Downregulation Of CCL2 Research Articles

Neutrophil infiltration associated genes on the prognosis and tumor immune microenvironment of lung adenocarcinoma.

The neutrophils exhibit both anti-tumor and pro-tumor effects in cancers. The correlation between neutrophils and tumor development in lung adenocarcinoma (LUAD) is still uncertain, possibly due to a lack of specific neutrophil infiltration evaluation methods. In this study, we identified 30 hub genes that were significantly associated with neutrophil infiltration in LUAD through data mining, survival analysis, and multiple tumor-infiltrating immune cells (TICs) analysis, including TIMER, CIBERSORT, QUANTISEQ, XCELL, and MCPCOUNTER. Consensus clustering analysis showed that these 30 hub genes were correlated with clinical features in LUAD. We further developed a neutrophil scoring system based on these hub genes. The neutrophil score was significantly correlated with prognosis and tumor immune microenvironment (TIME) in LUAD. It was also positively associated with PD-L1 expression and negatively associated with tumor mutational burden (TMB). When combined with the neutrophil score, the predictive capacity of PD-L1 and TMB for prognosis was significantly improved. Thus, the 30 hub genes might play an essential role in the interaction of neutrophils and LUAD, and the neutrophil scoring system might effectually assess the infiltration of neutrophils. Furthermore, we verified the expression of these 30 genes in the LUAD tumor tissues collected from our department. We further found that overexpressed TNFAIP6 and TLR6 and downregulated P2RY13, SCARF1, DPEP2, PRAM1, CYP27A1, CFP, GPX3, and NCF1 in LUAD tissue might be potentially associated with neutrophils pro-tumor effects. The following in vitro experiments demonstrated that TNFAIP6 and TLR6 were significantly overexpressed, and P2RY13 and CYP27A1 were significantly downregulated in LUAD cell lines, compared to BEAS-2B cells. Knocking down TNFAIP6 in A549 and PC9 resulted in the upregulation of FAS, CCL3, and ICAM-1, and the downregulation of CCL2, CXCR4, and VEGF-A in neutrophils when co-culturing with the conditioned medium (CM) from LUAD cells. Knocking down TNFAIP6 in LUAD also led to an elevated early apoptosis rate of neutrophils. Therefore, overexpressed TNFAIP6 in LUAD cancer cells might lead to neutrophils "N2" polarization, which exhibited pro-tumor effects. Further research based on the genes identified in this pilot study might shed light on neutrophils' effects on LUAD in the future.

Abstract 2192: HSF1 downregulation of CCL5 reduces CD8 + T cell trafficking in breast cancer

Abstract Breast cancer is the second leading cause of cancer-related death in women. The presence of cytotoxic immune cells, specifically CD8+ T cells, in breast tumors is associated with better patient outcomes. Understanding the mechanisms that regulate the infiltration of CD8+ T cells into breast tumors could improve the treatment and enhance patient overall survival. Utilizing a novel HSF1 activity gene signature, we observed that HSF1 activity was negatively associated with the presence of CD8+ T cells in breast cancer patients. Both CIBERSORT analysis of TCGA data as well as direct assessment of patient primary specimens demonstrated that patient tumors with high HSF1 activity had lower numbers of CD8+ T cells. To functionally test this relationship, HSF1 was knocked down in 4T1 breast cancer cells and grown orthotopically in BALB/c mice. Tumors with HSF1 knockdown showed lower tumor volumes and increased CD8+ T cell infiltration. We further tested the effect of CD8+ T cells on the growth of HSF1 knockdown tumors by depleting CD8+ T cells in BALB/c mice. HSF1 knockdown tumors were significantly larger with CD8+ T cell depletion suggesting a functional role for HSF1 to inhibit CD8+ T cell infiltration and protect the tumor from immune-mediated killing. To investigate the mechanism by which HSF1 affects CD8+ T cells, we investigated whether HSF1 affects chemotactic cytokines. We found that loss of HSF1 significantly increased secretion of CCL5, a known chemoattractant for CD8+ T cells. Loss of HSF1 also increased mRNA levels of CCL5, suggesting a transcriptional effect on CCL5. To test the importance of CCL5 in the phenotype of HSF1 knockdown, a transwell migration assay was performed wherein T cells from BALB/c mice were placed in the upper chamber of a transwell chamber and the lower chamber contained conditioned medium from 4T1 cells with knockdown of HSF1 with or without knockdown of CCL5. The lower chamber was subjected to flow cytometry for CD3/CD8 to identify CD8+ T cells and we observed that loss of HSF1 increased migration of CD8+ T cells and this was ablated with additional loss of CCL5. These results suggest CCL5 is a significant mediator for the recruitment of CD8+ T cells when HSF1 is lost in cancer cells. We propose the model whereby high HSF1 activity in breast cancer cells suppresses expression and secretion of the chemo-attractant cytokine CCL5 that ultimately leads to a decrease in CD8+ T cells in breast tumors microenvironment. Citation Format: Curteisha LeeAnn Jacobs, Sakhi Shan, Wen-Cheng Lu, Richard Carpenter. HSF1 downregulation of CCL5 reduces CD8 + T cell trafficking in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2192.

A Unique Cooperativity between Optineurin and CCL5 Triggers Early Cell Death during Herpes Simplex Virus-1 Infection of the Cornea

Abstract Induction of cell death is considered an effective strategy to control viral infections. Rapid killing of infected cells prevents propagation of viruses and thereby, effectively reduces the infection. Herpes Simplex Virus-1 (HSV-1) is known to cause ocular and nervous system infections. While studying an autophagy receptor protein, Optineurin (OPTN), we found evidence of a unique collaboration between OPTN and CCL5 to generate signal for early cell death in HSV-1 infected cells in the cornea. OPTN knockout mice tend to die due to rapid spread of HSV-1 infection to the brain causing encephalitis. Interestingly, the OPTN knockout animals also exhibit a marked reduction in CCL5 induction. Human transcriptomic profile dataset shows a strong positive correlation between CCL5 upregulation and OPTN expression during HSV infection. Our data shows that a well-coordinated activity of both, OPTN and CCL5, is needed to trigger an early cell death signal that mimics pyroptosis to prevent viral spread. Downregulation of CCL5 and/or OPTN causes interference with this signal and a corresponding delay in cell death. Our results provide new information on an anti-HSV1 chemokine signaling pathway that participates in innate immune control of HSV-1 spread and viral transmission.

Cannabinoid Receptor 2 Alters Social Memory and Microglial Activity in an Age-Dependent Manner.

Physiological brain aging is characterized by gradual, substantial changes in cognitive ability, accompanied by chronic activation of the neural immune system. This form of inflammation, termed inflammaging, in the central nervous system is primarily enacted through microglia, the resident immune cells. The endocannabinoid system, and particularly the cannabinoid receptor 2 (CB2R), is a major regulator of the activity of microglia and is upregulated under inflammatory conditions. Here, we elucidated the role of the CB2R in physiological brain aging. We used CB2R−/− mice of progressive ages in a behavioral test battery to assess social and spatial learning and memory. This was followed by detailed immunohistochemical analysis of microglial activity and morphology, and of the expression of pro-inflammatory cytokines in the hippocampus. CB2R deletion decreased social memory in young mice, but did not affect spatial memory. In fact, old CB2R−/− mice had a slightly improved social memory, whereas in WT mice we detected an age-related cognitive decline. On a cellular level, CB2R deletion increased lipofuscin accumulation in microglia, but not in neurons. CB2R−/− microglia showed an increase of activity markers Iba1 and CD68, and minor upregulation in tnfa and il6 expression and downregulation of ccl2 with age. This was accompanied by a change in morphology as CB2R−/− microglia had smaller somas and lower polarity, with increased branching, cell volume, and tree length. We present that CB2Rs are involved in cognition and age-induced microglial activity, but may also be important for microglial activation itself.

Mas receptor activation attenuates allergic airway inflammation via inhibiting JNK/CCL2-induced macrophage recruitment

BackgroundDefective absorption of acute allergic airway inflammation is involved in the initiation and development of chronic asthma. After allergen exposure, there is a rapid recruitment of macrophages around the airways, which promote acute inflammatory responses. The Ang-(1−7)/Mas receptor axis reportedly plays protective roles in various tissue inflammation and remodeling processes in vivo. However, the exact role of Mas receptor and their underlying mechanisms during the pathology of acute allergic airway inflammation remains unclear. ObjectiveWe investigated the role of Mas receptor in acute allergic asthma and explored its underlying mechanisms in vitro, aiming to find critical molecules and signal pathways. MethodsMas receptor expression was assessed in ovalbumin (OVA)-induced acute asthmatic murine model. Then we estimated the anti-inflammatory role of Mas receptor in vivo and explored expressions of several known inflammatory cytokines as well as phosphorylation levels of MAPK pathways. Mas receptor functions and underlying mechanisms were studied further in the human bronchial epithelial cell line (16HBE). ResultsMas receptor expression decreased in acute allergic airway inflammation. Multiplex immunofluorescence co-localized Mas receptor and EpCAM, indicated that Mas receptor may function in the bronchial epithelium. Activating Mas receptor through AVE0991 significantly alleviated macrophage infiltration in airway inflammation, accompanied with down-regulation of CCL2 and phosphorylation levels of MAPK pathways. Further studies in 16HBE showed that AVE0991 pre-treatment inhibited LPS-induced or anisomycin-induced CCL2 increase and THP-1 macrophages migration via JNK pathways. ConclusionOur findings suggested that Mas receptor activation significantly attenuated CCL2 dependent macrophage recruitments in acute allergic airway inflammation through JNK pathways, which indicated that Mas receptor, CCL2 and phospho-JNK could be potential targets against allergic airway inflammation.

Type 2 Innate Lymphoid Cells Induce CNS Demyelination in an HSV-IL-2 Mouse Model of Multiple Sclerosis.

SummaryWe previously reported that infection of different mouse strains with a recombinant HSV-1 expressing IL-2 (HSV-IL-2) caused CNS demyelination. Histologic examination of infected IL-2rα−/−, IL-2rβ−/−, and IL-2rγ−/− mice showed demyelination in the CNS of IL-2rα−/− and IL-2rβ−/− mice but not in the CNS of IL-2rγ−/−-infected mice. No demyelination was detected in mice infected with control virus. IL-2rγ−/− mice that lack type 2 innate lymphoid cells (ILC2s) and ILCs, play important roles in host defense and inflammation. We next infected ILC1−/−, ILC2−/−, and ILC3−/− mice with HSV-IL-2 or wild-type (WT) HSV-1. In contrast to ILC1−/− and ILC3−/− mice, no demyelination was detected in the CNS of ILC2−/−-sinfected mice. However, transfer of ILC2s from WT mice to ILC2−/− mice restored demyelination in infected recipient mice. CNS demyelination correlated with downregulation of CCL5 and CXCL10. This study demonstrates that ILC2s contribute to HSV-IL-2-induced CNS demyelination in a mouse model of multiple sclerosis.

Effect of Organosulfide Diallyl Trisulfide (DATS) from Garlic on Racially Different Triple Negative Breast Cancer Cells

In triple negative breast cancer (TNBC), inflammation and immune response play a major role in the progression and metastasis of the disease. TNF‐α is a first response cytokine in the developing of the tumor and is released by macrophages to destroy the tumor due to its toxic effect on cancer cells. If TNF‐α is unable to destroy the cancer cells, macrophages begin to mimic the characteristics of cancer, eventually turning into tumor‐associated macrophages (TAM). Meanwhile, diallyl trisulfide (DATS), an organosulfide found in garlic, has previously been reported to be effective in chemoprevention, apoptosis, and cell cycle arrest in DNA damaged normal breast epithelial cells, breast cancer cells, as well as other cancers. The objective of this study is to investigate the effects of the natural compound DATS on the release of TNF‐α‐induced tumor promoting cytokines in TNBC cells, specifically MDA‐MB‐231 and MDA‐MB‐468 (representing Caucasians and African Americans, respectively), and to evaluate those specifically associated with TAM recruitment. Cell viability, cytokine arrays, and specific ELISAs were performed to investigate DATS effects on TNBC cells. In cell viability studies, DATS showed a dose‐response effect in concentrations ranging from 0–200 μM in MDA‐MB‐231 and MDA‐MB‐468 cells. Based on these results, we established a concentration of 75 μM to be used for further experimentation. The cytokine arrays showed that TNF‐α (40 ng/mL) induced the up‐regulation of monocyte chemoattractant protein‐1 (MCP‐1/CCL2) in Caucasians and African Americans cell lines; however, interleukin‐6 (IL‐6) was only up regulated in MDA‐MB‐231 cells. DATS treatment showed to be effective in inhibiting CCL2 expression in MDA‐MB‐231, but not in MDA‐MB‐468 cells. In addition, IL‐6 also showed a down regulation after DATS treatment in Caucasian cells. In order to validate the results from the arrays, ELISA quantitative assays specific for CCL2 and IL‐6 were performed. The results confirmed that TNF‐α induces expression of CCL2 expression in both breast cancer cell lines, and DATS treatment leads to the down regulation of CCL2 in MDA‐MB‐231 only, with no significant effect in MDA‐MB‐468 cells. The data from this study showed that DATS has the ability to modulate the immune response involved with TAM and cancer progression through CCL2 down regulation in MDA‐MB‐231, but not in MDA‐MB‐468 cells. These findings may also provide a better understanding of the poor therapeutic response in African American patients with TNBC.Support or Funding InformationSupported by a grant from NIMHD U54 007582

CCL20 mediates the anti-tumor effect of vitamin D3 in p38MAPK/NF-κB signaling in colitis-associated carcinogenesis.

Vitamin D3 is beneficial in ameliorating or preventing inflammation and carcinogenesis. CCL20 is a potential therapeutic target in carcinogenesis, which mediates the protective effect of vitamin D or vitamin D analogue in autoimmune and cancer diseases. Here we aim to evaluate whether vitamin D3 plays a protective role in colitis-associated colorectal cancer (CAC) by affecting CCL20 and the molecular mechanism. Administration of azoxymethane (AOM) followed with dextran sulfate sodium (DSS) was used to simulate CAC in mouse. After 5-day DSS treatment, vitamin D3 supplementation was for 9 weeks at 60 IU/g/w. We found that dietary vitamin D3 significantly reduced the tumor number and tumor burden in mouse. In-vivo and -vitro, vitamin D3 reduced the levels of CCL20, phospho-p38 MAPK (p-p38) and phospho-NF-κB p65 (p-p65), and the transcriptional activity of NF-κB. Further studies showed that CCL20 mediated the inhibition of vitamin D3 in p38MAPK-mediated NF-κB signaling in vitro. Taken together, vitamin D3 effectively suppressed colonic carcinogenesis in AOM-DSS mouse model. Downregulation of CCL20 may contribute to the preventive effect of vitamin D3 on NF-κB activity. It may merit further clinical investigation as a therapeutic agent against CAC in humans.

CITED2 attenuates macrophage recruitment concordant with the downregulation of CCL20 in breast cancer cells.

The transcriptional co-regulator Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2 (CITED2) may promote breast tumor growth; however, the mechanisms by which its effects are mediated remain to be fully elucidated. Tumor-associated macrophages serve an important function in tumor development and progression and are recruited by chemotactic factors produced by cells within the tumor microenvironment. The present study assessed the effects of CITED2 silencing on macrophage recruitment in two xenograft mouse models of human breast cancer, one in which tumor growth was sensitive to CITED2 silencing (MDA-MB-231) and one in which it was insensitive (MDA-MB-468). The present study identified that silencing CITED2 significantly attenuated macrophage infiltration in MDA-MB-231 but not MDA-MB-468 orthotopic tumors, concordant with its effect on tumor growth. Correspondingly, conditioned media obtained from CITED2-silenced MDA-MB-231 cells exhibited a significantly decreased ability to induce macrophage recruitment by Transwell migration assay, whereas the chemotactic effect of MDA-MB-468 conditioned media was unaffected. Examining the expression of macrophage chemoattractants within orthotopic tumors and tumor cell-conditioned media revealed a significant decrease in C-C motif chemokine ligand (CCL)20 mRNA and protein expression following CITED2-silencing in MDA-MB-231 cells, compared with that in cells transfected with scramble shRNA. However, mRNA and protein expression was unaffected by CITED2-silencing in MDA-MB-468 cells. Furthermore, chromatin immunoprecipitation analysis revealed that CITED2 was localized to the CCL20 promoter in MDA-MB-231 cells, suggesting that it serves a direct function in its regulation, which is consistent with the effect of CITED2 silencing on CCL20 expression. Lastly, neutralizing CCL20 in the conditioned media of MDA-MB-231 cells significantly inhibited macrophage recruitment. Collectively, these results suggest that CITED2 is involved in modulating macrophage recruitment, representing a novel mechanism through which it may influence tumor growth. This may be partly mediated by regulating tumor cell production of the chemokine CCL20.

Oral Administration of Lactobacillus rhamnosus GG Ameliorates Salmonella Infantis-Induced Inflammation in a Pig Model via Activation of the IL-22BP/IL-22/STAT3 Pathway

The high rate of Salmonella enterica serovar Infantis (S. Infantis) infection poses significant risk for the development of non-typhoidal Salmonella gastroenteritis. However, efficient strategies to prevent or treat the infection remain elusive. Here, we explored the effect of the probiotic Lactobacillus rhamnosus GG (LGG) administration in preventing S. Infantis infection in a pig model. Probiotic LGG (1.0 × 1010 CFU/day) was orally administered to newly weaned piglets for 1 week before S. Infantis challenge. LGG pretreatment reduced the severity of diarrhea and alleviated intestinal inflammation caused by S. Infantis. Pre-administration of LGG excluded Salmonella from colonization of the jejunal mucosa but increased the abundance of Bifidobacterium in the feces. LGG promoted the expansion of CD4+ T-bet+ IFNγ+ T cells but attenuated S. Infantis-induced increases in the percentage of CD4+ IFNγ+ T cells and serum interleukin (IL)-22 levels in peripheral blood after S. Infantis challenge. In the small intestine, LGG pretreatment upregulated expression of the transcription factor T-bet but downregulated the S. Infantis-induced increase of CD4+ IFNγ+ T cells in Peyer's patches and IL-7Rα expression in the jejunum. Notably, LGG-treated pigs had enhanced expression of IL-22 and activated STAT3 in the ileum in response to S. Infantis infection. Pretreatment of pigs with LGG also elevated intestinal IL-22-binding protein production in response to S. Infantis challenge. In contrast, LGG consumption reduced the S. Infantis-induced increase in the number of CCL20-expressing cells in the jejunum. Our results suggest that the mechanism by which LGG ameliorates the intestinal inflammation caused by S. Infantis involves the upregulation of T-bet, activation of STAT3, and downregulation of CCL20.

Ellagic acid ameliorates cuprizone-induced acute CNS inflammation via restriction of microgliosis and down-regulation of CCL2 and CCL3 pro-inflammatory chemokines.

Ellagic acid (EA) is a natural phenol antioxidant with various therapeutic activities. However, the efficacy of EA has not been examined in neuro-inflammatory conditions. Microglia making the innate immune system of the central nervous system (CNS) and are imperative cellular mediators of neuro-inflammatory processes. In this study, neuro-protective effects of EA on cuprizone (Cup)-induced acute CNS inflammation evaluated. C57BL/6J mice were fed with chow containing 0.2 % Cup for 3 weeks to induce acute neuro-inflammation predominantly in the corpus callosum (CC). EA was administered at different doses (40 or 80 mg/kg body weight/day/i.p) from the first day of the Cup diet. Microglia activation (microgliosis) and expression of microglia related chemokines during Cup challenge were examined. Results shows that EA significantly decreased the number of activated microglia cells (Iba-1+ cells) and also restricted proliferation of these cell population (Iba-1+/Ki67+ cells) in dose dependent manner. Consequently, concentration of microglial pro-inflammatory chemokines including monocyte chemoattractant protein-1/Chemokine (C-C motif) ligand 2 (MCP-1/CCL2), and macrophage inflammatory protein 1-alpha/Chemokine (C-C motif) ligand 3 (MIP-1-α/CCL3) dramatically reduced in CC after EA treatment. According to this results, we conclude that EA is a suitable therapeutic agent for moderation brain damages in neuro-inflammatory diseases.

Effect of the Ketone Body Beta-Hydroxybutyrate on the Innate Defense Capability of Primary Bovine Mammary Epithelial Cells.

Negative energy balance and ketosis are thought to cause impaired immune function and to increase the risk of clinical mastitis in dairy cows. The present in vitro study aimed to investigate the effect of elevated levels of the predominant ketone body β-hydroxybutyrate on the innate defense capability of primary bovine mammary epithelial cells (pbMEC) challenged with the mastitis pathogen Escherichia coli (E. coli). Therefore, pbMEC of healthy dairy cows in mid- lactation were isolated from milk and challenged in culture with 3 mM BHBA and E. coli. pbMEC stimulated with E. coli for 6 h or 30 h showed an up-regulation of several innate immune genes, whereas co-stimulation of pbMEC with 3 mM BHBA and E. coli resulted in the down-regulation of CCL2, SAA3, LF and C3 gene expression compared to the challenge with solely the bacterial stimulus. These results indicated that increased BHBA concentrations may be partially responsible for the higher mastitis susceptibility of dairy cows in early lactation. Elevated levels of BHBA in blood and milk during negative energy balance and ketosis are likely to impair innate immune function in the bovine mammary gland by attenuating the expression of a broad range of innate immune genes.

Therapeutic Effects of Erythroid Differentiation Regulator 1 on Imiquimod-Induced Psoriasis-Like Skin Inflammation.

Psoriasis is a common skin disease accompanied by chronic inflammation. In previous studies, erythroid differentiation regulator 1 (ERDR1) was shown to have a negative correlation with proinflammatory cytokine IL-18. However, the role of ERDR1 in the inflammatory skin disease psoriasis has not been evaluated. In this study, to investigate the role of ERDR1 in psoriasis, recombinant ERDR1 was injected intraperitoneally into a psoriasis mouse model. Recombinant ERDR1 (rERDR1) significantly alleviated the symptoms of psoriasis-like skin inflammation and reduced the mRNA of various psoriasis-related markers, including keratin 14, S100A8, and Th17-related cytokines IL-17 and IL-22, suggesting that rERDR1 exerts therapeutic effects on psoriasis via the regulation of Th17 functions. Additionally, the expression of CCL20, a well-known Th17 attracting chemokine, was determined. CCL20 expression significantly decreased in the rERDR1-injected group compared with the vehicle (PBS)-injected group. CCR6 expression in the psoriatic lesional skin was also decreased by rERDR1 administration, implying the inhibition of CCR6-expressing Th17 cell chemotaxis via the downregulation of CCL20. Taken together, this study provides the first evidence that ERDR1 may be a potential therapeutic target for psoriasis.

The bisphosphonate zoledronic acid regulates key angiogenesis-related genes in primary human gingival fibroblasts

BackgroundOsteonecrosis of the jaws is recognised as a serious complication for patients receiving bisphosphonates. The anti-angiogenic effects of bisphosphonates have been implicated in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The purpose of this study was to determine the effects of zoledronic acid on cultured human gingival fibroblasts in relation to the modulation of genes associated with angiogenic regulation. MethodsPrimary cultures of fibroblasts were developed from gingival tissues excised during crown-lengthening surgery from three patients. Cells were cultured with and without 30μM zoledronic acid for 6, 12 and 24h and cellular proliferation and migration investigated using CellTiter-Blue and scratch wound assays, respectively. Gene expression was determined using semi-quantitative PCR array technology that allowed the analysis of 84 pathway-focused genes known to be important in the regulation of angiogenesis. ResultsZoledronic acid increased the proliferation of the gingival fibroblasts in a dose dependent manner with 12 and 24h of exposure. Scratch wounding of the human gingival fibroblasts and treatment with increasing doses and time exposure to zoledronic acid (ZA) inhibited their migration. Statistically significant increases in gene expression were found for RHOB, VEGFA, CD55 and BMP2 (p≤0.05) in response to 30μM zoledronic acid. CCL2 and IL6 genes were significantly downregulated (p≤0.05). ConclusionsThe regulation of the prenylated protein RHOB in this study was consistent with the known effects of zoledronic acid on the mevalonate pathway. The down regulation of CCL2 and IL6 and the upregulation of CD55 may be associated with suppression of inflammation. An increase in VEGFA and BMP2 gene expression suggests that fibroblasts respond to zoledronic acid by producing a proangiogenic environment.

Lenalidomide Promotes a Pro-Inflammatory Switch of Nurse-like Cells Derived from Chronic Lymphocytic Leukemia

Lenalidomide is an immunomodulatory agent clinically active in CLL patients. The specific mechanism of action is still undefined, but includes the modulation of microenvironment. In CLL patients, nurse-like cells (NLCs) differentiate from CD14+ mononuclear cells and nurture/protect CLL cells from apoptosis. NLCs resemble M2 macrophages with potent immunosuppressive functions.We examined the ability of lenalidomide to mediate a pro-inflammatory switch of NLCs affecting the protective microenvironment generated by CLL into tissues.NLCs were generated in presence or absence of lenalidomide: cell surface markers, phagocytosis and induction of T cell proliferation were analyzed after 10 days. NLCs activation was measured using a yellow tetrazolium MTT assay after 5 days of culture and NLCs proliferation was measured by CFSE staining. Microarray-based gene expression profiles of NLCs treated or not with lenalidomide were evaluated after 10 days and data were confirmed by real time PCR.Lenalidomide modifies the immunophenotype and the biological characteristics of NLCs. First, treatment with lenalidomide 0.5µM and 1µM increased the number of NLCs to 268% and 309% compared to untreated control (100%) respectively (p<0.05). To explain the high number of NLCs generated by lenalidomide, we analyzed cell activation and proliferation. We observed a strong increase in NLCs activation after treatment with lenalidomide that correlated with stimulation of NLCs proliferation from 44% to 55% (% of dividing cells) (p<0.05).In contrast with the high number of NLCs generated in presence of lenalidomide, we found that NLCs lost the ability to nurture and protect CLL cell from apoptosis reducing their viability from 54.2% to 44.5% (p<0.05), but they strongly attracted CLL cells reaching an increase of adhesion to 227% and 212% with the addition of 0.5µM and 1µM lenalidomide (p<0.05). Accordingly with these results, we investigated the ability of lenalidomide to interfere with leukemia-promoting activity of NLCs. Lenalidomide improved the ability of NLCs to engulf zymogen particles to 141% and 155% with dosage of 0.5µM and 1µM compared to control (100%) (p<0.05), further confirmed analyzing the uptake of FITC-dextran by NLCs that increased to 252% and 356% compared to untreated control (p<0.01). Moreover lenalidomide strongly improved the ability of NLCs to induce T cells proliferation from 19.5% to 35.0% (% of dividing cells) (p<0.05). Lastly, gene expression profiling showed a switch to a pro-inflammatory profile in NLCs induced by treatment with lenalidomide involving a modulation of pivotal genes for the immune response, activation/proliferation of T cells, complement activation as well as regulation of cellular movement, cytokine and chemokine activation. In particular, down-regulation of CCL2, CXCL12, IL-10, CD163 and up-regulation of IL-2 were apparent.Collectively, our data provide new insights into the mechanism of action of lenalidomide that reverts NLCs polarization from M2 to M1-skewed phenotype affecting the supporting and protective microenvironment generated by CLL into tissues. DisclosuresMaffei:Celgene: support for travel to congresses Other. Marasca:Celgene: Honoraria, Research Funding.

Uncontrollable stress causes downregulation of CCL2 in colon of the endometriosis rat model (898.4)

Background: Endometriosis is characterized by the growth of endometrial tissue in areas such as the peritoneal cavity and colon causing pain and discomfort. We have previously demonstrated that rats with explants of endometrial tissue in the peritoneal cavity subjected to stress showed a significant increase in the size of endometrial lesions. Chemokine ligand 2 (CCL2) has been found to be increased in plasma in women with endometriosis perhaps contributing to the inflammatory process but the effects of stress on this cytokine are still unclear. Aim: Examine the relationship between CCL2 expression and stress in a rat model of endometriosis. Methods: Endometriosis was surgically induced in female Sprague‐Dawley rats by transplanting uterine tissue onto the intestinal mesentery. Sham rats had sutures only. Animals were exposed to either an uncontrollable (ENDO‐UN) or controllable (ENDO‐C) stress protocol for 10 days, or no stress (ENDO‐NS). At sacrifice (day 60), all rats were examined for presence of endometrial cysts, and colons were collected for mRNA extraction and histology analysis. The relative expression of CCL2 was measured by PCR, and macrophage numbers were determined by immunofluorescence for CD68. Results: As might be expected CCL2 transcript levels were higher in colonic tissue from animals with ENDO‐NS compared to sham however they were significantly decreased in ENDO‐UN (p&lt;0.05). mRNA expression of CCL2 positively correlated with CD68 positive cells (p&lt;0.05). Surprisingly, higher levels of corticosterone and microscopic damage accompanied the lower levels of CCL2 in stressed animals. Conclusion: In this model uncontrollable stress decreases colonic CCL2 expression, possibly via corticosterone‐induced immunosuppression. It is unclear whether decreased CCL2 and/or increased corticosterone levels induced by stress contribute to an increased severity of colonic damage.Grant Funding Source: R15A006373, RR003050/MD007579

Galectin-9 ameliorates anti-GBM glomerulonephritis by inhibiting Th1 and Th17 immune responses in mice

Antiglomerular basement membrane glomerulonephritis (anti-GBM GN) is a Th1- and Th17-predominant autoimmune disease. Galectin-9 (Gal-9), identified as the ligand of Tim-3, functions in diverse biological processes and leads to the apoptosis of CD4(+)Tim-3(+) T cells. It is still unclear how Gal-9 regulates the functions of Th1 and Th17 cells and prevents renal injury in anti-GBM GN. In this study, Gal-9 was administered to anti-GBM GN mice for 7 days. We found that Gal-9 retarded the increase of Scr, ameliorated renal tubular injury, and reduced the formation of crescents. The infiltration of Th1 and Th17 cells into the spleen and kidneys significantly decreased in Gal-9-treated nephritic mice. The reduced infiltration of Th1 and Th17 cells might be associated with the downregulation of CCL-20, CXCL-9, and CXCL-10 mRNAs in the kidney. In parallel, the blood levels of IFN-γ and IL-17A declined in Gal-9-treated nephritic mice at days 21 and 28. In addition, an enhanced Th2 cell-mediated immune response was observed in the kidneys of nephritic mice after a 7-day injection of Gal-9. In conclusion, the protective role of Gal-9 in anti-GBM GN is associated with the inhibition of Th1 and Th17 cell-mediated immune responses and enhanced Th2 immunity in the kidney.

Intake of red wine in different meals modulates oxidized LDL level, oxidative and inflammatory gene expression in healthy people: a randomized crossover trial.

Several studies have found that adherence to the Mediterranean Diet, including consumption of red wine, is associated with beneficial effects on oxidative and inflammatory conditions. We evaluate the outcome of consumption of a McDonald's Meal (McD) and a Mediterranean Meal (MM), with and without the additive effect of red wine, in order to ascertain whether the addition of the latter has a positive impact on oxidized (ox-) LDL and on expression of oxidative and inflammatory genes. A total of 24 subjects were analyzed for ox-LDL, CAT, GPX1, SOD2, SIRT2, and CCL5 gene expression levels, before and after consumption of the 4 different meal combinations with washout intervals between each meal. When red wine is associated with McD or MM, values of ox-LDL are lowered (P < 0.05) and expression of antioxidant genes is increased, while CCL5 expression is decreased (P < 0.05). SIRT2 expression after MM and fasting with red wine is significantly correlated with downregulation of CCL5 and upregulation of CAT (P < 0.001). GPX1 increased significantly in the comparison between baseline and all conditions with red wine. We highlighted for the first time the positive effect of red wine intake combined with different but widely consumed meal types on ox-LDL and gene expression. Trial Registration. This trial is registered with ClinicalTrials.gov NCT01890070.

Simvastatin reduces CCL2 expression in monocyte-derived cells by induction of a repressive CCL2 chromatin state

Statins exert anti-inflammatory characteristics, besides their lipid lowering properties, and may display beneficial effects for the treatment of inflammatory diseases. One possible explanation is that statins interfere in the deregulated gene transcription patterns associated with immune-mediated diseases, although the precise mechanism is not fully understood. Besides gene regulatory proteins, epigenetic mechanisms play an important role in the orchestration of gene expression. Disturbances in the tightly controlled epigenetic mechanisms influence the cellular portrait of expressed genes resulting in the protein dysfunctions found in many inflammatory diseases. In this study, we found that simvastatin reduces secretion and gene expression of CCL2 in monocyte-derived immature dendritic cells and in type 1 macrophages, which is accompanied by increased levels of the 3meK27H3 and 3meK9H3 repressive histone marks and decreased levels of the permissive histone marks AcH3 and 3meK4H3 in CCL2 promoter chromatin. The repressive chromatin status of the CCL2 promoter region affected recruitment of the NF-κB p65 subunit, which controls CCL2 transcription. The down-regulation of CCL2 in these immune cells may therefore impact their chemotactic activity and reduce their recruitment to sites of tissue injury.

Abstract A063: Targeting the CCL2 chemokine pathway in breast tumors through intratumoral delivery of calcium cross linked TAT peptide: siRNA complexes

Abstract Increased expression of CCL2 correlates with poor patient survival and development of invasive breast cancer. CCL2 regulates mammary tumor progression by recruitment of tumor promoting macrophages. Our laboratory and others have shown that CCL2 signaling mediates stromal: tumor interactions to regulate breast cancer cell survival, invasion and metastasis. While the CCL2 signaling represents a novel and potentially effective therapeutic target in invasive breast cancer, it is necessary to develop new tools to specifically inhibit CCL2 signaling in vivo. siRNA knockdown could specifically target CCL2, but is unstable and toxic when delivered in vivo. The HIV-1 derived TAT peptide penetrates cell plasma membranes at high efficiency and contains nuclear translocation signals to enhance siRNA delivery to the nucleus. Recent studies from our research team have shown that CaCl2 condenses TAT/nucleic acids to nanoparticles facilitating cellular uptake. The goals of these studies are to design TAT nanoparticle formulas to target CCL2 protein expression in breast tumors. Using an in vitro luciferase reporter system, we identified specific N/P (TAT:siRNA) ratios and CaCl2 concentrations that would efficiently deliver siRNAs to PyVmT mammary tumor cells. Utilizing a novel ex vivo mammary tumor system, TAT/CaCl2 nanocomplexes carrying GFP plasmid or Cy3 labeled siRNAs were then injected into PyVmT mammary tumors. We observed visible GFP expression and Cy3 labeled siRNA uptake in over 50% of the PyVmT mammary tumor. Flow cytometry analysis showed that low N/P ratio resulted in efficient TAT/CaCl2/siRNA nanoparticle uptake in tumor epithelial cells. Normal mammary tissues did not show significant uptake of TAT nanoparticles. CCL2 knockdown was observed in tumor epithelial cells, but not stromal cells as determined by flow cytometry. Similar results were observed in cultured cells. At low N/P ratio, PyVmT tumor cells showed a 50% reduction in CCL2 expression upon delivery of TAT/CaCl2 nanoparticles carrying CCL2 siRNAs, while there was no significant effect on stromal cells as determined by ELISA. Downregulation of CCL2 in the tumor cells significantly decreased proliferation as determined by flow cytometry analysis. Importantly, treatment with TAT/siRNA nanoparticle did not significantly affect viability of normal cells. These data indicate this nanoparticle strategy represents a novel, feasible approach to target the CCL2 signaling in breast cancer, with important implications on therapeutic targeting. Citation Format: Wei Bin Fang, Nabil Alhakamy, Cory Berkland, Nikki Cheng. Targeting the CCL2 chemokine pathway in breast tumors through intratumoral delivery of calcium cross linked TAT peptide: siRNA complexes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A063.