Mesenchymal stromal cells (MSCs) tend to infiltrate into tumors and form a major component of the tumor microenvironment. Our previous work demonstrated that tumor necrosis factor α (TNFα)-activated MSCs significantly promoted tumor growth. However, the role of TNFα-treated MSCs in tumor metastasis remains elusive. Employing a lung metastasis model of murine breast cancer, we found that TNFα-activated MSCs strikingly enhanced tumor metastasis compared with normal MSCs. We analyzed the chemokine profiles and found that the expression of CCL5, CCR2 and CXCR2 ligands were enhanced in TNFα-activated MSCs. Using genetic or pharmacological strategies to inhibit CCL5 or CCR2, we demonstrated that CCL5 and CCR2 ligands were indispensable in supporting TNFα-activated MSCs to promote tumor metastasis. Analysis of immune cells revealed that CXCR2 ligands (CXCL1, CXCL 2 and CXCL5) expressed by TNFα-activated MSCs efficiently recruited CXCR2+ neutrophils into tumor. These neutrophils were responsible for the pro-metastatic effect of MSCs since inhibition of this chemotaxis abolished increased neutrophil recruitment and tumor metastasis. The interaction between neutrophils and tumor cells resulted in markedly elevated metastasis-related genes by tumor cells, including CXCR4, CXCR7, MMP12, MMP13, IL-6 and TGFβ. Importantly, in IL8high human breast cancer samples, we also observed similar alterations of gene expression. Collectively, our findings demonstrate that TNFα-activated MSCs promote tumor metastasis via CXCR2+ neutrophil recruitment.

Chemokines produced in the liver during hepatitis C virus (HCV) infection induce migration of activated T cells from the periphery to infected parenchyma. The milieu of chemokines secreted by infected hepatocytes is predominantly associated with the T-helper cell/Tc1 T cell (Th1/Tc1) response. These chemokines consist of CCL3 (macrophage inflammatory protein-1 alpha; MIP-1 alpha), CCL4 (MIP-1 beta), CCL5 (regulated on activation normal T cell expressed and secreted; RANTES), CXCL10 (interferon-gamma-inducible protein-10; IP-10), CXCL11 (interferon-inducible T-cell alpha chemoattractant; I-TAC), and CXCL9 (monokine induced by interferon gamma; Mig) and they recruit T cells expressing either CCR5 or CXCR3 chemokine receptors. Intrahepatic and peripheral blood levels of these chemokines are increased during chronic hepatitis C. The interaction between chemokines and their receptors is essential in recruiting HCV-specific T cells to control the infection. When the adaptive immune response fails in this task, non-specific T cells without the capacity to control the infection are also recruited to the liver, and these are ultimately responsible for the persistent hepatic damage. The modulation of chemokine receptor expression and chemokine secretion could be a viral escape mechanism to avoid specific T cell migration to the liver during the early phase of infection, and to maintain liver viability during the chronic phase, by impairing non-specific T cell migration. Some chemokines and their receptors correlate with liver damage, and CXCL10 (IP-10) and CXCR3 levels have shown a clinical utility as predictors of treatment response outcome. The regulation of chemokines and their receptors could be a future potential therapeutic target to decrease liver inflammation and to increase specific T cell migration to the infected liver.

In the present study, we investigated whether human adipose tissue derived stem cells (hASCs) could enhance tumor invasion and whether these hASCs could be a potential source of CCL5. We observed a significant increase in the number of breast cancer cells that invaded the matrigel when Co-cultured with hASCs. We found that hASCs produce CCL5 in the Co-culture and cancer cell invasion was diminished by an antibody against CCL5. Furthermore, cancer cell invasion in the Co-culture was associated with an elevated level of MMP-9 activity. We conclude that CCL5 plays a crucial role for tumor invasion in the interplay of tissue resident stem cells from the fat tissue and breast cancer cells.

Complement component C3a causes a robust degranulation in human mast cells. Whether C3a also stimulates chemokine production in human mast cells and what signaling pathway it activates is not known. In the present study, we demonstrate that CD34+ cell-derived primary mast cells and a human mast cell line LAD 2 express surface C3a receptors at similar levels. Furthermore, C3a caused approximately 50% internalization of cell surface C3a receptors in both cell types. We therefore used LAD 2 cells as a model to study C3a-induced biological responses and signaling in human mast cells. We found that C3a stimulated substantial degranulation and induced chemokine monocyte chemoattractant protein 1 (MCP-1/CCL2) and regulated upon activation, normal T-cell expressed and secreted (RANTES/CCL5) production in LAD 2 mast cells. C3a caused a rapid and sustained extracellular-signal-regulated kinase (ERK) phosphorylation and Akt phosphorylation in LAD 2 mast cells. Furthermore, U0126 and LY294002, which respectively inhibit MEK-induced ERK phosphorylation and PI3 kinase-mediated Akt phosphorylation had distinct effects on C3a-induced responses. Thus, U0126, which blocked C3a-induced RANTES/CCL5 production by 50.6+/-2.3%, inhibited MCP-1/CCL2 generation by 85.2+/-0.6%. In contrast, LY294002 had no effect on C3a-induced RANTES/CCL5 production but blocked MCP-1/CCL2 generation by 83.7+/-1.5%. These data demonstrate that C3a activates divergent signaling pathways to induce chemokine production in human mast cells.

Infection and systemic inflammation are risk factors for cerebrovascular diseases and poststroke infections impair outcome in stroke patients, although the mechanisms of their contribution are mostly unknown. No preclinical studies have identified how chronic infection affects ischemic brain damage and which key inflammatory mediators are involved. We used a well established model of gut infection (Trichuris muris) to study how chronic infection contributes to brain injury. We show that, in mice, infection that leads to a chronic Th1-polarized immune response dramatically (60%) exacerbates brain damage caused by experimental stroke. Chronic Th1-type infection resulted in systemic upregulation of proinflammatory mediators and profoundly altered stroke-induced early (40 min to 4 h) and late (48 h) inflammation in the brain and peripheral tissues. Using the same infection, we show that a Th1-, but not Th2-polarized response augments brain injury by increasing the Th1 chemokine CCL5 [regulated on activation, normal T-cell expressed and secreted (RANTES)] systemically. This infection-associated response paralleled altered regulatory T-cell response, accelerated platelet aggregation in brain capillaries, and increased microvascular injury and matrix metalloproteinase activation after stroke. Antibody neutralization of RANTES reversed the effect of chronic infection on brain damage, microvascular MMP-9 activation, and cellular inflammatory response. Our results suggest that chronic infection exacerbates ischemic brain damage via a RANTES-mediated systemic inflammatory response, which leads to delayed resolution of inflammation and augmented microvascular injury in the brain.

Astrocytes play a number of important physiological roles in CNS homeostasis. Inflammation stimulates astrocytes to secrete cytokines and chemokines that guide macrophages/microglia and T cells to sites of injury/inflammation. Herein, we describe how these processes are controlled by the suppressor of cytokine signaling (SOCS) proteins, a family of proteins that negatively regulate adaptive and innate immune responses. In this study, we describe that the immunomodulatory cytokine IFN-beta induces SOCS-1 and SOCS-3 expression in primary astrocytes at the transcriptional level. SOCS-1 and SOCS-3 transcriptional activity is induced by IFN-beta through IFN-gamma activation site (GAS) elements within their promoters. Studies in STAT-1alpha-deficient astrocytes indicate that STAT-1alpha is required for IFN-beta-induced SOCS-1 expression, while STAT-3 small interfering RNA studies demonstrate that IFN-beta-induced SOCS-3 expression relies on STAT-3 activation. Specific small interfering RNA inhibition of IFN-beta-inducible SOCS-1 and SOCS-3 in astrocytes enhances their proinflammatory responses to IFN-beta stimulation, such as heightened expression of the chemokines CCL2 (MCP-1), CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CCL5 (RANTES), and CXCL10 (IP-10), and promoting chemotaxis of macrophages and CD4(+) T cells. These results indicate that IFN-beta induces SOCS-1 and SOCS-3 in primary astrocytes to attenuate its own chemokine-related inflammation in the CNS.

OBJECTIVE

The recruitment of lymphocytes to tissues via endothelium has been studied extensively but less is known about the signals that direct migration and positioning within tissues. Liver myofibroblasts associate with lymphocytes in hepatitis and are positioned below the sinusoidal endothelium, through which lymphocytes are recruited to the liver. We investigated whether activated human liver myofibroblasts (aLMF) affect the migration and accumulation of lymphocytes within the inflamed liver.

METHODS

The ability of human aLMF and hepatic stellate cells to promote lymphocyte chemotaxis, adhesion, and migration was studied in vitro.

RESULTS

When cultured in vitro, aLMF from diseased human liver and hepatic stellate cells from noninflamed liver secrete a distinct profile of cytokines comprising interleukin (IL)-6, IL-12, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and the chemokines CCL2, CCL3, CCL5, CXCL8, CXCL9, and CXCL10. aLMF-conditioned media had chemotactic activity for lymphocytes, which partially was inhibited by pertussis toxin. IL-6, HGF, and VEGF all contributed to G-protein-coupled receptor-independent chemotaxis of lymphocytes. Lymphocytes adhered to aLMF via intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and a proportion of adherent cells migrated through the fibroblast monolayer, mediated by IL-6, HGF, and VEGF.

CONCLUSIONS

Human aLMF support G-protein coupled receptor-dependent and -independent lymphocyte adhesion and migration and thereby regulate the recruitment and positioning of lymphocytes in chronic hepatitis.

Ewing sarcoma is an aggressive round cell sarcoma with poor patient prognosis, particularly in cases of advanced-stage disease. Dynamic tumor-host immune interations within the tumor microenvironment may polarize in situ immune responses and shape tumor development and/or progression. To gain insight into the nature of tumour-host immune interactions within the Ewing sarcoma microenvironment, the presence and spatial distribution of infiltrating CD8(+) /CD4(+) T-lymphocytes were evaluated in therapy-naive Ewing sarcoma. Expression profiling of 40 different chemokines and several chemokine receptors was performed in therapy-naive tumours and cell lines by qPCR, immunohistochemistry, and flow cytometry. Considerable inter-tumour variation was observed regarding density, type, and distribution of infiltrating T-lymphocytes. Tumour-infiltrating T-cells contained significantly higher percentages of CD8(+) T-lymphocytes as compared to stroma-infiltrating cells, suggesting preferential migration of this T-cell type into tumour areas. Gene expression levels of several type 1-associated, pro-inflammatory chemokines (CXCR3- and CCR5-ligands CXCL9, CXCL10, and CCL5) correlated positively with infiltrating (CD8(+) ) T-lymphocyte numbers expressing corresponding chemokine receptors. Survival analyses demonstrated an impact of tumour-infiltrating, and not stroma-infiltrating, CD8(+) T-lymphocytes on tumour progression. At protein level, both tumour and stromal cells expressed the IFNγ-inducible chemokines CXCL9 and CXCL10. CCR5-ligand CCL5 was exclusively expressed by non-tumoural stromal/infiltrating cells. Together, our results indicate that an inflammatory immune microenvironment with high expression of type 1-associated chemokines may be critical for the recruitment of (CD8(+) ) T-lymphocytes expressing corresponding chemokine receptors. The observed impact of tumour-infiltrating (CD8(+) ) T-lymphocytes is consistent with a role for adaptive anti-tumour immunity in the prevention of Ewing sarcoma progression. Recognition of the merits and exploitation/induction of an inflammatory microenvironment may improve the efficacy of natural immune responses against, and (adoptive) immunotherapeutic approaches for, Ewing sarcoma.

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

Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-γ, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-α were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-α, IFN-γ, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-α and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-α, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus.

BACKGROUND

Chemokines play important roles in inflammation and in immune responses. This article will discuss the current literature on the C-C chemokine ligand 5 (CCL5), and whether it is a therapeutic target in the context of various allergic, autoimmune or infectious diseases.

METHODS

Small-molecule inhibitors, chemokine and chemokine receptor-deficient mice, antibodies and modified chemokines are the current tools available for CCL5 research, and there are several ongoing clinical trials targeting the CCL5 receptors, CCR1, CCR3 and CCR5. There are fewer studies specifically targeting the chemokine itself and clinical studies with anti-CCL5 antibodies are still to be carried out.

CONCLUSIONS

Although clinical trials are strongly biased toward HIV treatment and prevention with blockers of CCR5, the therapeutic potential for CCL5 and its receptors in other diseases is relevant. Overall, it is not likely that specific targeting of CCL5 will result in new adjunct strategies for the treatment of infectious diseases with a major inflammatory component. However, targeting CCL5 could result in novel therapies for chronic inflammatory diseases, where it may decrease inflammatory responses and fibrosis, and certain solid tumors, where it may have a role in angiogenesis.

In vitro generated OVA-specific IL-17-producing CD8 T effector cells (Tc17) from OT-1 mice, adoptively transferred into B16-OVA tumor-bearing mice, controlled tumor growth in early and late stage melanoma. IL-17, TNF, and IFN-gamma from the Tc17 effectors all played a role in an enhanced recruitment of T cells, neutrophils, and macrophages to the tumor. In addition, Tc17 cells and recently recruited, activated neutrophils produced further chemokines, including CCL3, CCL4, CCL5, CXCL9, and CXCL10, responsible for the attraction of type 1 lymphocytes (Th1 and Tc1) and additional neutrophils. Neutrophils were rapidly attracted to the tumor site by an IL-17 dependent mechanism, but at later stages the induction of the chemokine CXCL2 by Tc17-derived TNF and IFN-gamma contributed to sustain neutrophil recruitment. Approximately 10-50 times as many Tc17 effectors were required compared with Tc1 effectors to exert the same level of control over tumor growth. The recruitment of neutrophils was more prominent when Tc17 rather than Tc1 were used to control tumor and depletion of neutrophils resulted in a diminished capacity to control tumor growth.

Snail is primarily known as a transcriptional repressor that induces epithelial-mesenchymal transition by suppressing adherent proteins. Emerging evidence suggests that Snail can act as an activator; however, the mechanism and biological significance are unclear. Here, we found that CREB-binding protein (CBP) is the critical factor in Snail-mediated target gene transactivation. CBP interacts with Snail and acetylates Snail at lysine 146 and lysine 187, which prevents the repressor complex formation. We further identified several Snail-activated targets, including TNF-α, which is also the upstream signal for Snail acetylation, and CCL2 and CCL5, which promote the recruitment of tumor-associated macrophages. Here, we present our results on the mechanism by which Snail induces target gene transactivation to remodel the tumor microenvironment.

Although type I IFNs play critical roles in antiviral and antitumor activity, it remains to be elucidated how type I IFNs are produced in sterile conditions of the tumor microenvironment and directly affect tumor-infiltrating immune cells. Mouse de novo gliomas show increased expression of type I IFN messages, and in mice, CD11b(+) brain-infiltrating leukocytes (BIL) are the main source of type I IFNs that are induced partially in a STING (stimulator of IFN genes)-dependent manner. Consequently, glioma-bearing Sting(Gt) (/Gt) mice showed shorter survival and lower expression levels of Ifns compared with wild-type mice. Furthermore, BILs of Sting(Gt) (/Gt) mice showed increased CD11b(+) Gr-1(+) immature myeloid suppressor and CD25(+) Foxp3(+) regulatory T cells (Treg) and decreased IFNγ-producing CD8(+) T cells. CD4(+) and CD8(+) T cells that received direct type I IFN signals showed lesser degrees of regulatory activity and increased levels of antitumor activity, respectively. Finally, intratumoral administration of a STING agonist (cyclic diguanylate monophosphate; c-di-GMP) improved the survival of glioma-bearing mice associated with enhanced type I IFN signaling, Cxcl10 and Ccl5, and T-cell migration into the brain. In combination with subcutaneous OVA peptide vaccination, c-di-GMP increased OVA-specific cytotoxicity of BILs and prolonged their survival. These data demonstrate significant contributions of STING to antitumor immunity via enhancement of type I IFN signaling in the tumor microenvironment and suggest a potential use of STING agonists for the development of effective immunotherapy, such as the combination with antigen-specific vaccinations.

OBJECTIVE

To evaluate the efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating interleukin-1beta (IL-1beta)-induced production of the chemokines RANTES (CCL5), monocyte chemoattractant protein 1 (MCP-1/CCL2), epithelial neutrophil-activating peptide 78 (ENA-78/CXCL5), growth-regulated oncogene alpha (GROalpha/CXCL1), and matrix metalloproteinase 2 (MMP-2) activity in rheumatoid arthritis (RA) synovial fibroblasts.

METHODS

Fibroblasts obtained from RA synovium were grown, and conditioned medium was obtained. Cell viability was determined by MTT assay. RANTES, MCP-1, ENA-78, and GROalpha produced in culture supernatants were measured by enzyme-linked immunosorbent assay. MMP-2 activity was analyzed by gelatin zymography. Western blotting was used to study the phosphorylation of protein kinase C (PKC) isoforms and nuclear translocation of NF-kappaB.

RESULTS

EGCG was nontoxic to RA synovial fibroblasts. Treatment with EGCG at 10 microM or 20 microM significantly inhibited IL-1beta-induced ENA-78, RANTES, and GROalpha, but not MCP-1 production in a concentration-dependent manner. EGCG at 50 microM caused a complete block of IL-1beta-induced production of RANTES, ENA-78, and GROalpha, and reduced production of MCP-1 by 48% (P < 0.05). Zymography showed that EGCG blocked constitutive, IL-1beta-induced, and chemokine-mediated MMP-2 activity. Evaluation of signaling events revealed that EGCG preferentially blocked the phosphorylation of PKCdelta and inhibited the activation and nuclear translocation of NF-kappaB in IL-1beta-treated RA synovial fibroblasts.

CONCLUSIONS

These results suggest that EGCG may be of potential therapeutic value in inhibiting joint destruction in RA.

Chemokines and their receptors regulate the trafficking of leukocytes in hematopoiesis and inflammation, and thus are fundamental to the immune integrity of the host. In parallel, members of the chemokine system exert a large variety of functions that dictate processes of cancer development and progression. Chemokines can act as pro-tumoral or anti-tumoral regulators of malignancy by affecting cells of the tumor microenvironment (leukocytes, endothelial cells, fibroblasts) and the tumor cells themselves (migration, invasion, proliferation, resistance to chemotherapy). Several of the chemokines are generally skewed towards the cancer-promoting direction, including primarily the CCR5-CCL5 (RANTES) and the CXCR4-CXCL12 (SDF-1) axes. This review provides a general view of chemokines and chemokine receptors as regulators of malignancy, describing their multi-faceted activities in cancer. The tumor-promoting activities of the CCR5-CCL5 and CXCR4-CXCL12 pathways are enlightened, emphasizing their potential use as targets for personalized therapy. Indeed, novel blockers of chemokines and their receptors are constantly emerging, and two chemokine receptor inhibitors were recently approved for clinical use: Maraviroc for CCR5 and Plerixafor for CXCR4. The review addresses ongoing pre-clinical and clinical trials using these modalities and others in cancer. Then, challenges and opportunities of personalized therapy directed against chemokines and their receptors in malignancy are discussed, demonstrating that such novel personalized cancer therapies hold many challenges, but also offer hope for cancer patients.

In the present study, we investigated the involvement of macrophage-inflammatory protein-1alpha (MIP-1alpha)[CC chemokine ligand 3 (CCL3)], MIP-1beta[CCL4], regulated on activation, normal T expressed and secreted (RANTES)[CCL5], and CC chemokine receptors (CCRs) on neutrophil migration in murine immune inflammation. Previously, we showed that ovalbumin (OVA)-triggered neutrophil migration in immunized mice depends on the sequential release of tumor necrosis factor alpha (TNF-alpha) and leukotriene B(4)(LTB(4)). Herein, we show increased mRNA expression for MIP-1alpha[CCL3], MIP-1beta[CCL4], RANTES[CCL5], and CCR1 in peritoneal cells harvested from OVA-challenged, immunized mice, as well as MIP-1alpha[CCL3] and RANTES[CCL5] but not MIP-1beta[CCL4] proteins in the peritoneal exudates. OVA-induced neutrophil migration response was muted in immunized MIP-1alpha[CCL3](-/-) mice, but it was not inhibited by treatment with antibodies against RANTES[CCL5] or MIP-1beta[CCL4]. MIP-1alpha[CCL3] mediated neutrophil migration in immunized mice through induction of TNF-alpha and LTB(4) synthesis, as these mediators were detected in the exudates harvested from OVA-challenged immunized wild-type but not MIP-1alpha[CCL3](-/-) mice; administration of MIP-1alpha[CCL3] induced a dose-dependent neutrophil migration, which was inhibited by treatment with an anti-TNF-alpha antibody in TNF receptor 1 (p55(-/-))-deficient mice or by MK 886 (a 5-lipoxygenase inhibitor); and MIP-1alpha[CCL3] failed to induce LTB(4) production in p55(-/-) mice. MIP-1alpha[CCL3] used CCR1 to promote neutrophil recruitment, as OVA or MIP-1alpha[CCL3] failed to induce neutrophil migration in CCR1(-/-) mice, in contrast to CCR5(-/-) mice. In summary, we have demonstrated that neutrophil migration observed in this model of immune inflammation is mediated by MIP-1alpha[CCL3], which via CCR1, induces the sequential release of TNF-alpha and LTB(4). Therefore, whether a similar pathway mediates neutrophil migration in human immune-inflammatory diseases, the development of specific CCR1 antagonists might have a therapeutic potential.

BACKGROUND

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

OBJECTIVE

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

METHODS

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

RESULTS

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

CONCLUSIONS

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

The selective accumulation of different leucocyte populations during inflammation is regulated by adhesion molecules and chemokines expressed by vascular endothelium. This study examined how chemokine production and the expression of adhesion molecules and chemokine receptors vary between endothelia from different vascular beds. Human saphenous vein endothelium was compared with lung and dermal microvascular endothelia and with umbilical vein endothelium and a bone-marrow endothelial cell line. All endothelia produced CCL2 and CXCL8 constitutively, whereas CXCL10 and CCL5 were only secreted after tumour necrosis factor (TNF)-alpha or interferon (IFN)-gamma stimulation. In combination with TNF-alpha, IFN-gamma suppressed CXCL8 but enhanced CCL5 and CXCL10, whereas transforming growth factor (TGF)-beta reduced secretion of all chemokines. Basal chemokine secretion was higher from umbilical vein than other endothelial cells. Chemokine receptors, CXCR1, CXCR3 and CCR3, were present on all endothelia but highest on saphenous vein. CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 were also detected at variable levels on different endothelia. The variation between endothelia in chemokine secretion was much greater than the variations in adhesion molecules, both on resting cells and following cytokine stimulation. These results indicate that it is the tissue-specific variations in endothelial chemokine secretion rather than variations in adhesion molecules that can explain the different patterns of inflammation and leucocyte traffic seen in non-lymphoid tissues.

Although interleukin 1 (IL-1) induces expression of the transcription factor IRF1 (interferon-regulatory factor 1), the roles of IRF1 in immune and inflammatory responses and mechanisms of its activation remain elusive. Here we found that IRF1 was essential for IL-1-induced expression of the chemokines CXCL10 and CCL5, which recruit mononuclear cells into sites of sterile inflammation. Newly synthesized IRF1 acquired Lys63 (K63)-linked polyubiquitination mediated by the apoptosis inhibitor cIAP2 that was enhanced by the bioactive lipid S1P. In response to IL-1, cIAP2 and the sphingosine kinase SphK1 (the enzyme that generates S1P) formed a complex with IRF1, which led to its activation. Thus, IL-1 triggered a hitherto unknown signaling cascade that controlled the induction of IRF1-dependent genes that encode molecules important for sterile inflammation.

Inflammation in the central nervous system (CNS) is a complex process that involves a multitude of molecules and effectors, and it requires the transmigration of blood leukocytes across the blood-brain barrier (BBB) and the activation of resident immune cells. Cannabidiol (CBD), a non-psychotropic cannabinoid constituent of Cannabis sativa, has potent anti-inflammatory and immunosuppressive properties. Yet, how this compound modifies the deleterious effects of inflammation in TMEV-induced demyelinating disease (TMEV-IDD) remains unknown. Using this viral model of multiple sclerosis (MS), we demonstrate that CBD decreases the transmigration of blood leukocytes by downregulating the expression of vascular cell adhesion molecule-1 (VCAM-1), chemokines (CCL2 and CCL5) and the proinflammatory cytokine IL-1β, as well as by attenuating the activation of microglia. Moreover, CBD administration at the time of viral infection exerts long-lasting effects, ameliorating motor deficits in the chronic phase of the disease in conjunction with reduced microglial activation and pro-inflammatory cytokine production. Adenosine A2A receptors participate in some of the anti-inflammatory effects of CBD, as the A2A antagonist ZM241385 partially blocks the protective effects of CBD in the initial stages of inflammation. Together, our findings highlight the anti-inflammatory effects of CBD in this viral model of MS and demonstrate the significant therapeutic potential of this compound for the treatment of pathologies with an inflammatory component.

Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance.

We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided.

We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner.

We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.

BACKGROUND

Hepatocellular carcinoma (HCC) is a typical inflammation-associated cancer, but may also provoke antitumour immune responses whose significance and underlying mechanisms are incompletely understood.

OBJECTIVE

To characterise immune responses in the diethylnitrosamine (DEN)-liver cancer mouse model.

METHODS

Tumour development and immune cell functions upon DEN treatment were compared between C57BL/6 wild-type (WT), chemokine scavenging receptor D6-deficient, B cell- (Igh6), CD4 T cell- (MHC-II) and T-/B cell-deficient (Rag1) mice. Relevance for human HCC was tested by comparing gene array results from 139 HCC tissues.

RESULTS

The induction of premalignant lesions after 24 weeks and of HCC-like tumours after 42 weeks by DEN in mice was accompanied by significant leucocyte infiltration in the liver and upregulation of distinct intrahepatic chemokines (CCL2, CCL5, CXCL9). Macrophages and CD8 (cytotoxic) T cells were most prominently enriched in tumour-bearing livers, similar to samples from human HCC. Myeloid-derived suppressor cells (MDSC) increased in extrahepatic compartments of DEN-treated mice (bone marrow, spleen). The contribution of immune cell subsets for DEN-induced hepatocarcinogenesis was functionally dissected. In D6(-/-) mice, which lack the chemokine scavenging receptor D6, hepatic macrophage infiltration was significantly increased, but tumour formation and progression did not differ from that of WT mice. In contrast, progression of hepatic tumours (numbers, diameters, tumour load) was strikingly enhanced in T-/B cell-deficient Rag1(-/-) mice upon DEN treatment. When mice deficient for B cells (Igh6(-/-), μMT) or major histocompatibility complex II were used, the data indicated that T cells prevent initial tumour formation, while B cells critically limit growth of established tumours. Accordingly, in tumour-bearing mice antibody production against liver-related model antigen was enhanced, indicating tumour-associated B cell activation. In agreement, T and B cell pathways were differentially regulated in gene array analyses from 139 human HCC tissues and significantly associated with patients' survival.

CONCLUSIONS

Distinct axes of the adaptive immune system, which are also prognostic in human HCC, actively suppress DEN-induced hepatocarcinogenesis by controlling tumour formation and progression.

The opiates are well-established immunomodulatory factors, and recent evidence suggests that mu- and delta-opioid receptor ligands alter chemokine-driven chemotactic responses through the process of heterologous desensitization. In the present report, we sought to examine the capacity of mu- and delta-opioids to modulate the function of chemokine receptors CCR5 and CXCR4, the two major human immunodeficiency virus (HIV) coreceptors. We found that the chemotactic responses to the CCR1/5 ligand CCL5/regulated on activation, normal T expressed and secreted, but not the CXCR4 ligand stromal cell-derived factor-1alpha/CXCL12 were inhibited following opioid pretreatment. Studies were performed with primary monocytes and Chinese hamster ovary cells transfected with CCR5 and the micro-opioid receptor to determine whether cross-desensitization of CCR5 was a result of receptor internalization. Using radiolabeled-binding analysis, flow cytometry, and confocal microscopy, we found that the heterologous desensitization of CCR5 was not associated with a significant degree of receptor internalization. Despite this, we found that the cross-desensitization of CCR5 by opioids was associated with a decrease in susceptibility to R5 but not X4 strains of HIV-1. Our findings are consistent with the notion that impairment of the normal signaling activity of CCR5 inhibits HIV-1 coreceptor function. These results have significant implications for our understanding of the effect of opioids on the regulation of leukocyte trafficking in inflammatory disease states and the process of coreceptor-dependent HIV-1 infection. The interference with HIV-1 uptake by heterologous desensitization of CCR5 suggests that HIV-1 interaction with this receptor is not passive but involves a signal transduction process.