Human mesenchymal stem cells and derived extracellular vesicles induce regulatory dendritic cells in type 1 diabetic patients.

AIMS/HYPOTHESIS: Mesenchymal stem cells (MSCs) can exert an immunosuppressive effect on any component of the immune system, including dendritic cells (DCs), by direct contact, the release of soluble markers and extracellular vesicles (EVs). We evaluated whether MSCs and MSC-derived EVs have an immunomodulatory effect on monocyte-derived DCs in type 1 diabetes. METHODS: Bone marrow derived MSCs were characterised and EVs were obtained by ultracentrifugation. DCs were differentiated from CD14(+) cells, obtained from nine type 1 diabetic patients at disease onset, pulsed with antigen GAD65 and cultured with MSCs or EVs. Levels of DC maturation and activation markers were evaluated by flow cytometry. GAD65-pulsed DCs and autologous CD14(-) cell were co-cultured and IFN-γ enzyme-linked immunosorbent spot responses were assayed. Secreted cytokine levels were measured and Th17 and regulatory T cells were analysed. RESULTS: MSC- and EV-conditioned DCs acquired an immature phenotype with reduced levels of activation markers and increased IL-10 and IL-6 production. Conditioned DC plus T cell co-cultures showed significantly decreased IFN-γ spots and secretion levels. Moreover, higher levels of TGF-ß, IL-10 and IL-6 were detected compared with unconditioned DC plus T cell co-cultures. Conditioned DCs decreased Th17 cell numbers and IL-17 levels, and increased FOXP3(+) regulatory T cell numbers. EVs were internalised by DCs and EV-conditioned DCs exhibited a similar effect. CONCLUSIONS/INTERPRETATION: In type 1 diabetes, MSCs induce immature IL-10-secreting DCs in vitro, thus potentially intercepting the priming and amplification of autoreactive T cells in tissue inflammation. These DCs can contribute to the inhibition of inflammatory T cell responses to islet antigens and the promotion of the anti-inflammatory, regulatory responses exerted by MSCs.

B7h triggering inhibits the migration of tumor cell lines.

Vascular endothelial cells (ECs) and several cancer cells express B7h, which is the ligand of the ICOS T cell costimulatory molecule. We have previously shown that B7h triggering via a soluble form of ICOS (ICOS-Fc) inhibits the adhesion of polymorphonuclear and tumor cell lines to HUVECs; thus, we suggested that ICOS-Fc may act as an anti-inflammatory and antitumor agent. Because cancer cell migration and angiogenesis are crucial for metastasis dissemination, the aim of this work was to evaluate the effect of ICOS-Fc on the migration of cancer cells and ECs. ICOS-Fc specifically inhibited the migration of HUVECs, human dermal lymphatic ECs, and the HT29, HCT116, PC-3, HepG2, JR8, and M14 tumor cell lines expressing high levels of B7h, whereas it was ineffective in the RPMI7932, PCF-2, LM, and BHT-101 cell lines expressing low levels of B7h. Furthermore, ICOS-Fc downmodulated hepatocyte growth factor facilitated the epithelial-to-mesenchymal transition in HepG2 cells. Moreover, ICOS-Fc downmodulated the phosphorylation of focal adhesion kinase and the expression of ß-Pix in both HUVECs and tumor cell lines. Finally, treatment with ICOS-Fc inhibited the development of lung metastases upon injection of NOD-SCID-IL2Rγnull mice with CF-PAC1 cells, as well as C57BL/6 mice with B16-F10 cells. Therefore, the B7h-ICOS interaction may modulate the spread of cancer metastases, which suggests the novel use of ICOS-Fc as an immunomodulatory drug. However, in the B16-F10-metastasized lungs, ICOS-Fc also increased IL-17A/RORc and decreased IL-10/Foxp3 expression, which indicates that it also exerts positive effects on the antitumor immune response.

Triggering of B7h by the ICOS modulates maturation and migration of monocyte-derived dendritic cells.

B7h, expressed by several cell types, binds ICOS expressed by activated T cells. We have previously shown that B7h triggering by ICOS-Fc inhibits human endothelial cell adhesiveness. This work investigated the effect of ICOS-Fc on human monocyte-derived dendritic cells (DCs). We found that DCs matured with LPS in the presence of ICOS-Fc (mDCs(ICOS)) produced greater amounts of IL-23 and IL-10, and promoted a higher secretion of IL-17A and IL-17F in MLCs than did those DCs matured with LPS alone (mDCs). Moreover, mDCs(ICOS) pulsed with the keyhole limpet hemocyanin Ag during the maturation phase were better stimulators of Ag-specific MHC class I-, but not class II-restricted T cells than mDCs. This was probably due to promotion of cross-presentation because it was not detected when the Flu-MA(58-66) Ag was directly loaded on already matured DCs and mDCs(ICOS). Finally, ICOS-Fc inhibited the adhesion of both immature DCs and mDCs to vascular and lymphoid endothelial cells, their migratory activity, and the expression of the Rac-1 activator ß-Pix involved in cell motility. These data suggest that B7h stimulation modulates DC function with effects on their maturation and recruitment into tissues. This opens a novel view on the use of interactors of the ICOS:B7h system as immunomodulatory drugs.

Human mesenchymal stem cell-derived microvesicles modulate T cell response to islet antigen glutamic acid decarboxylase in patients with type 1 diabetes.

AIMS/HYPOTHESIS: Mesenchymal stem cells (MSCs) have been shown to abrogate in vitro the proinflammatory response in type 1 diabetes. The mechanism involves paracrine factors, which may include microvesicles (MVs). We evaluated whether MVs derived from heterologous bone-marrow MSCs exert an immunomodulatory effect on T cell responses against GAD (glutamic acid decarboxylase) antigen in type 1 diabetes. METHODS: MVs were purified from heterologous human MSCs by differential centrifugation. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with type 1 diabetes at disease onset, and responses to GAD65 stimulation were assessed by IFN-γ enzyme-linked immunosorbent spot analysis. Levels of cytokines and prostaglandin E2 (PGE2) were measured in the supernatant fraction, and T helper 17 (Th17) and regulatory T cell analysis was performed. RESULTS: MVs were internalised by PBMCs, as assessed by confocal microscopy and flow cytometry analyses. MVs significantly decreased IFN-γ spots and levels in GAD65-stimulated PBMCs, and significantly increased transforming growth factor-ß (TGF-ß), IL-10, IL-6 and PGE2 levels. Furthermore, MVs decreased the number of Th17 cells and the levels of IL-17, and increased FoxP3(+) regulatory T cells in GAD65-stimulated PBMCs. CONCLUSIONS/INTERPRETATION: These results provide evidence that MSC-derived MVs can inhibit in vitro a proinflammatory response to an islet antigenic stimulus in type 1 diabetes. The action of MVs involves PGE2 and TGF-ß signalling pathways and IL-10 secretion, suggesting a switch to an anti-inflammatory response of T cells.

Vaccination with ENO1 DNA prolongs survival of genetically engineered mice with pancreatic cancer.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) is an aggressive tumor, and patients typically present with late-stage disease; rates of 5-year survival after pancreaticoduodenectomy are low. Antibodies against α-enolase (ENO1), a glycolytic enzyme, are detected in more than 60% of patients with PDA, and ENO1-specific T cells inhibit the growth of human pancreatic xenograft tumors in mice. We investigated whether an ENO1 DNA vaccine elicits antitumor immune responses and prolongs survival of mice that spontaneously develop autochthonous, lethal pancreatic carcinomas. METHODS: We injected and electroporated a plasmid encoding ENO1 (or a control plasmid) into Kras(G12D)/Cre (KC) mice and Kras(G12D)/Trp53(R172H)/Cre (KPC) mice at 4 weeks of age (when pancreatic intraepithelial lesions are histologically evident). Antitumor humoral and cellular responses were analyzed by histology, immunohistochemistry, enzyme-linked immunosorbent assays, flow cytometry, and enzyme-linked immunosorbent spot and cytotoxicity assays. Survival was analyzed by Kaplan-Meier analysis. RESULTS: The ENO1 vaccine induced antibody and a cellular response and increased survival times by a median of 138 days in KC mice and 42 days in KPC mice compared with mice given the control vector. On histologic analysis, the vaccine appeared to slow tumor progression. The vaccinated mice had increased serum levels of anti-ENO1 immunoglobulin G, which bound the surface of carcinoma cells and induced complement-dependent cytotoxicity. ENO1 vaccination reduced numbers of myeloid-derived suppressor cells and T-regulatory cells and increased T-helper 1 and 17 responses. CONCLUSIONS: In a genetic model of pancreatic carcinoma, vaccination with ENO1 DNA elicits humoral and cellular immune responses against tumors, delays tumor progression, and significantly extends survival. This vaccination strategy might be developed as a neoadjuvant therapy for patients with PDA.

Chronic hypoxia reprograms human immature dendritic cells by inducing a proinflammatory phenotype and TREM-1 expression.

DCs are powerful antigen-presenting cells central in the orchestration of innate and acquired immunity. DC development, migration, and activities are intrinsically linked to the microenvironment. DCs migrate through pathologic tissues before reaching their final destination in the lymph nodes. Hypoxia, a condition of low partial oxygen pressure, is a common feature of many pathologic situations, capable of modifying DC phenotype and functional behavior. We studied human monocyte-derived immature DCs generated under chronic hypoxic conditions (H-iDCs). We demonstrate by gene expression profiling the upregulation of a cluster of genes coding for antigen-presentation, immunoregulatory, and pattern recognition receptors, suggesting a stimulatory role for hypoxia on iDC immunoregulatory functions. In particular, we show that H-iDCs express triggering receptor expressed on myeloid cells(TREM-1), a member of the Ig superfamily of immunoreceptors and an amplifier of inflammation. This effect is reversible because H-iDC reoxygenation results in TREM-1 down-modulation. TREM-1 engagement promotes upregulation of T-cell costimulatory molecules and homing chemokine receptors, typical of mature DCs, and increases the production of proinflammatory, Th1/Th17-priming cytokines/chemokines, resulting in increased T-cell responses. These results suggest that TREM-1 induction by the hypoxic microenvironment represents a mechanism of regulation of Th1-cell trafficking and activation by iDCs differentiated at pathologic sites.

Hypoxia modulates the gene expression profile of immunoregulatory receptors in human mature dendritic cells: identification of TREM-1 as a novel hypoxic marker in vitro and in vivo.

Dendritic cells (DCs) are a heterogeneous group of professional antigen-presenting cells functioning as sentinels of the immune system and playing a key role in the initiation and amplification of innate and adaptive immune responses. DC development and functions are acquired during a complex differentiation and maturation process influenced by several factors present in the local milieu. A common feature at pathologic sites is represented by hypoxia, a condition of low pO(2), which creates a unique microenvironment affecting cell phenotype and behavior. Little is known about the impact of hypoxia on the generation of mature DCs (mDCs). In this study, we identified by gene expression profiling a significant cluster of genes coding for immune-related cell surface receptors strongly up-regulated by hypoxia in monocyte-derived mDCs and characterized one of such receptors, TREM-1, as a new hypoxia-inducible gene in mDCs. TREM-1 associated with DAP12 in hypoxic mDCs, and its engagement elicited DAP12-linked signaling, resulting in ERK-1, Akt, and IκBα phosphorylation and proinflammatory cytokine and chemokine secretion. Finally, we provided the first evidence that TREM-1 is expressed on mDCs infiltrating the inflamed hypoxic joints of children affected by juvenile idiopathic arthritis, representing a new in vivo marker of hypoxic mDCs endowed with proinflammatory properties.

Exploring chitosan-shelled nanobubbles to improve HER2 + immunotherapy via dendritic cell targeting.

Immunotherapy is a valuable approach to cancer treatment as it is able to activate the immune system. However, the curative methods currently in clinical practice, including immune checkpoint inhibitors, present some limitations. Dendritic cell vaccination has been investigated as an immunotherapeutic strategy, and nanotechnology-based delivery systems have emerged as powerful tools for improving immunotherapy and vaccine development. A number of nanodelivery systems have therefore been proposed to promote cancer immunotherapy. This work aims to design a novel immunotherapy nanoplatform for the treatment of HER2 + breast cancer, and specially tailored chitosan-shelled nanobubbles (NBs) have been developed for the delivery of a DNA vaccine. The NBs have been functionalized with anti-CD1a antibodies to target dendritic cells (DCs). The NB formulations possess dimensions of approximately 300 nm and positive surface charge, and also show good physical stability up to 6 months under storage at 4 °C. In vitro characterization has confirmed that these NBs are capable of loading DNA with good encapsulation efficiency (82%). The antiCD1a-functionalized NBs are designed to target DCs, and demonstrated the ability to induce DC activation in both human and mouse cell models, and also elicited a specific immune response that was capable of slowing tumor growth in mice in vivo. These findings are the proof of concept that loading a tumor vaccine into DC-targeted chitosan nanobubbles may become an attractive nanotechnology approach for the future immunotherapeutic treatment of cancer.

Urinary Zinc Loss Identifies Prostate Cancer Patients.

Prostate Cancer (PCa) is one of the most common malignancies in men worldwide, with 1.4 million diagnoses and 310,000 deaths in 2020. Currently, there is an intense debate regarding the serum prostatic specific antigen (PSA) test as a diagnostic tool in PCa due to the lack of specificity and high prevalence of over-diagnosis and over-treatments. One of the most consistent characteristics of PCa is the marked decrease in zinc; hence the lost ability to accumulate and secrete zinc represents a potential parameter for early detection of the disease. We quantified zinc levels in urine samples collected after a standardized prostatic massage from 633 male subjects that received an indication for prostate biopsy from 2015 and 2019 at AOU Città della Salute e della Scienza di Torino Hospital. We observed that the mean zinc levels were lower in the urine of cancer patients than in healthy subjects, with a decreasing trend in correlation with the progression of the disease. The combination of zinc with standard parameters, such as PSA, age, digital rectal exploration results, and magnetic resonance findings, displayed high diagnostic performance. These results suggest that urinary zinc may represent an early and non-invasive diagnostic biomarker for prostate cancer.

Circulating autoantibodies to phosphorylated α-enolase are a hallmark of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and no diagnostic markers have, as of yet, been defined. In PDAC patients, α-enolase (ENOA) is up-regulated and elicits the production of autoantibodies. Here, we analyzed the autoantibody response to post-translational modifications of ENOA in PDAC patients. ENOA isolated from PDAC tissues and cell lines was characterized by two-dimensional electrophoresis (2-DE) Western blot (WB), revealing the expression of six different isoforms (named ENOA1,2,3,4,5,6) whereas only 4 isoforms (ENOA3,4,5,6) were detectable in normal tissues. As assessed by 2-DE WB, 62% of PDAC patients produced autoantibodies to the two more acidic isoforms (ENOA1,2) as opposed to only 4% of controls. Mass spectrometry showed that ENOA1,2 isoforms were phosphorylated on serine 419. ROC analysis demonstrated that autoantibodies to ENOA1,2 usefully complement the diagnostic performance of serum CA19.9 levels, achieving approximately 95% diagnostic accuracy in both advanced and resectable PDAC. Moreover, the presence of autoantibodies against ENOA1,2 correlated with a significantly better clinical outcome in advanced patients treated with standard chemotherapy. In conclusion, our results demonstrate that ENOA phosphorylation is associated with PDAC and induces specific autoantibody production in PDAC patients that may have diagnostic value.

The interferon-inducible gene IFI16 secretome of endothelial cells drives the early steps of the inflammatory response.

The IFN-inducible human IFI16 gene is highly expressed in endothelial cells as well as epithelial and hematopoietic tissues. Previous gene array analysis of human umbilical vein endothelial cells overexpressing IFI16 has revealed an increased expression of genes involved in inflammation and apoptosis. In this study, protein array analysis of the IFI16 secretome showed an increased production of chemokines, cytokines and adhesion molecules responsible for leukocyte chemotaxis. Functional analysis of the promoter for CCL20, the chemokine responsible for leukocyte recruitment in the early steps of inflammation, by site-specific mutation demonstrated that NF-κB is the main mediator of CCL20 induction at the transcriptional level. Finally, both Langerhans DC and B-lymphocyte migration triggered by supernatants from IFI16-overexpressing endothelial cells was partially inhibited by Ab inactivating CCL4, CCL5 and CCL20 chemokines. Altogether, these results demonstrate that the IFI16 gene, through its secretome, regulates proinflammatory activity of endothelial cells, thus corroborating its role in the early steps of inflammation.

Survival and migration of human dendritic cells are regulated by an IFN-alpha-inducible Axl/Gas6 pathway.

Axl, a prototypic member of the transmembrane tyrosine kinase receptor family, is known to regulate innate immunity. In this study, we show that Axl expression is induced by IFN-alpha during human dendritic cell (DC) differentiation from monocytes (IFN/DC) and that constitutively Axl-negative, IL-4-differentiated DC (IL-4/DC) can be induced to up-regulate Axl by IFN-alpha. This effect is inhibited by TLR-dependent maturation stimuli such as LPS, poly(I:C), TLR7/8 ligand, and CD40L. LPS-induced Axl down-regulation on the surface of human IFN-alpha-treated DC correlates with an increased proteolytic cleavage of Axl and with elevated levels of its soluble form. GM6001 and TAPI-1, general inhibitors of MMP and ADAM family proteases, restored Axl expression on the DC surface and diminished Axl shedding. Furthermore, stimulation of Axl by its ligand, Gas6, induced chemotaxis of human DC and rescued them from growth factor deprivation-induced apoptosis. Our study provides the first evidence that Gas6/Axl-mediated signaling regulates human DC activities, and identifies Gas6/Axl as a new DC chemotaxis pathway. This encourages one to explore whether dysregulation of this novel pathway in human DC biology is involved in autoimmunity characterized by high levels of IFN-alpha.

Low Levels of Urinary PSA Better Identify Prostate Cancer Patients.

Serum prostatic specific antigen (PSA) has proven to have limited accuracy in early diagnosis and in making clinical decisions about different therapies for prostate cancer (PCa). This is partially due to the fact that an increase in PSA in the blood is due to the compromised architecture of the prostate, which is only observed in advanced cancer. On the contrary, PSA observed in the urine (uPSA) reflects the quantity produced by the prostate, and therefore can give more information about the presence of disease. We enrolled 574 men scheduled for prostate biopsy at the urology clinic, and levels of uPSA were evaluated. uPSA levels resulted lower among subjects with PCa when compared to patients with negative biopsies. An indirect correlation was observed between uPSA amount and the stage of disease. Loss of expression of PSA appears as a characteristic of prostate cancer development and its evaluation in urine represents an interesting approach for the early detection of the disease and the stratification of patients.

Macrophages expressing TREM-1 are involved in the progression of HPV16-related oropharyngeal squamous cell carcinoma.

INTRODUCTION: Many types of research have been performed to improve the diagnosis, therapy, and prognosis of oropharyngeal carcinomas (OP-SCCs). Since they arise in lymphoid-rich areas and intense lymphocytic infiltration has been related to a better prognosis, a TREM-1 putative function in tumour progression and survival has been hypothesized. MATERIALS AND METHODS: Twenty-seven human papillomavirus (HPV) 16+ OP-SCC specimens have been analyzed to relate TREM-1 expression with histiocytic and lymphocytic markers, HPV presence and patients' outcome. RESULTS: No differences have been shown between intratumoral and stromal CD4+ cells, while intratumoral CD8+ lymphocytes are higher with respect to the tumour stroma (p = .0005). CD68+ cells are more than CD35+ and TREM-1+; their presence is related to CD35± and TREM-1± histiocytes (p = .005 and .026, respectively). Intratumoral CD4+ lymphocytes are higher in p16+ cases (11/27) than in p16- (p = .042); moreover, p16 positivity correlates to a better survival (p = .034). CD4+, CD8+ and CD35+ cells have no impact on survival, while CD68 expression heavily influences progression and bad outcome (p = .037). TREM-1 positivity also leads to worst overall survival (p = .001): peritumoral expression and death-cause relationship are always significant, particularly when the cause is OP-SCC (p = .000). CONCLUSION: While p16 shows to better stratify HPV16+ patients' outcome, TREM-1+ macrophages suggest their key importance in HPV-related OP-SCCs progression.KEY MESSAGESTREM-1 positivity correlates to the worst overall survival of HPV16-positive OPSCCs-affected patients.p16-positive HPV16 related OPSCCs patients have a better prognosis with respect to p16-negative ones.

The proapoptotic activity of the Interferon-inducible gene IFI16 provides new insights into its etiopathogenetic role in autoimmunity.

Several lines of evidence link Interferons (IFNs) with autoimmune disorders. Autoantibodies against the Interferon-inducible IFI16 protein, a member of the HIN-200 family constitutively expressed in endothelial cells and keratinocytes, have been identified in patients affected by autoimmune diseases including Systemic Lupus Erythematosus (SLE), Sjogren Syndrome (SjS), and Scleroderma (SSc). These findings point to a role for IFI16 in the etiopathogenesis of autoimmune diseases, but the exact mechanisms involved in the development of autoimmunity remain obscure. In this study, we report for the first time that endothelial cells overexpressing IFI16 undergo apoptosis via the activation of caspase 2 and caspase 3, and that a positive feedback loop appears to link these two caspases. The relevance of IFI16-mediated apoptosis is highlighted by the observation that IFI16 knock down by RNA interference in endothelial cells inhibits the activation of both caspase 2 and caspase 3 by IFN-beta priming and synthetic double-stranded RNA treatment. Expression of a dominant-negative mutant of IKK2 kinase or treatment with AS602868, an inhibitor of IKK2 activity, results in a strong reduction of NF-kB activation along with absence of caspase 2 and caspase 3 activation and apoptosis induction. Collectively, our findings provide new insights into the role of IFI16 in the pathogenesis of autoimmune diseases by demonstrating that in addition to the stimulation of pro-inflammatory molecules, IFI16 also leads to apoptosis in endothelial cells.

Lack of plasma protein hemopexin dampens mercury-induced autoimmune response in mice.

Several factors affect the autoimmune response, including iron-dependent modulation of T cells. Hemopexin is the plasma protein with the highest binding affinity to heme. It mediates heme-iron recovery in the liver, thus controlling heme-iron availability in peripheral cells. The aim of the present study was to investigate the role of hemopexin in the progress of an autoimmune response. To this end, we chose a mouse model of mercury-induced autoimmunity and evaluated the susceptibility of hemopexin-null mice to mercury treatment compared with wild-type controls. In this study we show that lack of hemopexin dampens mercury-induced autoimmune responses in mice. Hemopexin-null mice produced fewer antinuclear autoantibodies and had reduced deposits of immune complexes in the kidney after mercuric chloride treatment compared with wild-type mice. These features were associated with a reduction in activated T cells and lower absolute B cell number in spleen and impaired IgG1 and IgG2a production. In contrast, in hemopexin-null mice the response to OVA/CFA immunization was maintained. In addition, hemopexin-null mice had reduced transferrin receptor 1 expression in T cells, possibly due to the increase in heme-derived iron. Interestingly, CD4(+)T cells isolated from mercury-treated hemopexin-null mice show reduced IFN-gamma-dependent STAT1 phosphorylation compared with that of wild-type mice. Our data suggest that hemopexin, by controlling heme-iron availability in lymphocytes, modulates responsiveness to IFN-gamma and, hence, autoimmune responses.

An integrated humoral and cellular response is elicited in pancreatic cancer by alpha-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with a very poor 5-year survival rate. alpha-Enolase is a glycolytic enzyme that also acts as a surface plasminogen receptor. We find that it is overexpressed in PDAC and present on the cell surface of PDAC cell lines. The clinical correlation of its expression with tumor status has been reported for lung and hepatocellular carcinoma. We have previously demonstrated that sera from PDAC patients contain IgG autoantibodies to alpha-enolase. The present work was intended to assess the ability of alpha-enolase to induce antigen-specific T cell responses. We show that alpha-enolase-pulsed dendritic cells (DC) specifically stimulate healthy autologous T cells to proliferate, secrete IFN-gamma and lyse PDAC cells but not normal cells. In vivo, alpha-enolase-specific T cells inhibited the growth of PDAC cells in immunodeficient mice. In 8 out of 12 PDAC patients with circulating IgG to alpha-enolase, the existence of alpha-enolase-specific T cells was also demonstrated. Taken as a whole, these results indicate that alpha-enolase elicits a PDAC-specific, integrated humoral and cellular response. It is thus a promising and clinically relevant molecular target candidate for immunotherapeutic approaches as new adjuvants to conventional treatments in pancreatic cancer.

Transcriptome of hypoxic immature dendritic cells: modulation of chemokine/receptor expression.

Hypoxia is a condition of low oxygen tension occurring in inflammatory tissues. Dendritic cells (DC) are professional antigen-presenting cells whose differentiation, migration, and activities are intrinsically linked to the microenvironment. DCs will home and migrate through pathologic tissues before reaching their final destination in the lymph node. We studied the differentiation of human monocytes into immature DCs (iDCs) in a hypoxic microenvironment. We generated iDC in vitro under normoxic (iDCs) or hypoxic (Hi-DCs) conditions and examined the hypoxia-responsive element in the promoter, gene expression, and biochemical KEGG pathways. Hi-DCs had an interesting phenotype represented by up-regulation of genes associated with cell movement/migration. In addition, the Hi-DC cytokine/receptor pathway showed a dichotomy between down-regulated chemokines and up-regulated chemokine receptor mRNA expression. We showed that CCR3, CX3CR1, and CCR2 are hypoxia-inducible genes and that CCL18, CCL23, CCL26, CCL24, and CCL14 are inhibited by hypoxia. A strong chemotactic response to CCR2 and CXCR4 agonists distinguished Hi-DCs from iDCs at a functional level. The hypoxic microenvironment promotes the differentiation of Hi-DCs, which differs from iDCs for gene expression profile and function. The most prominent characteristic of Hi-DCs is the expression of a mobility/migratory rather than inflammatory phenotype. We speculate that Hi-DCs have the tendency to leave the hypoxic tissue and follow the chemokine gradient toward normoxic areas where they can mature and contribute to the inflammatory process.

Lactoferrin, a major defense protein of innate immunity, is a novel maturation factor for human dendritic cells.

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.

Human dendritic cells differentiated in hypoxia down-modulate antigen uptake and change their chemokine expression profile.

Dendritic cells (DCs) are the most potent antigen-presenting cells and fine-tune the immune response. We have investigated hypoxia's effects on the differentiation and maturation of DCs from human monocytes in vitro, and have shown that it affects DC functions. Hypoxic immature DCs (H-iDCs) significantly fail to capture antigens through down-modulation of the RhoA/Ezrin-Radixin-Moesin pathway and the expression of CD206. Moreover, H-iDCs released higher levels of CXCL1, VEGF, CCL20, CXCL8, and CXCL10 but decreased levels of CCL2 and CCL18, which predict a different ability to recruit neutrophils rather than monocytes and create a proinflammatory and proangiogenic environment. By contrast, hypoxia has no effect on DC maturation. Hypoxic mature DCs display a mature phenotype and activate both allogeneic and specific T cells like normoxic mDCs. This study provides the first demonstration that hypoxia inhibits antigen uptake by DCs and profoundly changes the DC chemokine expression profile and may have a critical role in DC differentiation, adaptation, and activation in inflamed tissues.