Valencene (VAL) isolated from Cyperus rotundus possesses various biological effects such as antiallergic and antimelanogenesis activity. We investigated the effect of VAL on atopic dermatitis (AD) skin lesions and their molecular mechanisms. We topically applied VAL to 1-chloro-2,4-dinitrobenzene (DNCB) sensitized NC/Nga mice. Modified scoring atopic dermatitis index, scratching behavior, and histological/immunohistochemical staining were used to monitor disease severity. RT-PCR, western blotting, and enzyme-linked immunosorbent assay were used to determine the level of IgE, proinflammatory cytokines/chemokines production, and skin barrier proteins expression. Topical application of VAL significantly reduced AD-like symptoms and recovered decreased expression of filaggrin in DNCB-sensitized NC/Nga mice. The levels of serum IgE, IL-1β, IL-6, and IL-13 in skin/splenic tissue were reduced. In vitro studies using TNF-α and IFN-γ treated HaCaT cells revealed that VAL inhibited the exaggerated expression of Th2 chemokines including TARC/CCL17, MDC/CCL22, and proinflammatory chemokines such as CXCL8, GM-CSF, and I-CAM through blockade of the NF-κB pathway. In addition, expression of the skin barrier protein, involucrin, was also increased by VAL treatment. VAL inhibited the production and expression of proinflammatory cytokines IL-1β and IL-6 in LPS-stimulated RAW 264.7 cells. These results suggest that VAL may serve as a potential therapeutic option for AD.

BACKGROUND

Mycosis fungoides (MF) is a malignant lymphoma characterized by expansion of CD4(+) memory T-cell clones. Infiltrating cells express CCR4, which is attracted to CC chemokine ligands 17 and 22 (thymus and activation-regulated chemokine [TARC]/CCL17 and TARC/CCL22). Bath-psoralen plus ultraviolet A (PUVA) is effective against MF. In patients with psoriasis, bath-PUVA induces circulating regulatory T cells (Tregs), which suppress effector T cells. To understand the mechanisms in MF, we analyzed lesion-infiltrating cells before and after bath-PUVA therapy.

METHODS

Thirteen patients with MF (12 stage IB, 1 stage III; mean age 69.2 years, range 35-87 years; 6 men, 7 women) were recruited.

RESULTS

Immunohistochemical analysis revealed that lesion CCR4-positive (CCR4(+)) cells and Tregs significantly decreased from 105.1 ± 164.8 cells/10(-2) mm(2) to 31.4 ± 39.0 cells/10(-2) mm(2) and from 78.1 ± 67.8 cells/10(-2) mm(2) to 24.7 ± 25.0 cells/10(-2) mm(2), respectively. Serum TARC levels significantly correlated with infiltrating CD3(+) (r = 0.997), CCR4(+) (r = 0.991), and forkhead box P3-positive (Foxp3(+)) cells (r = 0.843). Circulating Tregs before bath-PUVA therapy were not significantly different from those in healthy volunteers. Bath-PUVA did not significantly change the percentage of circulating Tregs.

CONCLUSIONS

Bath-PUVA decreased CCR4(+) cells and Tregs in MF lesions but did not induce circulating Tregs, which might suppress effector T cells. Direct effects through skin lesions might eliminate both pathogenetically relevant cells and Tregs. Systemic immunosuppression was not induced.

There is growing evidence that chemokines recruit leukocytes in allergic, inflammatory and immune responses. CC chemokine receptor 4 (CCR4) is implicated as a preferential marker for T helper 2 cells, and the cells selectively respond to CC chemokine ligand 17 (CCL17) and CCL22. We searched for compounds having a profile as a CCR4 antagonist from an in-house library and have previously reported that 3-{2-[(2R)-2-phenyl-4-(4-pyridin-4-ylbenzyl)morpholin-2-yl]ethyl}quinazoline-2,4(1H,3H)-dione (named RS-1154) was capable of significantly inhibiting the binding of [(125) I]CCL17 to human CCR4-expressing CHO cells. From further synthesis of its derivatives, we newly focused on 3-(isobutyrylamino)-N-{2-[(2R)-2-phenyl-4-(4-pyridin-4-ylbenzyl)morpholin-2-yl]ethyl}benzamide (RS-1269), which showed potency comparable to RS-1154 in inhibiting CCL17-induced migration of DO11.10 mice-derived T helper 2 cells with an IC(50) value of 5.5 nM in vitro. We then investigated the pharmacological effects of RS-1269 on ovalbumin-induced ear swelling and lipopolysaccharide-induced endotoxic shock in mice. The ear thickness was significantly decreased by oral administration of RS-1269 at the dose of 30 mg/kg. Treatment with lipopolysaccharide significantly increased the serum level of tumour necrosis factor-α. Compared with an anti-CCL17 antibody, RS-1269 significantly inhibited the production at the dose of 100 mg/kg. These results raise the possibility that RS-1269 or one of its derivatives has potential to serve as a prototype compound to develop therapeutic agents for atopic dermatitis and inflammatory diseases.

Upon inflammation, circulating monocytes leave the bloodstream and migrate into the tissues, where they differentiate after exposure to various growth factors, cytokines or infectious agents. The best defined macrophage polarization types are M1 and M2. However, the platelet-derived CXC chemokine CXCL4 induces the polarization of macrophages into a unique phenotype. In this study, we compared the effect of CXCL4 and its variant CXCL4L1 on the differentiation of monocytes into macrophages and into immature monocyte-derived dendritic cells (iMDDC). Differently to M-CSF and CXCL4, CXCL4L1 is not a survival factor for monocytes. Moreover, the expression of the chemokine receptors CCR2, CCR5 and CXCR3 was significantly higher on CXCL4L1-treated monocytes compared to M-CSF- and CXCL4-stimulated monocytes. IL-1 receptor antagonist (IL-1RN) expression was upregulated by CXCL4 and downregulated by CXCL4L1, respectively, whereas both chemokines reduced the expression of the mannose receptor (MRC). Furthermore, through activation of CXCR3, CXCL4L1-stimulated monocytes released significantly higher amounts of CCL2 and CXCL8 compared to CXCL4-treated monocytes, indicating more pronounced inflammatory traits for CXCL4L1. In contrast, in CXCL4L1-treated monocytes, the production of CCL22 was lower. Compared to iMDDC generated in the presence of CXCL4L1, CXCL4-treated iMDDC showed an enhanced phagocytic capacity and downregulation of expression of certain surface markers (e.g. CD1a) and specific enzymes (e.g. MMP-9 and MMP-12). CXCL4 and CXCL4L1 did not affect the chemokine receptor expression on iMDDC and cytokine production (CCL2, CCL18, CCL22, CXCL8, IL-10) by CXCL4- or CXCL4L1-differentiated iMDDC was similar. We can conclude that both CXCL4 and CXCL4L1 exert a direct effect on monocytes and iMDDC. However, the resulting phenotypes are different, which suggests a unique role for the two CXCL4 variants in physiology and/or pathology.

BACKGROUND

Tipping the balance toward regulatory T cells (Tregs) through adoptive cell therapy has shown promise to induce transplantation tolerance. Although such strategy has been explored in many mice organ transplantation studies, less knowledge was available in rat systems. Furthermore, the behaviors of the transferred cells have not been well studied in real-time fashion.

METHODS

Tregs from naïve LEW rats were purified in two steps with the autoMACS system. Immunosuppression potential of these cells was examined with mixed lymphocyte reaction. Following stimulation by the alloantigen in vitro, the purified Tregs were infused into the recipients of vascularized composite allotransplantation (VCA). Secondary allogeneic skin grafting challenge was performed on the recipients with long-term survived VCA. Live optical imaging was performed to track luciferase-expressing Tregs following infusion to the VCA recipients. Expression of relevant molecules was studied by flow cytometry or quantitative RT-PCR.

RESULTS

Rat Tregs were enriched following two-step cell sorting and showed immunosuppressive capacity. Upon infusion into the VCA recipients that have been treated with antilymphocyte serum and short-term Cyclosporin A, the antigen-stimulated Tregs significantly prolonged VCA survival and induced donor-specific tolerance. Tracking of the infused bioluminescent Tregs showed their specific homing to lymph nodes, and then to the VCAs. Following secondary skin grafting, Tregs specifically gathered at the donor-derived skin that was not rejected by the recipient. The in vivo migratory pattern coincided with the altered expression of cell surface molecules of CD62L, CD103, CD134, and CD278, following donor-antigen stimulation. Elevated expression of CCR4 and CCL22 in allograft may also participate in recruiting Tregs for maintenance of VCA survival and promoting donor-specific tolerance.

CONCLUSIONS

Sorted Tregs induced donor-specific tolerance to VCA in rats. Live cell tracking demonstrated that activated CD4+CD25+FoxP3+ Tregs targeted primarily to the lymph nodes and VCA. The Tregs migrated to the secondary grafted donor skin and contributed to the maintenance of donor-specific tolerance. These behaviors were associated with phenotypic changes induced by donor antigen stimulation. Increased expression of CCR4 and CCL22 in VCA skin may also be relevant.

Exposure to disinfectants and disinfection byproducts (DBPs) due to swimming in chlorinated water has been associated with allergic and respiratory health effects, including asthma.

Biological mechanisms contributing to these associations are largely unknown. We hypothesized a potential pathway involving modulation of the immune system.

We assessed levels of immune markers (CCL11, CCL22, CXCL10, CRP, EGF, GCSF, IL-8, IL-17, IL-1RA, MPO, VEGF, Periostin) in serum collected from 30 women and 29 men before and after 40min of swimming in a chlorinated pool. Exposure to DBPs was assessed by measuring bromodichloromethane, bromoform, chloroform, and dibromochloromethane in exhaled breath before and after swimming. Covariate data including information on physical activity was available through questionnaires and measurements. We assessed the association between indicators of swimming in a chlorinated pool and changes in serum immune marker concentrations using linear regression with bivariate normal distributions and adjusted for multiple comparisons by applying the Benjamini-Hochberg procedure.

We observed a significant decrease in serum concentrations of IL-8 (-12.53%; q=2.00e-03), CCL22 (-7.28%; q=4.00e-04), CCL11 (-7.15%; q=9.48e-02), CRP (-7.06%; q=4.68e-05), and CXCL10 (-13.03%; q=6.34e-14) and a significant increase in IL-1RA (20.16%; q=4.18e-06) from before to after swimming. Associations with quantitative measurements of DBPs or physical activity were similar in direction and strength. Most of the observed associations became non-significant when we adjusted the effects of exposure to DBPs for physical activity or vice-versa.

Our study indicates that swimming in a chlorinated pool induces perturbations of the immune response through acute alterations of patterns of cytokine and chemokine secretion. The observed effects could not be uniquely attributed to either exposure to DBPs or physical activity. Evidence in the literature suggests that observed decreases in immune markers are possibly due to an immunosuppressive effect of DBPs, while the increase in IL-1RA might be due to physical activity.

Canine epitheliotropic cutaneous lymphoma (cECL) is characterized by infiltration of neoplastic lymphocytes in the skin with a specific tropism for the epidermis. Migration of lymphocytes is strictly controlled by interactions between chemokines and chemokine receptors, which may be involved in the pathogenesis of cECL. In this study, we investigated mRNA transcription levels of several chemokines (CCL17, CCL19, CCL21, CCL22, CCL27, CCL28 and CXCL10) and chemokine receptors (CCR4, CCR7, CCR10 and CXCR3) in lesional skin of cECL by quantitative real-time RT-PCR. To examine the subsets of accumulating neoplastic lymphocytes, we also investigated transcription levels of type-1 (IFN-γ, IL-12p35, IL-12p40 and LT-α) and type-2 (IL-4 and IL-13) cytokines and cytotoxic markers (perforin and granzyme B). We found that the lesional skin had higher mRNA transcription of CCL19, CXCL10, CCR4, CCR7, CCR10 and CXCR3 and lower transcription of CCL27 than healthy dog skin (p<0.05). In addition, transcription levels of type-1 cytokine and cytotoxic markers in lesional skin were significantly higher than those in healthy dog skin. These results indicate that the transcription of some chemokines and chemokine receptors, which are necessary for skin-homing, epitheliotropism and peripheral segregation of T-cells, is upregulated in the lesional skin of cECL. In addition, our results also indicate that the subset of neoplastic lymphocytes in cECL is most likely type-1 cytotoxic T-cells.

Crohn's disease (CD) and ulcerative colitis (UC) are the most widely known types of inflammatory bowel diseases (IBD) and have been paid more attention due to their increasing incidence and a substantial increase in the risk of colorectal cancer (CRC). However, the phenotype and, more importantly, the function in the regulation of mucosal inflammation by different macrophages are poorly understood, even though macrophages constitute a major subset of intestinal myeloid cells. The results firstly showed that the subset of peritoneal CD11b+CD169+ macrophages increased and CCL22 expression level decreased significantly during the DSS-induced colitis. DSS-induced colitis was alleviated in CD169-DTR mice at least partially due to the deletion CD169+ macrophages. Moreover, the CCL22 expression level in peritoneal macrophages from CD169-DTR mice was much higher than that from WT mice with DSS-induced colitis. And, the cell-sorting result revealed that CD11b+CD169+ macrophage cells did not express CCL22 dominantly. Further experiment in vivo demonstrated that treatment with recombinant murine CCL22 (rmCCL22) ameliorated the clinical symptoms of DSS-induced colitis. All these data indicated that macrophage subset of CD11b+CD169+ from peritoneal cavity played critical role probably together with low levels of CCL22 in DSS-induced colitis.

UNASSIGNED

In China in 2009, esophageal cancer was the fifth most commonly diagnosed malignancy and the fourth leading cause of malignancy-related death. Accumulating evidence indicates that genetic factors might play an important role in esophageal squamous cell carcinoma (ESCC) carcinogenesis.

UNASSIGNED

In total, we recruited 629 ESCC patients and 686 controls. Genetic variations in the S100A14, MLH1, SMAD7 and CCL22/MDC genes were measured using the ligation detection reaction method.

UNASSIGNED

When the S100A14 rs11548103 GG genotype was considered as the reference group, the GA genotype associated with decreased risk of ESCC (GA vs. GG: adjusted OR = 0.73, 95% CI = 0.57-0.93, p = 0.009). In the dominant model, GA/AA variants were associated with a significantly decreased risk of ESCC compared with the GG genotype (GA/AA vs. GG: adjusted OR = 0.76, 95% CI = 0.61-0.95, p = 0.018). Logistic regression analyses showed that the MLH1 rs1800734 C>T, SMAD7 rs12953717 C>T and CCL22/MDC rs4359426C>A polymorphisms were not associated with the risk of ESCC in any of the models tested.

UNASSIGNED

Our findings indicated that, in a Chinese population, rs11548103 might contribute to a decreased risk of ESCC. Further studies are need to confirm these data with results from a lager cohort and different ethnic origins.

BACKGROUND

CCL22/MDC is a CC chemokine with a critical role in regulation of the immune balance in physiological condition. CCL22/CCR-4 ligation has been documented to participate in the migration of regulatory T (Treg) cells and Th2 lymphocytes to the site of breast tumors; circumstances that are known to be associated with poor prognosis.

OBJECTIVE

To investigate the association of a single nucleotide polymorphism (SNP) in CCL22 gene; 16C/A (rs4359426; Asp2Ala), with susceptibility to breast cancer in a sample of Iranian population.

METHODS

161 patients with pathologically confirmed breast carcinoma (mean age 49.3 ± 11.5 yrs) and 178 age-matched healthy women (mean age: 49.3 ± 12.9 yrs) were studied. CCL22 genotypes were investigated by the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. Data was verified by direct automated sequencing. Arlequin analysis showed no deviation from Hardy-Weinberg equilibrium.

RESULTS

The most frequent genotype in both patient and control groups was wild type CC genotype with frequency of 146 out of 161 (90.7%) among patients and 153 out of 178 (86.0%) in control group (p=0.24). The frequency of CA genotype was 15 (9.3%) and 23 (12.9%) in patients and controls, respectively (p=0.38). No AA genotype was observed among patients but this genotype was observed with the frequency of 2 out of 178 (1.1%) in control subjects. The minor allele frequency (MAF) was 0.07 in the population.

CONCLUSIONS

No correlation was found between the investigated genotypes and clinicopathological characteristics of the patients. Conclusively, results of this investigation do not support the association of 16C/A SNP (rs4359426; Asp2Ala) in CCL22 gene with susceptibility to, and progression of, breast cancer in Iranian population.

Although therapies for advanced melanoma have been greatly improved by the development of immune checkpoint inhibitors and BRAF/mitogen-activated protein kinase kinase inhibitors, there are still many concerns about the administration of these novel drugs. Therefore, to combine these therapies sequentially at appropriate time points of the disease is important. In this report, we report two cases in which dabrafenib and trametinib therapy for advanced melanoma failed but were successfully controlled by nivolumab monotherapy, and investigated the sera sCD163, CCL22 and CXCL10 as biomarkers for tumor progression. Interestingly, the sera levels of sCD163, CXCL10 and CCL22, both of which are produced by activated tumor-associated macrophages, were increased in parallel with the tumor progression in each case. Because this report presents only two cases, further data will need to be accumulated to provide more fundamental insights into the usefulness of these biomarkers for predicting disease progression in melanoma.

Cytokines, including chemokines, are small secreted proteins, which specifically effect on the interactions and communications between cells. Pro-inflammatory cytokines are produced predominantly by activated macrophages and are involved in the upregulation of inflammatory reactions. Dysregulation of cytokines is associated with post-traumatic stress disorder (PTSD). Here, we use both before-and-after and case-control studies to search for potential chemokine biomarkers associated with PTSD onset, risk, and resilience as well as stress responses in US military service members deployed to Iraq and Afghanistan. Blood samples and scores of the PTSD Checklist (PCL) were obtained from soldiers pre- and post deployment (pre, post). Forty chemokines were measured using the Bio-Plex Pro Human Chemokine Panel Assays. The before-and-after analysis showed potential markers (CCL2, CCL15, CCL22, CCL25, CXCL2, and CXCL12) are associated with PTSD onset, and CCL3, CXCL11, and CXCL16 are related to stress response. The case-control study demonstrated that CCL13, CCL20, and CXCL6 were possible PTSD risk markers, and CX3CL1 might be a resilience marker. In addition, CCL11, CCL13, CCL20, and CCL25 were correlated with the PCL scores, indicating their association with PTSD symptom severity. Our data, for the first time, suggest that these dysregulated chemokines may serve as biomarkers for PTSD onset, risk, and resilience as well as stress responses, and may benefit developing approaches not only for PTSD diagnosis but also for PTSD treatment.

The chemokine gene cluster [CCL22, CX3CL1, CCL17] (previously known as [SCYA22, SCYD1, SCYA17]) is a candidate locus for one of the susceptibility genes for inflammatory bowel disease that are located in the peri-centromeric region of chromosome 16. Screening for sequence variation at this locus led to the detection of 14 single nucleotide polymorphisms (SNPs). An efficient experimental and computational approach was developed to estimate allele frequencies and pairwise linkage disequilibrium relationships between SNPs at this locus, and to test them for association with inflammatory bowel disease. The 12 common SNPs were assigned to 5 distinct linkage disequilibrium groups. Genotyping of one SNP from each linkage disequilibrium group in a large cohort of families with inflammatory bowel disease did not provide convincing evidence of association with either Crohn's disease or ulcerative colitis. We describe an efficient experimental design from SNP screening to association testing. This strategy can be used to test candidate genes for involvement in susceptibility to complex disease.

Type 1 diabetes (T1D) is an autoimmune disease characterized by destruction of pancreatic β cells. One of the promising therapeutic approaches in T1D is the transplantation of islets; however, it has serious limitations. To address these limitations, immunotherapeutic strategies have focused on restoring immunologic tolerance, preventing transplanted cell destruction by patients' own immune system. Macrophage-derived chemokines such as chemokine-ligand-22 (CCL22) can be utilized for regulatory T cell (Treg) recruitment and graft tolerance. Stellate cells (SCs) have various immunomodulatory functions: recruitment of Tregs and induction of T-cell apoptosis. Here, we designed a unique immune-privileged microenvironment around implantable islets through overexpression of CCL22 proteins by SCs. We prepared pseudoislets with insulin-secreting mouse insulinoma-6 (MIN6) cells and human SCs as a model to mimic naive islet morphology. Our results demonstrated that transduced SCs can secrete CCL22 and recruit Tregs toward ​the implantation site in vivo. This study is promising to provide a fundamental understanding of SC-islet interaction and ligand synthesis and transport from SCs at the graft site for ensuring local immune tolerance. Our results also establish a new paradigm for creating tolerable grafts for other chronic diseases such as diabetes, anemia, and central nervous system (CNS) diseases, and advance the science of graft tolerance.

Chitin is a polysaccharide that provides structure and rigidity to the cell walls of fungi and insects. Mammals possess multiple chitinases, which function to degrade chitin, thereby supporting a role for chitinases in immune defense. However, chitin degradation has been implicated in the pathogenesis of asthma. Here, we determined the impact of acidic mammalian chitinase (AMCase) (Chia) deficiency on host defense during acute exposure to the fungal pathogen Aspergillus fumigatus as well as its contribution to A. fumigatus-associated allergic asthma. We demonstrate that chitin in the fungal cell wall was detected at low levels in A. fumigatus conidia, which emerged at the highest level during hyphal transition. In response to acute A. fumigatus challenge, Chia-/- mice unexpectedly demonstrated lower A. fumigatus lung burdens at 2 days postchallenge. The lower fungal burden correlated with decreased lung interleukin-33 (IL-33) levels yet increased IL-1β and prostaglandin E2 (PGE2) production, a phenotype that we reported previously to promote the induction of IL-17A and IL-22. During chronic A. fumigatus exposure, AMCase deficiency resulted in lower dynamic and airway lung resistance than in wild-type mice. Improved lung physiology correlated with attenuated levels of the proallergic chemokines CCL17 and CCL22. Surprisingly, examination of inflammatory responses during chronic exposure revealed attenuated IL-17A and IL-22 responses, but not type 2 responses, in the absence of AMCase. Collectively, these data suggest that AMCase functions as a negative regulator of immune responses during acute fungal exposure and is a contributor to fungal asthma severity, putatively via the induction of proinflammatory responses.

Inflammatory skin disease are caused by multiple factors, including susceptibility genes, and immunologic and environmental factors, and are characterized by an increase in epidermal thickness and the infiltration of macrophages, keratinocytes, mast cells, eosinophils and other inflammatory cells. Keratinocytes may serve an important role in the pathogenesis of inflammatory skin diseases. The traditional herbal decoction Hyangso‑san (HSS) has been used to treat symptoms of the common cold, including headache, pantalgia, fever and chills. However, to the best of our knowledge, there is no evidence regarding whether HSS has an effect on inflammatory skin diseases. The present study investigated the anti‑skin inflammation activity of HSS using the HaCaT human keratinocyte cell line. The mRNA expression and production of inflammatory chemokines, including C‑C motif chemokine ligand 22 (CCL22), CCL5, CCL17, and interleukin (IL)‑8, was measured using reverse transcription polymerase chain reaction and ELISA analyses. Moreover, we evaluated the effect of HSS on signal transducer and activator of transcription 1 (STAT1) pathway in HaCaT cells. The cells were stimulated with tumor necrosis factor‑α (TNF‑α) and interferon‑γ (IFN‑γ) to induce an inflammatory reaction. In the TNF‑α‑ and IFN‑γ‑stimulated cells, the production and expression of inflammatory chemokines were observed, including CCL22, CCL5, CCL17 and IL‑8. In addition, stimulation with TNF‑α and IFN‑γ increased the phosphorylation and nuclear translocation of STAT1 in HaCaT cells. By contrast, HSS extract treatment inhibited TNF‑α‑ and IFN‑γ‑induced STAT1 activation. Results from the present study indicated that HSS exhibited inhibitory effects on TNF‑α‑ and IFN‑γ‑mediated chemokine production and expression by targeting STAT1 in keratinocytes. Overall, the results indicated that HSS may be a potential candidate therapeutic drug for inflammatory skin diseases such as atopic dermatitis.

The combination of radiation with immune-checkpoint inhibitors was reported in some cancers to have synergic effects both locally and distally. Our aim was to assess this combined therapy on both radiated and non-radiated bladder tumors and to characterize the immune landscape within the tumor microenvironment. Murine bladder cancer cells (MB49) were injected s.c. in both flanks of C57BL/6 mice. Mice were randomly assigned to the following treatments: placebo, antiPD-L1 (4 intra-peritoneal injections over 2 weeks), radiation to right flank (10Gy in two fractions), or radiation+anti-PD-L1. Tumor digestion, flow cytometry, and qPCR were performed. Log-rank analysis was used for statistical significance. Radiation+antiPD-L1 group demonstrated statistically significant slower tumor growth rate both in the radiated and non-irradiated tumors (p<0.001). Survival curves demonstrated superior survival in the combination group compared to each treatment alone (p=0.02). Flow cytometry showed increased infiltration of immuno-suppressive cells as well as cytotoxic lymphocytes (CTL) in the radiation and combination groups (p=0.04). Ratio of immunosuppressive cells to CTL shifted in favor of cytotoxic activity in the combination arm (p<0.001). The qPCR analysis revealed downregulation of immunosuppressive genes (CCL22, IL22 and IL13), as well as upregulation of markers of CTL activation (CXCL9, GZMA and GZMB) within both the radiated and distant tumors within the combination group. Combining radiation with immune checkpoint inhibitor provided better response in the radiated tumors and also the distant tumors along with a shift within the tumor microenvironment favoring cytotoxic activity. These findings demonstrate a possible abscopal effect in urothelial carcinoma with combination therapy.

Effective hazard screening will require the development of high-throughput or in vitro assays for the identification of potential sensitizers. The goal of this preliminary study was to identify potential biomarkers that differentiate the response to allergens vs non-allergens following an acute exposure in naïve individuals. Female BALB/c mice received a single intratracheal aspiration exposure to Metarhizium anisopliae crude antigen (MACA) or bovine serum albumin (BSA) in Hank's Balanced Salt Solution (HBSS) or HBSS alone. Mice were terminated after 1, 3, 6, 12, 18 and 24 h. Bronchoalveolar lavage fluid (BALF) was evaluated to determine total and differential cellularity, total protein concentration and LDH activity. RNA was isolated from lung tissue for microarray analysis and qRT-PCR. MACA administration induced a rapid increase in BALF neutrophils, lymphocytes, eosinophils and total protein compared to BSA or HBSS. Microarray analysis demonstrated differential expression of genes involved in cytokine production, signaling, inflammatory cell recruitment, adhesion and activation in 3 and 12 h MACA-treated samples compared to BSA or HBSS. Further analyses allowed identification of approximately 100 candidate biomarker genes. Eleven genes were selected for further assessment by qRT-PCR. Of these, 6 demonstrated persistently increased expression (Ccl17, Ccl22, Ccl7, Cxcl10, Cxcl2, Saa1), while C3ar1 increased from 6-24 h. In conclusion, a single respiratory exposure of mice to an allergenic mold extract induces an inflammatory response which is distinct in phenotype and gene transcription from the response to a control protein. Further validation of these biomarkers with additional allergens and irritants is needed. These biomarkers may facilitate improvements in screening methods.

Intense lipolysis triggers an inflammatory response within adipose tissue characterized by adipose tissue macrophage (ATM) infiltration; however, the mechanisms triggering this process are poorly characterized in transition dairy cows. The aim of this study was to determine the association between ATM infiltration and body fat mobilization in the transition period, markers of excessive lipolysis, and adipose tissue expression of genes related to chemotactic and inflammatory responses. Subcutaneous adipose tissue samples were taken from the tailhead of 9 multiparous Holstein cows, 27 ± 2.2 d (far-off) and 10 ± 1.5 d (close-up) before and 9 ± 0.3 d after calving (fresh). Blood samples were collected by coccygeal venipuncture 2 h before adipose sample collections. Body condition score (BCS) was assessed independently by 3 experienced technicians at every time point. Based on BCS loss intensity between the close-up and fresh period, cows were divided into 2 groups: low BCS loss (LBCSL, change in BCS <0.25 units, n = 5) and high BCS loss (HBCSL, change in BCS >0.25 units, n = 4). Although none of the LBCSL cows had a health event, all cows in the HBCSL group suffered from one or more clinical disorder (retained placenta, milk fever, or ketosis) in the transition period. The number of ATM was determined by immunohistochemistry, and expression of selected chemotactic and inflammatory genes was determined by reverse-transcription quantitative real-time PCR in subcutaneous adipose tissue samples. The proportion of ATM in subcutaneous adipose tissue increased in HBCSL during the postpartum period. The proportion of ATM was not associated with serum β-hydroxybutyrate or free fatty acid concentrations on the day of adipose tissue collection. The ATM infiltration in the fresh period was associated with local expression of the chemotactic genes, C-C motif chemokine ligand 22 (CCL22), osteopontin (SPP1), and the receptor for SPP1, cluster of differentiation 44 (CD44). This supports a potential chemotactic role of CCL22 and SPP1 for ATM in bovine adipose tissue. None of the genes encoding pro- or anti-inflammatory mediators, tumor necrosis factor (TNF), IL6, and IL10 were associated with the proportion of ATM. Our results indicate that ATM infiltration of subcutaneous adipose tissue is associated with body fat mobilization in early-lactation dairy cows and supports a role for ATM in the adaptation of adipose tissues to the metabolic challenges of the transition period.

We evaluated the utility of chemokine MCP-3 and MDC/CCL22 as molecular adjuvants of DNA vaccines for botulinum neurotoxin serotype A (BoNT/A) in a Balb/c mouse model. Notably, the immunogenicity of the DNA vaccine against BoNT/A was not enhanced using a fusion of the AHc-C antigen with the MCP-3 or MDC/CCL22. Nevertheless, the potency of the DNA vaccine was significantly modulated and enhanced by co-administration of the AHc-C antigen with MCP-3 or MDC/CCL22. This strategy elicited high levels of humoral immune responses and protection against BoNT/A. The enhanced potency was further boosted by co-administration of the AHc-C antigen with both MCP-3 and MDC/CCL22 in Balb/c mice, but not by co-administration of AHc-C antigen with the MCP-3-MDC/CCL22 fusion. Co-immunization with both the MCP-3 and MDC/CCL22 constructs induced the highest levels of humoral immunity and protective potency against BoNT/A. Our results indicated that MCP-3 and MDC/CCL22 are effective molecular adjuvants of the immune responses induced by the AHc-C-expressing DNA vaccine when delivered by co-administration of the individual chemokines, but not when delivered in the form of a chemokine/antigen fusion. Thus, we describe an alternative strategy to the design and optimization of DNA vaccine constructs based on co-administration of the antigen with the chemokine rather than in the form of a chemokine/antigen fusion.

BACKGROUND

Chungsimyeonja-eum (CSYJE) is an herbal prescription used in traditional Oriental medicine for treating cerebral infarction by reducing ischemic damage. However, the effects of CSYJE on inflammation have not been verified scientifically.

METHODS

Anti-inflammatory effects of CSYJE was investigated to dertermine the inhibitory effects of CSYJE against inflammation using RAW 264.7 mouse macrophages and HaCaT human keratinocytes. To measure the effects of CSYJE on inflammatory mediators and cytokines/chemokines, we used the following methods: cell viability assay, enzyme-linked immunosorbent assay (ELISA), western blotting, immunocytochemistry. RAW 264.7 cells were pretreated with CSYJE (250, 500, or 1000 μg/mL) for 4 h and treated with lipopolysaccharide (LPS) for additional 20 h. HaCaT cells were stimulated with tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) (TI), and CSYJE (125, 250, or 500 μg/mL) for 24 h.

RESULTS

CSYJE suppressed the production of nitric oxide (NO, IC50 1000 μg/mL), prostaglandin E2 (PGE2, IC50 = 12.1 μg/mL), and interleukin (IL)-6 (IC50 = 248 μg/mL) in LPS-stimulated RAW 264.7 cells. CSYJE suppressed the effects of TI on the production of thymus and activation-regulated chemokine (TARC, IC50 = 330.2 μg/mL), macrophage-derived chemokine (MDC/CCL22, IC50 = 52.5 μg/mL), regulated on activation, normal T-cell expressed and secreted (RANTES/CCL5, IC50 = 372.9 μg/mL), and IL-8 (IC50 = 345.1 μg/mL) in HaCaT cells. CSYJE inhibited TI-stimulated STAT1 phosphorylation in a dose-dependent manner and nuclear translocation at 500 μg/mL in HaCaT cells.

CONCLUSIONS

Our results suggest a possible therapeutic application of CSYJE for treating inflammatory diseases.

Pyropia yezoensis, a red alga, is popular and harvested a lot in East Asia and is famous for its medicinal properties attributable to its bioactive compounds including amino acids (porphyra-334 and shinorine, etc.), polysaccharides, phytosterols, and pigments, but its anti-inflammatory effect and mechanism of anti-atopic dermatitis (AD) have not been elucidated. In this study, we investigate the anti-AD effect of P. yezoensis extract (PYE) on mRNA and protein levels of the pro-inflammatory chemokines, thymus, and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), in human HaCaT keratinocyte cells treated to interferon (IFN)-γ or tumor necrosis factor (TNF)-α (10 ng/mL each). The effect of the PYE on extracellular signal-regulated kinase (ERK) and other mitogen-activated protein kinases (MAPKs) was related to its suppression of TARC and MDC production by blocking NF-κB activation in HaCaT cells. Furthermore, astaxanthin and xanthophyll from P. yezoensis were identified as anti-AD candidate compounds. These results suggest that the PYE may improve AD and contained two carotenoids by regulating pro-inflammatory chemokines.

Banhasasim-tang (BHSST) is a Korean traditional herbal formula comprising eight medicinal herbs. The aim of the present study was to investigate the anti-inflammatory effect of BHSST using macrophage and keratinocyte cell lines. First, we evaluated the effects of BHSST on inflammatory mediator and cytokine production in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. BHSST markedly inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin- (IL-) 6. BHSST significantly suppressed the protein expression of toll-like receptor 4 (TLR4) and phosphorylated nuclear factor-kappa B (NF-κB) p65 in RAW 264.7 cells. Second, we examined whether BHSST influences the production of chemokines and STAT1 phosphorylation in tumor necrosis factor-α/interferon-γ TI-stimulated HaCaT keratinocytes. BHSST significantly suppressed the production of RANTES/CCL5, TARC/CCL17, MDC/CCL22, and IL-8 in TI-stimulated HaCaT cells. BHSST also suppressed TI-induced phosphorylation of STAT1 in HaCaT cells. These results suggest that BHSST may be useful as an anti-inflammatory agent, especially for inflammatory skin diseases.

Atopic dermatitis (AD) is a chronic inflammatory skin disease which is often associated with Staphylococcus aureus (S. aureus) colonization. S. aureus ingredients are potential ligands to activate the Toll-like receptor 2 (TLR2) and drive inflammatory cytokine or chemokine production. However, the role of TLR2-mediated chemokine expression in AD development has not been systematically investigated. In this study, we sought to determine the mode of TLR2-mediated chemokine expression in AD patients. Human peripheral blood mononuclear cells (PBMCs) were isolated from AD patients and healthy controls. Upon incubation with TLR2 ligands Pam3CSK4 and PGN, mRNA expression of chemokines, including CCL1, CCL5, CCL8, CCL13, CCL17, CCL18, CCL22, and CCL27, were determined by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The results showed that basal mRNA expression of CCL17 in PBMCs from AD patients was upregulated compared with healthy controls, while those of CCL8 and CCL13 were downregulated. When stimulated with TLR2 ligands, the mRNA expression of CCL5, CCL8, CCL13, CCL18, and CCL22 in PBMCs from AD patients was significantly higher than those from healthy controls. The different basal chemokine mRNA expression profiles indicate the different immune status in patients with AD compared with healthy controls. Excessive chemokine mRNA expression induced by TLR2 activation is associated with the development of AD.