analysis with S. thermophilus THS, B. breve Bb99, and L. lactis subsp. cremoris ARH74 indicated that these bacteria induced the expression of moDC maturation markers HLA class II and CD86 as efficiently as pathogenic bacteria. However, these bacteria differed in their ability to induce moDC cytokine gene expression. S. thermophilus induced the expression of pro-inflammatory (TNF-alpha, IL-12, IL-6, and CCL20) and Th1 type (IL-12 and IFN-gamma) cytokines, while B. breve and L. lactis were also potent inducers of anti-inflammatory IL-10. Mitogen-activated protein kinase (MAPK) p38, phosphatidylinositol 3 (PI3) kinase, and nuclear factor-kappa B (NF-kappaB) signaling pathways were shown to be involved in bacteria-induced cytokine production.

CONCLUSIONS

Our results indicate that potentially probiotic bacteria are able to induce moDC maturation, but their ability to induce cytokine gene expression varies significantly from one bacterial strain to another.

We previously reported that tumor-infiltrating interleukin (IL)-17A-producing cells play a protective role in human esophageal squamous cell carcinoma (ESCC). However, the potential mechanisms involved remain unclear. In the present study, we investigated the effects of IL-17A on immune cell recruitment and function in ESCC. In vitro chemotaxis assays using the ESCC cell lines EC109 and KYSE30 demonstrated that although IL-17A showed no significant direct effects on the migration of T cells, natural killer (NK) cells as well as dendritic cells (DCs), it could induce ESCC tumor cells to produce inflammatory chemokines, for example, CXCL9, CXCL10 and CCL2, CCL20, which are associated with the migration of T cells, NK cells, and DCs, respectively. In addition, IL-17A enhanced the cytotoxic effects of NK cells against tumor cells by augmenting the expression of cytotoxic molecules, for example, tumor necrosis factor-α, interferon-γ, Perforin, and Granzyme B and activation receptors, for example, NKp46, NKp44, NTB-A, and NKG2D on NK cells. Furthermore, immunohistochemical analysis revealed that the density of IL-17A-producing cells was positively and significantly associated with the density of CD1a DCs in tumor tissues. With the analyses of 181 ESCC patients, we found a correlation of higher number of tumor-infiltrating CD1a DCs with significantly improved overall survival of patients with ESCC. This study provides further understanding of the roles of Th17 cells in ESCC, which may contribute to the development of novel cancer immunotherapy strategies.

OBJECTIVE

Th17 cells are believed to be the primary effector cells in the pathogenesis of dry eye disease (DED). However, the mechanisms by which Th17 cells migrate from the lymphoid tissues to the ocular surface are unknown. The purpose of this study was to investigate the role of the C-C chemokine receptor 6/C-C chemokine ligand 20 (CCR6/CCL20) chemokine axis in mediating Th17 cell migration in DED.

METHODS

DED was induced by housing C57BL/6 mice in a low-humidity environment supplemented with scopolamine treatment. Th17 cell expression of CCR6 was evaluated using flow cytometry and ocular surface expression of CCL20 was measured using PCR and ELISA assays. CCL20 neutralizing antibody was administered subconjunctivally to DED mice and disease severity, including the frequency of conjunctival Th17 cells, was evaluated.

RESULTS

CCR6 is preferentially expressed by Th17 cells in both normal and DED mice and DED significantly upregulates ocular surface expression of CCL20. Disruption of CCR6/CCL20 binding with CCL20 neutralizing antibody decreases T-cell migration in vitro and reduces Th17 cell infiltration of the conjunctiva when administered in vivo, significantly improving clinical signs of DED. These changes were accompanied by a decrease in ocular surface inflammatory cytokine levels and corneal CD11b+ cell frequencies. Treatment also significantly reduced the generation of Th17 cells.

CONCLUSIONS

Local neutralization of CCL20 decreases Th17 cell infiltration of the ocular surface in DED, leading to improvement in clinical signs of disease. This suggests that CCR6/CCL20 interactions direct Th17 cell migration in DED and that disruption of this axis may be a novel therapeutic approach to this condition.

In a novel paradigm of T cell differentiation, type 17 T helper (Th17) cells may play a significant role in endometriosis, a chronic inflammatory disease. However, the mechanism regulating the accumulation of Th17 cells in endometriotic tissues remains unknown. We hypothesized that Th17 cells migrate to endometriotic tissues through an interaction of the chemokine CC chemokine ligand (CCL)20 and its receptor CCR6. Using endometriotic tissues from women with endometriosis, we demonstrated, by flow cytometry, that Th17 cells in endometriotic tissues express CC chemokine receptor (CCR)6. Immunohistochemistry also revealed that CCL20 was expressed in the epithelial cells and stromal cells beneath the epithelium of endometriotic tissues. CCR6+ cells were small and round and scattered in the stroma in which abundant CCL20+ cells were detected. CCL20 caused selective migration of Th17 cells in the peripheral blood in a migration assay. IL-1β, TNF-α, and IL-17A increased the secretion of CCL20 in cultured endometriotic stromal cells. Inhibitors of p38- and p42/44-MAPKs, and stress-activated protein kinase/c-Jun kinase suppressed the secretion of CCL20 increased by IL-1β, TNF-α, and IL-17A. This suggests that the CCL20/CCR6 system is involved in the migration of Th17 cells to endometriotic tissues and that proinflammatory cytokines contribute to the development of endometriosis via up-regulation of CCL20 secretion from endometriotic stromal cells.

When injected subcutaneously, mouse plasmacytoma (MOPC315) grew rapidly in situ, and metastatic cells became detectable first in the lymph nodes (LNs) and bone marrow, and later in the liver and lungs. We studied MOPC315 cell migration by tracking metastatic cells labelled with green fluorescent protein (GFP). We measured the levels of their chemokine receptor mRNA (by semiquantitative and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), because chemokines can regulate organ predilection of metastasis. Freshly sorted metastatic cells and tumour cell lines derived from the liver of BALB/c mice overexpressed functional CCR6 and CCR7 molecules compared with primary tumour. Preincubation with the CCR6 ligand (CCL20) induced liver-sorted tumour cells to preferentially colonize the liver, demonstrating an association between liver metastasis and CCR6 expression in the mouse. Because the liver is a common site for metastasis, second only to draining LNs, we wished to ascertain whether this finding could be generalized, i.e. whether other cancers can use the similar mechanism of metastasis to the liver, and whether it holds true for humans. We found that CCR6 is overexpressed in small liver metastases of colon, thyroid and ovarian carcinomas compared with normal liver. Because human liver constitutively expresses CCL20, it could attract and select CCR6+ cancer cells. We suggest that chemotaxis via CCR6 might be a common mechanism by which malignant cancers metastasize to the liver. As metastasis in patients with cancer poses the biggest peril for survival, inhibition of CCR6 signalling, either during or after medical or surgical treatment, might be useful in preventing liver metastasis.

Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein β (C/EBPβ) as a critical regulator of CCL20 gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBPβ converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBPβ as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBPβ in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBPβ-inducible and constitutive CCL20 gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity.

The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated.

To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells.

IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments.

IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival.

Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.