Human herpesvirus 7 (HHV-7) is a member of the subfamily Betaherpesvirinae that exhibits a restricted cell tropism, preferentially infecting CD4+ T cells in vitro. HHV-7 encodes two functional chemokine receptors, U12 and U51. The human chemokines that act as ligands for these receptors have been identified as CCL22 (the natural ligand for CCR4) and CCL19 (the natural ligand for CCR7). It was found that murine L1.2 cells co-expressing CCR4 or CCR7 and U12 responded to both CCL22 and CCL19 in calcium-mobilization assays, but migrated in response only to the appropriate ligand for the expressed cellular receptor. Similar results were obtained with L1.2 cells co-expressing CCR4 or CCR7 with U51. These results suggest that the HHV-7 U12 and U51 receptors can function in concert with CCR4 and CCR7 in host-cell signalling pathways.

The random amino acid copolymer poly(Y,E,A,K)(n) (Copaxone®) is widely used in multiple sclerosis treatment and a second generation copolymer poly(Y,F,A,K)(n) with enhanced efficacy in experimental autoimmune encephalomyelitis in mice has been described. A major mechanism through which copolymers function to ameliorate disease is the generation of immunosuppressive IL-10-secreting regulatory T cells entering the CNS. In addition, the antigen presenting cell to which these copolymers bind through MHC Class II proteins may have an important role. Here, both CCL22 (a Th2 cell chemoattractant) in large amounts and CXCL13 in much smaller amounts are shown to be secreted after administration of YFAK to mice and to a smaller extent by YEAK parallel to their serum concentrations. Moreover, bone marrow-derived macrophages secrete CCL22 in vitro in response to YFAK and to higher concentrations of YEAK. Strikingly, these chemokines are also secreted into serum of MHC Class II -/- mice, indicating that an innate immune receptor on these cells also has an important role. Thus, both the innate and the adaptive immune systems are involved in the mechanism of EAE amelioration by YFAK. The enhanced ability of YFAK to stimulate the innate immune system may account for its enhanced efficacy in EAE treatment.

OBJECTIVE

This study aimed to identify CD4+ T-cell-derived microparticles (MPs) and investigate their roles in rheumatoid arthritis (RA).

METHODS

Synovial fluids from 34 RA, 33 osteoarthritis patients and 42 healthy individuals were analyzed by flow cytometry. Human fibroblast-like synoviocytes and peripheral blood mononuclear cells were cultured with or without isolated MPs, chemokines and cytokines were measured by ELISA.

RESULTS

CD4+CD161+CD39+ and CD4+CD39+CD73+ MPs were abundantly present in RA patients, which were positively or negatively correlated with RA features, respectively. Chemokines CCL20, CCL17 and CCL22, and cytokines IL-17 and IL-10 were influenced by these MPs in human fibroblast-like synoviocytes (HFLS) or PMBCs.

CONCLUSIONS

CD4+ T-cell-derived CD161+CD39+ and CD39+CD73+ MPs could serve as new reciprocal biomarkers for RA evaluation.

The only clinically available curative treatment of type 1 diabetes mellitus is replacement of the pancreatic islets by allogeneic transplantation, which requires immunosuppressive therapies. Regimens used today are associated with serious adverse effects and impaired islet engraftment and function. The aim of the current study was to induce local immune privilege by accumulating immune-suppressive regulatory T-lymphocytes (Tregs) at the site of intramuscular islet transplantation to reduce the need of immunosuppressive therapy during engraftment. Islets were cotransplanted with a plasmid encoding the chemokine CCL22 into the muscle of MHC-mismatched mice, after which pCCL22 expression and leukocyte recruitment were studied in parallel with graft functionality. Myocyte pCCL22 expression and secretion resulted in local accumulation of Tregs. When islets were cotransplanted with pCCL22, significantly fewer effector T-lymphocytes were observed in close proximity to the islets, leading to delayed graft rejection. As a result, diabetic recipients cotransplanted with islets and pCCL22 intramuscularly became normoglycemic for 10 consecutive days, while grafts cotransplanted with control plasmid were rejected immediately, leaving recipients severely hyperglycemic. Here we propose a simple method to initially shield MHC-mismatched islets by the recruitment of endogenous Tregs during engraftment in order to improve early islet survival. Using this approach, the very high doses of systemic immunosuppression used initially following transplantation can thereby be avoided.

We investigated expressions of -CC chemokine ligand 2 (CCL2) and CCL5 in tumor samples from 147 breast cancer (BCa) patients and correlated with transforming growth factor-β (TGF-β) expression. We observed an inverse correlation of TGF-β expression with CCL2, CCL5 expression in early stages of BCa. On contrary, in late stages, CCL2, not CCL5, expression was found to be directly proportional with TGF-β expression. TGF-β stimulated MDA-MB-231 cells to express CCL2, however, downregulated both CCL2 and CCL5 in MCF-7. Interestingly, a significant swing of Th1-Th2 ratio towards Th2 is seen within the primary tumors expressing moderate/high-CCL2-low/negative-CCL5. We observed that CCL2-CCR2 interaction induces monocytes/macrophages to secrete Th2-attracting chemokine CCL22 in vitro. Therefore, CCL2 secreted from the tumor microenvironment may attract and interact with monocytes/macrophages, and favor Th2 accumulation by inducing CCL22 secretion. Study in 4T1-BALB/c BCa mouse model demonstrated significant (p<0.05) decrease in CCL2, CCL5 and CCL22 levels and reduction in lung metastatic nodule numbers upon administering TGF-β inhibitor. These findings collectively indicate that TGF-β regulates CCL2 and CCL5 expression in a stage-dependent manner during BCa progression, which in turn, determines Th1-Th2 balance within the tumor microenvironment.

Manipulation of regulatory T cell (Treg) migration by islet expression of the chemokine CCL22 prevents diabetes in NOD mice and delays recurrent autoimmunity in syngeneic islet transplants. We sought to determine whether attracting Tregs with CCL22 also prevents islet allograft rejection. Isolated Bl/6 mouse islets were transduced overnight with adenovirus expressing CCL22 (Ad-CCL22) downstream of the CMV promoter. Islets were transplanted under the renal capsule of Balb/c recipients made diabetic by streptozotocin. To assess immunologic tolerance, graft-bearing kidneys from recipients of CCL22-expressing islet grafts were removed, and mice received a second transplant of naive islets from the same donor strain or third-party islets into the contralateral kidney. Adenoviral expression of CCL22 conferred prolonged protection of islet allografts in MHC-mismatched, diabetic recipients, maintaining normoglycemia in 75% of recipients for at least 80 days. Increased frequency of Treg cells was observed in islet grafts transduced with Ad-CCL22 compared with untreated grafts. Normoglycemic recipients of CCL22-expressing islet grafts showed complete absence of antidonor antibodies and no lymphocyte proliferation after exposure to donor splenocytes. After removal of the primary graft at day 80, mice that received a second transplant with untreated islets from the same donor strain did not reject the grafts, suggesting the development of tolerance. Expression of CCL22 recruits Treg cells to transplanted islets, prevents activation of alloreactive T-cells and islet allograft failure and induces alloantigen-specific tolerance. Manipulation of Treg cells by CCL22 in transplanted islets may be a novel therapeutic strategy for diabetes.

Identification of HLA-restricted CD8+ T cell epitopes is important to study RSV-induced immunity and illness. We algorithmically analyzed the sequence of the fusion protein (F) of respiratory syncytial virus (RSV) and generated synthetic peptides that can potentially bind to HLA-A*0201. Four out of the twenty-five 9-mer peptides tested: peptides 3 (F33-41), 13 (F214-222), 14 (F273-281), and 23 (F559-567), were found to bind to HLA-A*0201 with moderate to high affinity and were capable of inducing IFN-γ and IL-2 secretion in lymphocytes from HLA-A*0201 transgenic (HLA-Tg) mice pre-immunized with RSV or recombinant adenovirus expressing RSV F. HLA-Tg mice were immunized with these four peptides and were found to induce both Th1 and CD8+ T cell responses in in vitro secondary recall. Effector responses induced by these peptides were observed to confer differential protection against live RSV challenge. These peptides also caused better recovery of body weight loss induced by RSV. A significant reduction of lung viral load was observed in mice immunized with peptide 23, which appeared to enhance the levels of inflammatory chemokines (CCL17, CCL22, and IL-18) but did not increase eosinophil infiltration in the lungs. Whereas, significant reduction of infiltrated eosinophils induced by RSV infection was found in mice pre-immunized with peptide 13. Our results suggest that HLA-A2-restricted epitopes of RSV F protein could be useful for the development of epitope-based RSV vaccine.

BACKGROUND

The purpose of this study was to investigate the presence of CD4(+) CD25(+) FOXP3(+) regulatory T (Treg) cells both in peripheral blood and local tumors in patients with nasal inverted papilloma (NIP).

METHODS

By using flow cytometry, the frequencies of CD4(+) CD25(+) FOXP3(+) Treg cells in both peripheral blood and tissues from 18 patients with NIP and 8 control subjects were determined. CCL22 and CCL17 proteins in NIP tumors were analyzed by using enzyme-linked immunosorbent assay. The suppressive capacity of Treg cells was estimated by WST-8 and enzyme-linked immunosorbent assay (ELISA; interferon-gamma [IFN-γ] and interleukin-4 [IL-4]) analysis.

RESULTS

Patients with NIP showed increased CD4(+) CD25(+) FOXP3(+) Treg cell frequencies in tumor tissues and CD4(+) T cell fraction rather than in peripheral blood. CCL22 increased in NIP tumors. Phytohemagglutinin (PHA)-induced proliferation and cytokine production of CD4(+) CD25(-) T cells were suppressed equally well by CD4(+) CD25(high) cells from both patients with NIP and controls.

CONCLUSIONS

Our study demonstrated the increased frequencies of CD4(+) CD25(+) FOXP3(+) Treg cells in NIP tumors, which might be influenced by CCL22.

Pancreatic cancer is an aggressive malignancy that is unresponsive to conventional radiation and chemotherapy. Therefore, development of novel immune therapeutic strategies is urgently needed. L-4F, an Apolipoprotein A-I (ApoA-I) mimetic peptide, is engineered to mimic the anti-inflammatory and anti-oxidative functionalities of ApoA-I. In this work, H7 cells were orthotopically implanted in C57BL/6 mice and treated with L-4F. Then, pancreatic cancer progression and the inflammatory microenvironment were investigated in vivo. The cytotoxicity of L-4F toward H7 cells was assessed in vitro. Furthermore, we investigated the effects of L-4F on macrophage polarization by analyzing the polarization and genes of mouse bone marrow-derived macrophages in vitro. The results show that L-4F substantially reduced the tumorigenicity of H7 cells. L-4F inhibited inflammation by reducing the accumulation of inflammatory cells, such as IL-17A-, IL-4-, GM-CSF-, IL-1β-, and IL-6-producing cells and Th1 and Th17. Notably, L-4F also decreased the percentage of macrophages in tumor tissues, especially M2 macrophages (CD11b+F4/80+CD206+), which was also confirmed in vitro. Additionally, the expression of the M2 marker genes Arg1, MRC1, and CCL22 and the inflammatory genes IL-6, iNOS, and IL-12 was decreased by L-4F, indicating that L-4F prevents M2 type macrophage polarization. However, L-4F could not directly attenuate H7 cell invasion or proliferation and did not induce apoptosis. In addition, L-4F potently down-regulated STAT3, JNK and ERK signaling pathways but not affects the phosphorylation of p38 in RAW 264.7 cells. These results suggest that L-4F exhibits an effective therapeutic effect on pancreatic cancer progression by inhibiting tumor-associated macrophages and inflammation.

BACKGROUND

Patients with atopic dermatitis (AD) are frequently colonized with Staphylococcus aureus (S. aureus), one-third of them producing α-toxin, which is correlated with the severity of eczema in AD. Staphylococcus aureus colonizes in patients with psoriasis as well. Distinct expression of chemokine (C-C motif) ligand (CCL) and chemokine (C-X-C motif) ligand (CXCL) chemokines has been documented in both diseases. In this study, we investigated the effects of sublytic α-toxin concentrations on human macrophages that accumulate in the skin of patients with AD and psoriasis.

METHODS

IFN-γ-induced protein of 10-kDa (IP-10)/CXCL10 and macrophage-derived chemokine (MDC)/CCL22 production were evaluated at the mRNA or at the protein level using qRT-PCR or ELISA, respectively. Cell surface markers' expression and chemotaxis were determined by flow cytometry and Boyden chamber technique, respectively.

RESULTS

Sublytic concentrations of α-toxin strongly induced CXCL10 in macrophages at both the mRNA and the protein levels and significantly up-regulated MHC class II expression. Supernatants of α-toxin-stimulated macrophages induced the migration of human CD4+ lymphocytes via the CXCL10 receptor (CXCR3). Macrophages from patients with AD produced lower levels of CXCL10 compared to cells from patients with psoriasis as well as healthy controls in response to α-toxin. α-Toxin did not lead to a large variation in CCL22 production in macrophages from all three groups.

CONCLUSIONS

Staphylococcal α-toxin contributes to Th1 polarization by induction of CXCL10 in macrophages. Macrophages from patients with AD and psoriasis responded to α-toxin in the induction of Th1-related chemokine CXCL10 diversely, which could favour the recruitment of distinct leucocyte subsets into the skin.

Previously, using the Illumina HumanHT-12 microarray we found that β-defensin 131 (DEFB131), an antimicrobial peptide, is upregulated in the human prostate epithelial cell line RWPE-1 upon stimulation with lipoteichoic acid (LTA; a gram-positive bacterial component), than that in the untreated RWPE-1 cells. In the current study, we aimed to investigate the role of DEFB131 in RWPE-1 cells during bacterial infection. We examined the intracellular signaling pathways and nuclear responses in RWPE-1 cells that contribute to DEFB131 gene induction upon stimulation with LTA. Chromatin immunoprecipitation was performed to determine whether NF-κB directly binds to the DEFB131 promoter after LTA stimulation in RWPE-1 cells. We found that DEFB131 expression was induced by LTA stimulation through TLR2 and p38MAPK/NF-κB activation, which was evident in the phosphorylation of both p38MAPK and IκBα. We also found that SB203580 and Bay11-7082, inhibitors of p38MAPK and NF-κB, respectively, suppressed LTA-induced DEFB131 expression. The chromatin immunoprecipitation assay showed that NF-κB directly binds to the DEFB131 promoter, suggesting that NF-κB is a direct regulator, and is necessary for LTA-induced DEFB131 expression in RWPE-1 cells. Interestingly, with DEFB131 overexpression in RWPE-1 cells, the accumulation of mRNA and protein secretion of cytokines (IL-1α, IL-1β, IL-6, and IL-12α) and chemokines (CCL20, CCL22, and CXCL8) were significantly enhanced. In addition, DEFB131-transfected RWPE-1 cells markedly induced chemotactic activity in THP-1 monocytes. We concluded that DEFB131 induces cytokine and chemokine upregulation through the TLR2/NF-κB signaling pathway in RWPE-1 cells during bacterial infection and promotes an innate immune response.

Chemokine receptor antagonists appear to access two distinct binding sites on different members of this receptor family. One class of CCR4 antagonists has been suggested to bind to a site accessible from the cytoplasm while a second class did not bind to this site. In this report, we demonstrate that antagonists representing a variety of structural classes bind to two distinct allosteric sites on CCR4. The effects of pairs of low-molecular weight and/or chemokine CCR4 antagonists were evaluated on CCL17- and CCL22-induced responses of human CCR4(+) T cells. This provided an initial grouping of the antagonists into sets which appeared to bind to distinct binding sites. Binding studies were then performed with radioligands from each set to confirm these groupings. Some novel receptor theory was developed to allow the interpretation of the effects of the antagonist combinations. The theory indicates that, generally, the concentration-ratio of a pair of competing allosteric modulators is maximally the sum of their individual effects while that of two modulators acting at different sites is likely to be greater than their sum. The low-molecular weight antagonists could be grouped into two sets on the basis of the functional and binding experiments. The antagonistic chemokines formed a third set whose behaviour was consistent with that of simple competitive antagonists. These studies indicate that there are two allosteric regulatory sites on CCR4.

Chemokine receptor CCR4 and its ligands (CCL17 and CCL22) are important for the recruitment of memory T cells into the skin in various cutaneous immune diseases. However, information on CCR4 and its ligands in contact hypersensitivity is relatively limited. In this study, we investigated the expression of CCR4, CCL17, and CCL22 in a mouse model of contact hypersensitivity to oxazolone. Contact sensitization to oxazolone increased the proportions of memory CD4+ T cells in the draining lymph nodes, spleen, and peripheral blood. Although CCR4+ mRNA and CCR4+ cells were detectable in naive mouse lymph nodes, they significantly increased in the sensitized mice. The majority of CCR4+ cells in both control and sensitized mouse lymph nodes were CD4+ T cells. In the skin of naive mice, the mRNAs for CCR4, CCL17, and CCL22 were detectable, but only CCL17 and CCL22 proteins were constitutively expressed in the skin, particularly in the epidermis. Interestingly, the mRNAs for CCR4 and its two ligands were significantly elevated in the inflamed skin of mice with contact hypersensitivity to oxazolone. Furthermore, a subpopulation of cells that infiltrated the skin was CCR4+ cells. Finally, the expression of CCL17 and CCL22 proteins was significantly enhanced in the epidermis of inflamed skin. Thus, our study provides direct evidence for the presence of CCR4 and its ligands in mouse contact hypersensitivity.

Diesel exhausts and their associated organic compounds may be involved in the recent increase in the prevalence of allergic disorders, through their ability to favor a type 2 immune response. Type 2 T cells have been shown to be preferentially recruited by the chemokines eotaxin (CCL11), macrophage-derived chemokine (MDC, CCL22), and thymus activation-regulated chemokine (CCL17) through their interaction with CCR3 and CCR4, respectively, whereas type 1 T cells are mainly recruited by IFN-gamma-induced protein-10 (CXCL10) through CXCR3 binding. The aim of the study was to evaluate the effect of diesel exposure on the expression of chemokines involved in type 1 and 2 T cell recruitment. PBMC and alveolar macrophages from house dust mite allergic patients were incubated with combinations of diesel extracts and Der p 1 allergen, and chemokine production was analyzed. Diesel exposure alone decreased the constitutive IP-10 production, while it further augmented allergen-induced MDC production, resulting in a significantly increased capacity to chemoattract human Th2, but not Th1 clones. Inhibition experiments with anti-type 1 or type 2 cytokine Abs as well as cytokine mRNA kinetic evaluation showed that the chemokine variations were not dependent upon IL-4, IL-13, or IFN-gamma expression. In contrast, inhibition of the B7:CD28 pathway using a CTLA-4-Ig fusion protein completely inhibited diesel-dependent increase of allergen-induced MDC production. This inhibition was mainly dependent upon the CD86 pathway and to a lesser extent upon the CD80 pathway. These results suggest that the exposure to diesel exhausts and allergen may likely amplify a deleterious type 2 immune response via a differential regulation of chemokine production through the CD28 pathway.

Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES) proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25) in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF) inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

BACKGROUND

Chemokines are critical mediators of T-cell homing into inflamed skin. The complex nature of this multicellular response makes it difficult to analyse mechanisms mediating the early responses in vivo.

OBJECTIVE

To visualize directly T-cell homing into inflamed skin and its inhibition by blockades using a unique noninvasive confocal microscopy.

METHODS

A mouse model of allergic contact dermatitis was used. T cells from oxazolone-sensitized and -challenged Balb/c mice were first analysed phenotypically in vitro. CD4 T cells were then labelled with a tracker dye and transferred into Balb/c-SCID mice. The recipient mice were challenged with oxazolone and CD4 T-cell homing into inflamed skin was visualized.

RESULTS

T cells with the skin homing receptors CCR4 and CCR10 were increased in the affected skin and draining lymph nodes, and effectively attracted by their specific chemokines CCL17, CCL22 and CCL27 in vitro. Using in vivo imaging, T-cell migration into the inflamed skin was observed at 2 h after application, peaking at 12 h and continuing for 48 h. Simultaneous systemic administration of neutralizing antibodies against CCR4 ligands (CCL17 and CCL22) and CCR10 ligand (CCL27) led to a significant suppression of T-cell migration and skin inflammation.

CONCLUSIONS

Our data indicate that these tissue-selective adhesion molecules and chemokine/receptor pathways act in concert to attract specialized T-cell populations to mediate cutaneous inflammation. The in vivo imaging technique can be applicable to other models of cutaneous diseases to help with better understanding of the pathogenesis and monitoring the therapeutic effects.

OBJECTIVE

To investigate differentially expressed genes in eyecup and retina of the ELOVL4 transgenic mouse, a model of Stargardt-like macular dystrophy (STGD3).

METHODS

We examined gene and protein expression in known pathways relevant to retinal degeneration using PCR arrays, Western blotting, and immunohistochemistry. Investigations were performed on ELOVL4 transgenic mice at 9 months, when 50% of rod (but no cone) photoreceptors had degenerated. Age-matched wild-type littermates served as controls.

RESULTS

Significant expression level changes were found in only 17 of the 252 genes examined. Nine were upregulated (Fgf2, Fgfr1, Ntf5, Cbln1, Ngfr, Ntrk1, Trp53, Tlr6, and Herpud1), and eight were downregulated (Ccl22, Ccr3, Il18rap, Nf1, Ccl11, Atf6β, Rpn1, and Serp1). Overexpression of FGF2 was detected at 1 month, before rod loss onset, and was maintained at high levels until cone loss (18 months). By 9 months, FGF2 overexpression was seen in photoreceptor cell bodies. Increased glial fibrillary acidic protein (GFAP) expression due to glial cell reactivity followed the same time course. Levels of NGFR/p75NTR remained invariant. Although present in rod outer segments at 1 month, the macrophage chemoattracting chemokine CCL22 became undetectable by 9 months, a likely consequence of progressive rod outer segment truncation.

CONCLUSIONS

At a mid-degeneration stage, major changes in gene expression in the ELOVL4 transgenic mouse retina included upregulation of Fgf2 and Fgfr1 and downregulation of Ccl22. Modulation of FGF2 occurred very early, concomitant with an increase in GFAP expression. Future studies will address which factors upstream of Fgf2 could provide potential therapeutic targets to slow photoreceptor degeneration in STGD3.

PI-2301 is an immunomodulator that could be an alternative therapy for MS. A placebo-controlled, multiple-ascending dose, double-blind study was performed in patients with secondary-progressive MS. Treatment was given subcutaneously once weekly for 8 weeks, followed by a 4-week open-label treatment period with active drug. The most common adverse event was transient injection site reactions. Non-significant trend for increases in serum levels of IL-3, IL-13, and CCL22 over time were suggestive of a beneficial T(H)2 immune response in subjects dosed with PI-2301 at 3 and 10 mg. MRI data indicated a non-significant trend for a reduction of lesion numbers in subjects treated with 1 and 3 mg PI-2301.

BACKGROUND

The presence of Tregs in tumors is associated with compromised tumor-specific immune responses and has a clear negative impact on survival of cancer patients. Thus, downregulation of Tregs is considered as a promising cancer immunotherapeutic approach. We have reported previously that neem leaf glycoprotein (NLGP) prophylaxis restricts tumor growth in mice by immune activation. In continuation, here, involvement of NLGP in the modulation of Tregs in association with tumor growth restriction is investigated.

RESULTS

NLGP downregulates CD4+CD25+Foxp3+ Tregs within tumors. NLGP-mediated downregulation of CCR4 along with its ligand CCL22 restricts Treg migration at the tumor site. NLGP is not apoptotic to Tregs but significantly downregulates the expression of Foxp3, CTLA4 and GITR. It also reverses the functional impairment of T-effector cells by Tregs, in terms of IFN-γ secretion, cellular proliferation and tumor cell cytotoxicity. NLGP also facilitates reconditioning of tumor microenvironment (hostile) by increasing IFN-γ and IL-12 but decreasing IL-10, TGF-β, VEGF and IDO, creating an antitumor niche. Interaction between Foxp3, p-NFATc3 and p-Smad2/3, needed for successful Treg function, is also inhibited by NLGP.

CONCLUSIONS

All of these coordinated events might result in inhibition of Treg associated-tumor growth and therefore increased survivability of mice having NLGP treatment before or/and after tumor inoculation. Thus, the possibility of NLGP being an excellent tool as a T-cell anergy breaker by abrogating the suppressor functions of Tregs in cancer needs to be explored further in the clinic.

Recruitment of immune cells to tumors is a complex process crucial for both inflammation-driven tumor progression and specific anti-tumor cytotoxicity. Chemokines control the directed migration of immune cells, and their actions are partly controlled by nonsignaling chemokine decoy receptors. The role of the receptors such as D6, Duffy antigen receptor for chemokines and ChemoCentryx chemokine receptor in immunity to tumors is still unclear. Using real-time PCR, we detected significantly decreased expression of D6 mRNA in colon tumors compared to unaffected mucosa. D6 protein was expressed by lymphatic endothelium and mononuclear cells in the colon lamina propria and detected by immunohistochemistry in two out of six tissue samples containing high D6 mRNA levels, whereas no staining was observed in any tissue samples expressing low mRNA levels. When examining the density of lymphatic vessels in colon tumors, we detected a marked increase in vessels identified by the lymphatic endothelial marker Lyve-1, excluding passive regulation of D6 due to decreased lymphatic vessel density. In parallel, the Treg-recruiting chemokine CCL22, which is sequestered by D6, was threefold increased in tumor tissue. Furthermore, we could show that low D6 expression correlated to more invasive tumors and that tumor location influences D6 expression, which is lower in the more distal parts of the colon. The data support that regulation of D6 by colon tumors results in altered levels of proinflammatory CC chemokines, thereby shaping the local chemokine network to favor tumor survival. This may have implications for the design of future immunotherapy for colon cancer.

BACKGROUND

An incomplete understanding of bone forming cells during wound healing and ectopic calcification has led to a search for circulating cells that may fulfill this function. Previously, we showed that monoosteophils, a novel lineage of calcifying/bone-forming cells generated by treatment of monocytes with the natural peptide LL-37, are candidates. In this study, we have analyzed their gene expression profile and bone repair function.

RESULTS

Human monoosteophils can be distinguished from monocytes, macrophages and osteoclasts by their unique up-regulation of integrin α3 and down-regulation of CD14 and CD16. Monoosteophils express high mRNA and protein levels of SPP1 (osteopontin), GPNMB (osteoactivin), CHI3L1 (cartilage glycoprotein-39), CHIT1 (Chitinase 1), MMP-7, CCL22 and MAPK13 (p38MAPKδ). Monocytes from wild type, but not MAPK13 KO mice are also capable of monoosteophil differentiation, suggesting that MAPK13 regulates this process. When human monoosteophils were implanted in a freshly drilled hole in mid-diaphyseal femurs of NOD/SCID mice, significant bone repair required only 14 days compared to at least 24 days in control treated injuries.

CONCLUSIONS

Human derived monoosteophils, characterized as CD45(+)α3(+)α3β(+)CD34(-)CD14(-)BAP (bone alkaline phosphatase)(-) cells, can function in an animal model of bone injury.

Heme oxygenase-1 (HO-1) has protective effects on liver damage induced by noxious stimuli. The mechanism of action of HO-1 is not well understood. In the present study, we investigate the effect of HO-1 in a model of fulminant hepatic failure induced by Propionibacterium acnes and lipopolysaccharide (LPS). The expression of HO-1 mRNA and protein in the liver was increased after repeated administration of the HO-1 inducer cobalt protoporphyrin IX. We found that HO-1 protected mice from acute liver damage induced by P. acnes/LPS and prolonged survival. On the contrary, administration of the HO-1 inhibitor zinc protoporphyrin IX increased liver damage induced by P. acnes/LPS. Subsequently, to investigate the underlying mechanisms of HO-1 in the acute liver injury model, we primed mice with P. acnes only. We found that the expression of HO-1 mRNA and protein in dendritic cells (DCs) was increased after the administration of cobalt protoporphyrin IX. HO-1 decreased the mature markers major histocompatibility complex II and CD80 on liver DCs. The expression of CCR7, CCL2, and CCL22 mRNA, which are expressed by mature DCs, was also reduced. These liver DCs could not efficiently stimulate CD4+ T cell activation and proliferation. Consequently, HO-1 inhibited the activation, proliferation, and T helper 1 polarization of liver-infiltrating CD4+ T cells and reduced the production of serum alanine aminotransferase and proinflammatory cytokines such as interferon-γ and tumor necrosis factor-α. Taken together, our data suggest that HO-1 alleviates P. acnes/LPS-induced fulminant hepatic failure, probably by inhibiting DC-induced adaptive responses.

Recent studies have shown that sparse distribution of regulatory T cells (Tregs) in the skin might be involved in the onset of severe cutaneous adverse drug reactions such as Stevens-Johnson syndrome and toxic epidermal necrolysis. Treg migration toward epithelial cells is regulated by certain chemokines, including TARC/CCL17 and MDC/CCL22. In this study, we analyzed the effect of allopurinol (APN), a drug known to cause severe adverse reactions, on the expression of factors affecting Treg migration and the mechanisms involved. APN inhibited the tumor necrosis factor (TNF)-α- and interferon (IFN)-γ-associated expression of TARC/CCL17 and MDC/CCL22 mRNA in HaCaT cells in a dose-dependent manner. Consistent with this, APN also suppressed TNF-α- and IFN-γ-induced production of TARC/CCL17 and MDC/CCL22 proteins and the migration of C-C chemokine receptor type 4-positive cells. Activity of the transcription factors NF-κB and STAT1, which are involved in TARC/CCL17 and MDC/CCL22 expression, was also investigated. APN inhibited activation of NF-κB, but not that of STAT1. Furthermore, it restricted p38 MAPK phosphorylation. These results suggest that APN inhibits TNF-α- and IFN-γ-induced TARC/CCL17 and MDC/CCL22 production through downregulation of p38 MAPK and NF-κB signaling, resulting in the sparse distribution of Tregs in the skin of patients with APN-associated Stevens-Johnson syndrome/toxic epidermal necrolysis.