The mechanisms of action of marketed TNF-blocking drugs in lesional tissues are still incompletely understood. Because psoriasis plaques are accessible to repeat biopsy, the effect of TNF/lymphotoxin blockade with etanercept (soluble TNFR) was studied in ten psoriasis patients treated for 6 months. Histological response, inflammatory gene expression, and cellular infiltration in psoriasis plaques were evaluated. There was a rapid and complete reduction of IL-1 and IL-8 (immediate/early genes), followed by progressive reductions in many other inflammation-related genes, and finally somewhat slower reductions in infiltrating myeloid cells (CD11c+ cells) and T lymphocytes. The observed decreases in IL-8, IFN-gamma-inducible protein-10 (CXCL10), and MIP-3alpha (CCL20) mRNA expression may account for decreased infiltration of neutrophils, T cells, and dendritic cells (DCs), respectively. DCs may be less activated with therapy, as suggested by decreased IL-23 mRNA and inducible NO synthase mRNA and protein. Decreases in T cell-inflammatory gene expression (IFN-gamma, STAT-1, granzyme B) and T cell numbers may be due to a reduction in DC-mediated T cell activation. Thus, etanercept-induced TNF/lymphotoxin blockade may break the potentially self-sustaining cycle of DC activation and maturation, subsequent T cell activation, and cytokine, growth factor, and chemokine production by multiple cell types including lymphocytes, neutrophils, DCs, and keratinocytes. This results in reversal of the epidermal hyperplasia and cutaneous inflammation characteristic of psoriatic plaques.

Tumor development and progression are multifactorial processes, regulated by a large variety of intrinsic and microenvironmental factors. A key role in cancer is played by members of the chemokine superfamily. Chemokines and their receptors are expressed by tumor cells and by host cells, in primary tumors and in specific metastatic loci. The effects of chemokines on tumorigenesis are diverse: While some members of the superfamily significantly support this process, others inhibit fundamental events required for tumor establishment and metastasis. The current review describes the multifaceted roles of chemokines in malignancy, addressing four major aspects of their activities: (1) inducing leukocyte infiltration to tumors and regulating immune functions, with emphasis on tumor-associated macrophages (and the chemokines CCL2, CCL5), T cells (and the chemokines CXCL9, CXCL10) and dendritic cells (and the chemokines CCL19, CCL20, CCL21); (2) directing the homing of tumor cells to specific metastatic sites (the CXCL12-CXCR4 axis); (3) regulating angiogenic processes (mainly the ELR(+)-CXC and non-ELR-CXC chemokines); (4) acting directly on the tumor cells to control their malignancy-related functions. Together, these different chemokine functions establish a net of interactions between the tumor cells and their microenvironment, and partly dictate the fate of the malignancy cascade.

Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing interspecies differences in the transcriptional responses of primary human and mouse macrophages to the Toll-like receptor (TLR)-4 agonist lipopolysaccharide (LPS). By using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5' ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologues were identified as "divergently regulated"). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated orthologues were enriched for genes encoding cellular "inputs" such as cell surface receptors (e.g., TLR6, IL-7Rα) and functional "outputs" such as inflammatory cytokines/chemokines (e.g., CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pretreatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. Divergently regulated genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced interspecies promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change.

Th9 cells are a subset of CD4+ Th cells that produce the pleiotropic cytokine IL-9. IL-9/Th9 can function as both positive and negative regulators of immune response, but the role of IL-9/Th9 in tumor immunity is unknown. We examined the role of IL-9/Th9 in a model of pulmonary melanoma in mice. Lack of IL-9 enhanced tumor growth, while tumor-specific Th9 cell treatment promoted stronger antitumor responses in both prophylactic and therapeutic models. Th9 cells also elicited strong host antitumor CD8+ CTL responses by promoting Ccl20/Ccr6-dependent recruitment of DCs to the tumor tissues. Subsequent tumor antigen delivery to the draining LN resulted in CD8+ T cell priming. In agreement with this model, Ccr6 deficiency abrogated the Th9 cell-mediated antitumor response. Our data suggest a distinct role for tumor-specific Th9 cells in provoking CD8+ CTL-mediated antitumor immunity and indicate that Th9 cell-based cancer immunotherapy may be a promising therapeutic approach.

Microenvironment molecular cues direct T helper (Th) cell differentiation; however, Th17 fate determination is still imprecisely understood in humans. To assess the role of prostaglandin E(2) (PGE(2)) in Th expansion, we activated peripheral blood mononuclear cells by CD3 cross-linking. In the presence of exogenous PGE(2), peripheral blood mononuclear cells produced higher interleukin-17 (IL-17), C-C chemokine ligand 20 (CCL20)/macrophage inflammatory protein 3alpha (MIP-3alpha), CXC chemokine ligand 8 (CXCL8)/IL-8, and lower interferon-gamma and IL-22 levels than in control cultures. Exogenous PGE(2) and IL-23 synergized in inducing IL-17, whereas indomethacin and IL-23 blockade drastically reduced IL-17 but not interferon-gamma production. Furthermore, IL-1 but not tumor necrosis factor was absolutely required for IL-17 production. PGE(2) doubled the frequency of CD4+ T cells producing IL-17 and within the CD4+ subset enhanced C-C chemokine receptor 6 (CCR6) and CCR4 while decreasing CXC chemokine receptor 3 (CXCR3) expression. Furthermore, in CD4+ T-cell lines, the production of IL-17 segregated with the CCR6+ subset. In the presence of CCR6+ compared with CXCR3+ Th cells, monocytes/macrophages produced much higher levels of matrix metalloproteinase-1, -3, and -9 but similar levels of CXCL10 and IL-1beta. These results identify PGE(2) and IL-23 as participating in the expansion of CD4+ T cells endowed with high IL-17 production capacity, which in turn favors monocyte production of mediators important for host defense and tissue destruction.

Langerhans cells (LCs) are epidermal dendritic cells with incompletely understood origins that associate with hair follicles for unknown reasons. Here we show that in response to external stress, mouse hair follicles recruited Gr-1(hi) monocyte-derived precursors of LCs whose epidermal entry was dependent on the chemokine receptors CCR2 and CCR6, whereas the chemokine receptor CCR8 inhibited the recruitment of LCs. Distinct hair-follicle regions had differences in their expression of ligands for CCR2 and CCR6. The isthmus expressed the chemokine CCL2; the infundibulum expressed the chemokine CCL20; and keratinocytes in the bulge produced the chemokine CCL8, which is the ligand for CCR8. Thus, distinct hair-follicle keratinocyte subpopulations promoted or inhibited repopulation with LCs via differences in chemokine production, a feature also noted in humans. Pre-LCs failed to enter hairless skin in mice or humans, which establishes hair follicles as portals for LCs.

OBJECTIVE

Adipose tissue (AT) plays a major role in the low-grade inflammatory state associated with obesity. The aim of the present study was to characterize the human AT lymphocytes (ATLs) and to analyze their interactions with adipocytes.

RESULTS

Human ATL subsets were characterized by flow cytometry in subcutaneous ATs from 92 individuals with body mass index (BMI) ranging from 19 to 43 kg/m(2) and in paired biopsies of subcutaneous and visceral AT from 45 class II/III obese patients. CD3(+) ATLs were composed of effector and memory CD4(+) helper and CD8(+) cytotoxic T cells. The number of ATLs correlated positively with BMI and was higher in visceral than subcutaneous AT. Mature adipocytes stimulated the migration of ATLs and released the chemokine CCL20, the receptor of which (CCR6) was expressed in ATLs. The expression of adipocyte CCL20 was positively correlated with BMI and increased in visceral compared to subcutaneous adipocytes. ATLs expressed inflammatory markers and released interferon gamma (IFN gamma). Progenitor and adipocyte treatment with ATL-conditioned media reduced the insulin-mediated upregulation of lipogenic enzymes, an effect involving IFN gamma.

CONCLUSIONS

Therefore, crosstalk occurs between adipocytes and lymphocytes within human AT involving T cell chemoattraction by adipocytes and modulation of lipogenesis by ATLs.

BACKGROUND

Tumor-associated macrophages (TAMs) remodel the colorectal cancer (CRC) microenvironment. Yet, findings on the role of TAMs in CRC seem to be contradictory compared with other cancers. FoxP3(+) regulatory T (Treg)-cells dominantly infiltrate CRC. However, the underlying molecular mechanism in which TAMs may contribute to the trafficking of Treg-cells to the tumor mass remains unknown.

RESULTS

CRC was either induced by N-methyl-N-nitrosourea (MNU) and H. pylori or established by subcutaneous injection of mouse colorectal tumor cell line (CMT93) in mice. CMT93 cells were co-cultured with primary macrophages in a transwell apparatus. Recruitment of FoxP3 green fluorescence protein positive (FoxP3(GFP+)) Treg-cells was assessed using the IVIS Imaging System or immunofluorescence staining. A role for macrophages in trafficking of Treg-cells and in the development of CRC was investigated in CD11b diphtheria toxin receptor (CD11b-DTR) transgenic C57BL/6J mice in which macrophages can be selectively depleted. Treg-cells remarkably infiltrated solid tumor, and predominantly expressed the homing chemokine receptor (CCR) 6 in the induced CRC model. Both CMT93 cancer cells and macrophages produced a large amount of CCL20, the sole ligand of CCR6 in vitro and in vivo. Injection of recombinant mouse CCL20 into tumor sites promoted its development with a marked recruitment of Treg-cells in the graft CRC model. Conditional macrophage ablation decreased CCL20 levels, blocked Treg-cell recruitment and inhibited tumor growth in CD11b-DTR mice grafted with CMT93.

CONCLUSIONS

TAMs recruit CCR6(+) Treg-cells to tumor mass and promote its development via enhancing the production of CCL20 in a CRC mouse model.

Human intestinal epithelial cells secrete an array of chemokines known to signal the trafficking of neutrophils and monocytes important in innate mucosal immunity. We hypothesized that intestinal epithelium may also have the capacity to play a role in signaling host adaptive immunity. The CC chemokine macrophage inflammatory protein (MIP)-3alpha/CCL20 is chemotactic for immature dendritic cells and CD45RO(+) T cells that are important components of the host adaptive immune system. In these studies, we demonstrate the widespread production and regulated expression of MIP-3alpha by human intestinal epithelium. Several intestinal epithelial cell lines were shown to constitutively express MIP-3alpha mRNA. Moreover, MIP-3alpha mRNA expression and protein production were upregulated by stimulation of intestinal epithelial cells with the proinflammatory cytokines tumor necrosis factor-alpha or interleukin-1alpha or in response to infection with the enteric bacterial pathogens Salmonella or enteroinvasive Escherichia coli. In addition, MIP-3alpha was shown to function as a nuclear factor-kappaB target gene. In vitro findings were paralleled in vivo by increased expression of MIP-3alpha in the epithelium of cytokine-stimulated or bacteria-infected human intestinal xenografts and in the epithelium of inflamed human colon. Mucosal T cells, other mucosal mononuclear cells, and intestinal epithelial cells expressed CCR6, the cognate receptor for MIP-3alpha. The constitutive and regulated expression of MIP-3alpha by human intestinal epithelium is consistent with a role for epithelial cell-produced MIP-3alpha in modulating mucosal adaptive immune responses.

T cells infiltrate the kidney in both human and experimental glomerulonephritis, and several lines of evidence indicate that T cell-mediated tissue damage plays an important role in the immunopathogenesis of renal inflammatory diseases. However, the functions of the different T cell subsets, particularly the recently identified interleukin-17 (IL-17)-producing T cells (Th17 cells), are incompletely understood in glomerulonephritis. Here, we identified renal IL-17-producing T cells in the T cell-mediated model of nephrotoxic nephritis in mice. In vitro, IL-17 enhanced the production of the proinflammatory chemokines CCL2/MCP-1, CCL3/MIP-1alpha, and CCL20/LARC, which are implicated in the recruitment of T cells and monocytes, in mouse mesangial cells. To determine the function of Th17 cells in renal inflammation, we induced nephrotoxic nephritis in IL-23 p19(-/-) mice, which have reduced numbers of Th17 cells, and in IL-17(-/-) mice, which are deficient in the effector cytokine IL-17 itself. In comparison with nephritic wild-type mice, IL-23 p19(-/-) mice demonstrated less infiltration of Th17 cells, and both IL-23 p19(-/-) and IL-17(-/-) mice developed less severe nephritis as measured by renal function, albuminuria, and frequency of glomerular crescent formation. These results demonstrate that the IL-23/IL-17 pathway significantly contributes to renal tissue injury in experimental glomerulonephritis. Targeting the IL-23/Th17 axis may be a promising therapeutic strategy for the treatment of proliferative and crescentic glomerulonephritis.

T(H)17 cells are the newest member of the T(H) cell family and are characterized by their ability to produce specific cytokines such as IL-17, IL-22, IL-17F, and CCL20. In this review, conditions for the differentiation of T(H)17 cells are defined in both murine and human systems, with discussion of T(H)17-specific cytokines and transcription factors. Functionally, T(H)17 cells contribute to host defense as a new effector T(H) cell subset with a role in protection against extracellular bacteria through activities on immune and nonimmune cells. Their activities, however, are also pivotal in the development of autoimmune diseases under pathologic conditions. T(H)17 cells are also beginning to be associated with the development and pathophysiology of allergic diseases, such as allergic contact dermatitis, atopic dermatitis, and asthma. Lymphoid tissue inducer-like cells and natural killer-like cells, termed RORgammat(+)NKp46(+) or NK-22 cells, might also play a role in allergic diseases because of their propensity to produce IL-17 and IL-22.

Growth in three-dimensional (3D) architectures has been suggested to play an important role in tumor expansion and in the resistance of cancers to treatment with drugs or cytokines or irradiation. To obtain an insight into underlying molecular mechanisms, we addressed gene expression profiles of NA8 melanoma cells cultured in bidimensional monolayers (2D) or in 3D multicellular tumor spheroids (MCTS). MCTS containing 10-30,000 cells were generated upon overnight culture in poly-Hydroxyethylmethacrylate (polyHEMA) coated plates. Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long-term culture (>10 days), however, MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with>>/=threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant,>>/=threefold, upregulation of the expression of >100 transcripts while 73 were>>/=threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO-alpha, -beta, and gamma), IL-8, CCL20 (MIP-3alpha), and Angiopoietin-like 4 were significantly upregulated, whereas basic FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real-time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process.

CCL20, like human beta-defensin (hBD)-2, is a potent chemoattractant for CCR6-positive immature dendritic cells and T cells in addition to recently found antimicrobial activities. We previously demonstrated that IL-17 is the most potent cytokine to induce an apical secretion and expression of hBD-2 by human airway epithelial cells, and the induction is JAK/NF-kappaB-dependent. Similar to hBD-2, IL-17 also induced CCL20 expression, but the nature of the induction has not been elucidated. Compared with a panel of cytokines (IL-1alpha, 1beta, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, IFN-gamma, GM-CSF, and TNF-alpha), IL-17 was as potent as IL-1alpha, 1beta, and TNF-alpha, with a time- and dose-dependent phenomenon in stimulating CCL20 expression in both well-differentiated primary human and mouse airway epithelial cell culture systems. The stimulation was largely dependent on the treatment of polarized epithelial cultures from the basolateral side with IL-17, achieving an estimated 4- to 10-fold stimulation at both message and protein levels. More than 90% of induced CCL20 secretion was toward the basolateral compartment (23.02 +/- 1.11 ng/chamber/day/basolateral vs 1.82 +/- 0.82 ng/chamber/day/apical). Actinomycin D experiments revealed that enhanced expression did not occur at mRNA stability. Inhibitor studies showed that enhanced expression was insensitive to inhibitors of JAK/STAT, p38, JNK, and PI3K signaling pathways, but sensitive to inhibitors of MEK1/2 and NF-kappaB activation, suggesting a MEK/NF-kappaB-based mechanism. These results suggest that IL-17 can coordinately up-regulate both hBD-2 and CCL20 expressions in airways through differentially JAK-dependent and -independent activations of NF-kappaB-based transcriptional mechanisms, respectively.

Langerhans cells (LCs) represent a subset of immature dendritic cells (DCs) specifically localized in the epidermis and other mucosal epithelia. As surrounding keratinocytes can produce interleukin (IL)-15, a cytokine that utilizes IL-2Rgamma chain, we analyzed whether IL-15 could skew monocyte differentiation into LCs. Monocytes cultured for 6 d with granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-15 differentiate into CD1a(+)HLA-DR(+)CD14(-)DCs (IL15-DCs). Agents such as lipopolysaccharide (LPS), tumor necrosis factor (TNF)alpha, and CD40L induce maturation of IL15-DCs to CD83(+), DC-LAMP(+) cells. IL15-DCs are potent antigen-presenting cells able to induce the primary (mixed lymphocyte reaction [MLR]) and secondary (recall responses to flu-matrix peptide) immune responses. As opposed to cultures made with GM-CSF/IL-4 (IL4-DCs), a proportion of IL15-DCs expresses LC markers: E-Cadherin, Langerin, and CC chemokine receptor (CCR)6. Accordingly, IL15-DCs, but not IL4-DCs, migrate in response to macrophage inflammatory protein (MIP)-3alpha/CCL20. However, IL15-DCs cannot be qualified as "genuine" Langerhans cells because, despite the presence of the 43-kD Langerin, they do not express bona fide Birbeck granules. Thus, our results demonstrate a novel pathway in monocyte differentiation into dendritic cells.

Human macrophage inflammatory protein-3alpha (MIP-3alpha; CCL20) is a CC-type chemokine that binds to and activates CC chemokine receptor-6 (CCR6). Although MIP-3alpha does not share the binding site of CCR6 with any other chemokine, human beta-defensin-1 and -2, small cationic antimicrobial peptides, have also been found to bind to and activate CCR6. Conversely, we have found that MIP-3alpha possesses antibacterial activity of greater potency than human beta-defensin-1 and -2 against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, while having no activity against the fungus Candida albicans. There is no clear sequence similarity between beta-defensins and the chemokine MIP-3alpha, beyond an abundance of cationic residues and the presence of disulfide bonds. Nonetheless, there are structural similarities between these three proteins that allow their overlap of chemotactic and antimicrobial activities. In this report, we describe the x-ray crystal structure of human MIP-3alpha refined to a resolution of 1.7 A and compare it with the crystal structures of human beta-defensin-1 and -2. Molecules of MIP-3alpha and the beta-defensins seem to share few structural motifs that are likely associated with their common biological activities.

BACKGROUND

Regulatory T cells (Tregs) are highly prevalent in tumor tissue and can suppress effective anti-tumor immune responses. However, the source of the increased tumor-infiltrating Tregs and their contribution to cancer progression remain poorly understood.

RESULTS

We here investigated the frequency, phenotype and trafficking property of Tregs and their prognostic value in patients with hepatocellular carcinoma (HCC). Our results showed that FoxP3(+) Tregs highly aggregated and were in an activated phenotype (CD69(+)HLA-DR(high)) in the tumor site, where they can suppress the proliferation and INF-γ secretion of CD4(+)CD25(-) T cells. These tumor-infiltrating Tregs could be selectively recruited though CCR6-CCL20 axis as illustrated by (a) high expression of CCR6 on circulating Tregs and their selective migration to CCR6 ligand CCL20, and (b) correlation of distribution and expression between tumor-infiltrating Tregs and intratumoral CCL20. In addition, we found that the number of tumor-infiltrating Tregs was associated with cirrhosis background (P = 0.011) and tumor differentiation (P = 0.003), and was an independent prognostic factor for overall survival (HR = 2.408, P = 0.013) and disease-free survival (HR = 2.204, P = 0.041). The increased tumor-infiltrating Tregs predicted poorer prognosis in HCC patients.

CONCLUSIONS

The CCL20-CCR6 axis mediates the migration of circulating Tregs into tumor microenvironment, which in turn results in tumor progression and poor prognosis in HCC patients. Thus, blocking CCL20-CCR6 axis-mediated Treg migration may be a novel therapeutic target for HCC.

Initially, children were thought to be spared from disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, a month into the epidemic, a novel multisystem inflammatory syndrome in children (MIS-C) emerged. Herein, we report on the immune profiles of nine MIS-C cases. All MIS-C patients had evidence of prior SARS-CoV-2 exposure, mounting an antibody response with intact neutralization capability. Cytokine profiling identified elevated signatures of inflammation (IL-18 and IL-6), lymphocytic and myeloid chemotaxis and activation (CCL3, CCL4, and CDCP1), and mucosal immune dysregulation (IL-17A, CCL20, and CCL28). Immunophenotyping of peripheral blood revealed reductions of non-classical monocytes, and subsets of NK and T lymphocytes, suggesting extravasation to affected tissues. Finally, profiling the autoantigen reactivity of MIS-C plasma revealed both known disease-associated autoantibodies (anti-La) and novel candidates that recognize endothelial, gastrointestinal, and immune-cell antigens. All patients were treated with anti-IL-6R antibody and/or IVIG, which led to rapid disease resolution.

Keywords: COVID-19; Kawasaki-like; MIS-C; PIMS; SARS-CoV-2; autoimmunity; dysfunction; immune; pediatrics.

OBJECTIVE

Several lines of evidence support a role for Toll-like receptor (TLR) signaling to protect the intestine from pathogenic infection. We hypothesized that TLR signaling at the level of the intestinal epithelium is critical for mucosal immune responses.

METHODS

We generated transgenic mice that express a constitutively active form of TLR4 in the intestinal epithelium (V-TLR4 mice). Lamina propria cellularity was evaluated by immunostaining and flow cytometry. Immunoglobulin (Ig) A levels in the stool and serum were measured by enzyme-linked immunosorbent assay. Chemokine and cytokine expression were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.

RESULTS

V-TLR4 transgenic mice reproduced normally and had a normal life span. Constitutive activity of TLR4 in the intestinal epithelium promoted recruitment of B cells and an increase in fecal IgA levels. Intestinal epithelial cells of V-TLR4 mice expressed higher levels of CCL20 and CCL28, chemokines known to be involved in B-cell recruitment, and of a proliferation-inducing ligand (APRIL), a cytokine that promotes T-cell-independent class switching of B cells to IgA. The changes in B-cell numbers and IgA levels were blocked by simultaneous expression in intestinal epithelial cells of M3, a herpes virus protein that binds and inhibits multiple chemokines.

CONCLUSIONS

TLR signaling in the intestinal epithelial cells significantly elevated the production of IgA in the intestine. This effect was mediated by TLR-induced expression of a specific set of chemokines and cytokines that promoted both recruitment of B cells into the lamina propria and IgA class switching of B cells.

Chronic obstructive pulmonary disease (COPD) is mainly caused by cigarette smoking, and is characterized by an increase in inflammatory cells in the airways and pulmonary tissue. The chemokine receptor CCR6 and its ligand MIP-3alpha/CCL20 may be involved in the recruitment of these inflammatory cells. To investigate the role of CCR6 in the pathogenesis of COPD, we analyzed the inflammatory responses of CCR6 knockout (KO) and wild-type mice upon cigarette smoke (CS) exposure. Both subacute and chronic exposure to CS induced an increase in cells of the innate and adaptive immune system in the bronchoalveolar lavage, both in CCR6 KO and wild-type mice. However, the accumulation of dendritic cells, neutrophils, and T lymphocytes, which express CCR6, was significantly attenuated in the CCR6 KO mice, compared with their wild-type littermates. In the lung tissue of CCR6 KO mice, there was an impaired increase in dendritic cells, activated CD8(+) T lymphocytes, and granulocytes. Moreover, this attenuated inflammatory response in CCR6 KO mice offered a partial protection against pulmonary emphysema, which correlated with an impaired production of MMP-12. Importantly, protein levels of MIP-3alpha/CCL20, the only chemokine ligand of the CCR6 receptor, and MCP-1/CCL2 were significantly increased upon CS exposure in wild-type, but not in CCR6 KO mice. In contrast, CCR6 deficiency had no effect on the development of airway wall remodeling upon chronic CS exposure. These results indicate that the interaction of CCR6 with its ligand MIP-3alpha contributes to the pathogenesis of CS-induced pulmonary inflammation and emphysema in this murine model of COPD.

BACKGROUND

Patients with idiopathic pulmonary arterial hypertension (IPAH) present circulating autoantibodies against vascular wall components. Pathogenic antibodies may be generated in tertiary (ectopic) lymphoid tissues (tLTs).

OBJECTIVE

To assess the frequency of tLTs in IPAH lungs, as compared with control subjects and flow-induced PAH in patients with Eisenmenger syndrome, and to identify local mechanisms responsible for their formation, perpetuation, and function.

METHODS

tLT composition and structure were studied by multiple immunostainings. Cytokine/chemokine and growth factor expression was quantified by real-time polymerase chain reaction and localized by immunofluorescence. The systemic mark of pulmonary lymphoid neogenesis was investigated by flow cytometry analyses of circulating lymphocytes.

RESULTS

As opposed to lungs from control subjects and patients with Eisenmenger syndrome, IPAH lungs contained perivascular tLTs, comprising B- and T-cell areas with high endothelial venules and dendritic cells. Lymphocyte survival factors, such as IL-7 and platelet-derived growth factor-A, were expressed in tLTs as well as the lymphorganogenic cytokines/chemokines, lymphotoxin-α/-β, CCL19, CCL20, CCL21, and CXCL13, which might explain the depletion of circulating CCR6(+) and CXCR5(+) lymphocytes. tLTs were connected with remodeled vessels via an ER-TR7(+) stromal network and supplied by lymphatic channels. The presence of germinal center centroblasts, follicular dendritic cells, activation-induced cytidine deaminase, and IL-21(+)PD1(+) follicular helper T cells in tLTs together with CD138(+) plasma cell accumulation around remodeled vessels in areas of immunoglobulin deposition argued for local immunoglobulin class switching and ongoing production.

CONCLUSIONS

We highlight the main features of lymphoid neogenesis specifically in the lungs of patients with IPAH, providing new evidence of immunological mechanisms in this severe condition.

Pericytes and mesenchymal stem cells (MSCs) are ontogenically related, and in fact, no significant phenotypic differences could be observed by flow cytometry. Transcriptome analysis of human pericytes and MSCs revealed that 43 genes were up-regulated more than 10-fold in pericytes compared with MSCs. Identification of Toll-like receptor 4 (TLR4) as one of the most abundant RNA species in pericytes with respect to MSCs and confirmation of TLR4 expression on the cell surface led us to obtain a comprehensive overview of the expression program of lipopolysaccharide (LPS)-stimulated pericytes. Transcriptional profiling of LPS-treated cells revealed that 22 genes were up-regulated more than 5-fold. Of them, 10 genes encoded chemokines and cytokines (CXCL10, CCL20, IL8, CXCL1, IL6, CCL2, IL1B, CXCL2, IL1A, and CXCL6), and three genes encoded adhesion molecules (ICAM1, VCAM1, and SELE). LPS induced nuclear translocation of the transcription factor NF-κB in stimulated pericytes. Moreover, inhibition of NF-κB activation by SC-514 blocked LPS-induced up-regulation of a subset of chemokine genes, confirming the key role of NF-κB in LPS signaling in pericytes. At the protein level, we assessed the secretion of the proinflammatory cytokines and chemokines IL-6, IL-8, CXCL1, CXCL2, CXCL3, and CCL2 not only after LPS treatment but also in HMGB1-stimulated pericytes. Up-regulation of the adhesion molecules ICAM-1 and VCAM-1 resulted in an increased adhesion of peripheral blood leukocytes to an LPS-treated pericyte monolayer. The role of pericytes in the inflammatory context has been scarcely addressed; according to these results, pericytes should be considered as active players in the inflammatory cascade with potential physiopathological implications.

Th17 cells have been named after their signature cytokine IL-17 and accumulating evidence indicates their involvement in the induction and progression of inflammatory diseases. In addition to IL-17 single-producing T cells, IL-17/IFN-gamma double-positive T cells are found in significantly elevated numbers in inflamed tissues or blood from patients with chronic inflammatory disorders. Because IFN-gamma is the classical Th1-associated cytokine, the origin and roles of these subsets remain elusive. In this paper, we show that not only IL-17(+)/IFN-gamma(+) but also IFN-gamma(+) (IL-17(-)) cells arise under Th17-inducing condition and have distinct properties from the Th1 lineage. In fact, these populations displayed characteristics reminiscent to IL-17 single-producing cells, including production of IL-22, CCL20, and induction of antimicrobial gene expression from epithelial cells. Live sorted IL-17(+) and Th17-IFN-gamma(+) cells retained expression of IL-17 or IFN-gamma after culture, respectively, whereas the IL-17(+)/IFN-gamma(+) population was less stable and could also become IL-17 or IFN-gamma single-producing cells. Interestingly, these Th17 subsets became "Th1-like" cells in the presence of IL-12. These results provide novel insights into the relationship and functionality of the Th17 and Th1 subsets and have direct implications for the analysis and relevance of IL-17 and/or IFN-gamma-producing T cells present in patients' peripheral blood and inflamed tissues.

BACKGROUND

Bacterial vaginosis increases the susceptibility to sexually transmitted infections and negatively affects women's reproductive health.

METHODS

To investigate host-vaginal microbiota interactions and the impact on immune barrier function, we colonized 3-dimensional (3-D) human vaginal epithelial cells with 2 predominant species of vaginal microbiota (Lactobacillus iners and Lactobacillus crispatus) or 2 prevalent bacteria associated with bacterial vaginosis (Atopobium vaginae and Prevotella bivia).

RESULTS

Colonization of 3-D vaginal epithelial cell aggregates with vaginal microbiota was observed with direct attachment to host cell surface with no cytotoxicity. A. vaginae infection yielded increased expression membrane-associated mucins and evoked a robust proinflammatory, immune response in 3-D vaginal epithelial cells (ie, expression of CCL20, hBD-2, interleukin 1β, interleukin 6, interleukin 8, and tumor necrosis factor α) that can negatively affect barrier function. However, P. bivia and L. crispatus did not significantly upregulate pattern-recognition receptor-signaling, mucin expression, antimicrobial peptides/defensins, or proinflammatory cytokines in 3-D vaginal epithelial cell aggregates. Notably, L. iners induced pattern-recognition receptor-signaling activity, but no change was observed in mucin expression or secretion of interleukin 6 and interleukin 8.

CONCLUSIONS

We identified unique species-specific immune signatures from vaginal epithelial cells elicited by colonization with commensal and bacterial vaginosis-associated bacteria. A. vaginae elicited a signature that is consistent with significant disruption of immune barrier properties, potentially resulting in enhanced susceptibility to sexually transmitted infections during bacterial vaginosis.

Psoriasis is an immune-mediated skin disease characterized by lymphocytic infiltration and altered keratinocyte differentiation. Using immunohistochemical techniques we found that the cellular infiltrate in acute psoriatic plaques includes 5-8% CD3(-)CD56(+) natural killer (NK) cells, mostly localized in the mid and papillary dermis. NK lymphocytes isolated from punch biopsy specimens of psoriatic plaques showed a CD56(bright)CD16(-)CD158b(-) phenotype, failed to express the skin homing cutaneous lymphocyte-associated antigen and released abundant IFN-gamma upon stimulation. Supernatants from psoriatic NK cells induced MHC class II and ICAM-1 expression and release of CXCL10 and CCL5 by cultured psoriatic keratinocytes. Skin NK cells expressed high levels of the chemokines receptors CXCR3 and CCR5, intermediate amounts of CXCR1, CCR6 and CCR8, and low levels of CCR1, CCR2, CCR4, CCR7 and CX3CR1. In addition, they promptly migrated in vitro toward CXCL10, CCL5, supernatants of IFN-gamma-activated psoriatic keratinocytes and, to a lower extent, CCL20 and CCL4. In contrast, they failed to migrate toward CXCL8, CCL1, CCL2, CCL3, CCL17, CCL19 and CX3CL1. Taken together, our results implicate NK lymphocytes as newly identified protagonists in the pathogenesis of psoriasis. Their distinctive homing properties should be taken into account in the design of specific therapy aimed at blocking pathogenic cell accumulation in the skin.