Immunomodulatory Effects of Cinobufagin on Murine Lymphocytes and Macrophages

Cinobufagin (CBG), a major bioactive component of the traditional Chinese medicine ChanSu, has been reported to have potent pharmacological activity. In this study, we aimed to elucidate the effects of CBG on the activity of immune cells in mice. Peritoneal macrophages and splenocytes from mice were prepared and cultured in RPMI1640 supplemented with 10% fetal bovine serum. Concanavalin (ConA), lipopolysaccharide (LPS), and CBG (0.0125, 0.05, 0.15, or 0.25 μg/mL) were added to the culture medium, and the phagocytic activity of macrophages was detected by MTT assays. Additionally, lymphocyte secretion of interleukin- (IL-)2 and IL-10 was detected by enzyme-linked immunosorbent assay, and the cell cycle distribution and cell surface markers were detected by flow cytometry. Our results demonstrated that CBG promoted lymphocyte proliferation; this effect was suppressed by combined treatment with ConA or LPS. Moreover, CBG also significantly improved the CD4+/CD8+ratio in spleen lymphocytes and increased the percentage of spleen lymphocytes in the S phase. Finally, we found that CBG enhanced the secretion of IL-2 and IL-10 and increased the phagocytosis ability of macrophages. In summary, CBG could enhance activity of immune cells.

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A TLR4 agonist improves immune checkpoint blockade treatment by increasing the ratio of effector to regulatory cells within the tumor microenvironment

Scientific Reports ◽

10.1038/s41598-021-94837-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

A. Farias ◽

A. Soto ◽

F. Puttur ◽

C. J. Goldin ◽

S. Sosa ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Growth ◽

Therapeutic Effect ◽

Immune Cells ◽

Immune Checkpoint ◽

Combined Treatment ◽

Immune Checkpoint Blockade ◽

Checkpoint Blockade ◽

Regulatory Cells ◽

Ifn Γ

AbstractBrucella lumazine synthase (BLS) is a homodecameric protein that activates dendritic cells via toll like receptor 4, inducing the secretion of pro-inflammatory cytokines and chemokines. We have previously shown that BLS has a therapeutic effect in B16 melanoma-bearing mice only when administered at early stages of tumor growth. In this work, we study the mechanisms underlying the therapeutic effect of BLS, by analyzing the tumor microenvironment. Administration of BLS at early stages of tumor growth induces high levels of serum IFN-γ, as well as an increment of hematopoietic immune cells within the tumor. Moreover, BLS-treatment increases the ratio of effector to regulatory cells. However, all treated mice eventually succumb to the tumors. Therefore, we combined BLS administration with anti-PD-1 treatment. Combined treatment increases the outcome of both monotherapies. In conclusion, we show that the absence of the therapeutic effect at late stages of tumor growth correlates with low levels of serum IFN-γ and lower infiltration of immune cells in the tumor, both of which are essential to delay tumor growth. Furthermore, the combined treatment of BLS and PD-1 blockade shows that BLS could be exploited as an essential immunomodulator in combination therapy with an immune checkpoint blockade to treat skin cancer.

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Effect ofTityus serrulatusvenom on cytokine production and the activity of murine macrophages

Mediators of Inflammation ◽

10.1080/09629350210308 ◽

2002 ◽

Vol 11 (1) ◽

pp. 23-31 ◽

Cited By ~ 27

Author(s):

Vera L. Petricevich

Keyword(s):

Cytokine Production ◽

Peritoneal Macrophages ◽

Interferon Γ ◽

Enzyme Linked Immunosorbent Assay ◽

No Production ◽

Dependent Manner ◽

Immune Functions ◽

Macrophage Activity ◽

Ifn Γ

The purpose of this study was to investigate the effects ofTityus serrulatusvenom (TSV) on murine peritoneal macrophages evaluated in terms of activation. The effects of crude TSV were analysed by detection of cytokines, oxygen intermediate metabolites (H2O2) and nitric oxide (NO) in supernatants of peritoneal macrophages. Several functional bioassays were employed including anin vitromodel for envenomating: cytotoxicity of TSV was assessed using the lyses percentage. Tumor necrosis factor (TNF) activity was assayed by measuring its cytotoxic activity on L-929 cells, and interleukin-6 (IL-6) and interferon-γ (IFN-γ) were assayed by enzyme-linked immunosorbent assay, whereas NO levels were detected by Griess colorimetric reactions in culture supernatant of macrophages incubated with TSV and subsequently exposed to either lipopolysaccharide or IFN-γ. Incubation of macrophages with TSV increased production of IL-6 and IFN-γ in a dose-dependent manner. TNF production was not detected in supernatants treated with TSV at any concentration. The increase in IL-6 secretion was not associated with concentration-dependent cytoxicity of TSV on these cells. These data suggest that the cytotoxicity does not appear to be the main cause of an increased cytokine production by these cells. Although NO is an important effector molecule in macrophage microbicidal activity, the inducing potential of the test compounds for its release was found to be very moderate, ranging from 125 to 800 mM. Interestingly, NO levels of peritoneal macrophages were increased after IFN-γ. Moreover, NO production had an apparent effect on macrophage activity. The results obtained here also shown that the TSV induces an important elevation in H2O2release. These results combined with NO production suggest that TSV possesses significant immunomodulatory activities capable of stimulating immune functionsin vitro.

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Anticancer Effects of Tacrolimus on Induced Hepatocellular Carcinoma in Mice

Current Molecular Pharmacology ◽

10.2174/1874467214666210531164546 ◽

2021 ◽

Vol 14 ◽

Author(s):

Shireen Sami Mahmoud ◽

Samia Hussein ◽

Hayam Rashed ◽

Eman M. A. Abdelghany ◽

Alaa I. Ali

Keyword(s):

Hepatocellular Carcinoma ◽

Cyclin D1 ◽

Liver Enzymes ◽

Combined Treatment ◽

Treated Group ◽

Nuclear Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Gamma Glutamyl Transferase ◽

Anticancer Effects ◽

Glutamyl Transferase

Background: Tacrolimus is a calcineurin inhibitor widely used for immunological disorders. However, there is a significant controversy regarding its effect on the liver. The present study was conducted to evaluate the anticancer effects of tacrolimus on an induced murine hepatocellular carcinoma (HCC) model and its possible hepatotoxicity at standard therapeutic doses. Methods: Fifty-four male mice were divided into five groups: a control healthy group, control HCC group, tacrolimus-treated group, doxorubicin (DOXO)-treated group, and combined tacrolimus- and DOXO-treated group. The activity of liver enzymes, including alkaline phosphatase, gamma-glutamyl transferase, lactate dehydrogenase, alanine transaminase, and aspartate transaminase, was determined. Serum vascular endothelial growth factor (VEGF) was measured using an enzyme-linked immunosorbent assay. A quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to measure the expression of proliferating cell nuclear antigen (PCNA), Bax, and p53 mRNA. Immunohistochemical staining for cyclin D1 and VEGF was performed. Results: Mice that received combined treatment with tacrolimus and DOXO exhibited the best improvement in all parameters when compared with the groups that received DOXO or tacrolimus alone (p < 0.001). Conclusion: The combination of DOXO and tacrolimus was more effective in the management of HCC compared with either agent alone. This improvement was detected by the reduction of liver enzymes and the improvement of the histopathological picture. The involved mechanisms included significant apoptosis induction demonstrated by upregulation of bax along with a reduction in angiogenesis demonstrated by downregulation of VEGF. This was accompanied by inhibition of cell cycle progression mediated by upregulated p53 and downregulated PCNA and cyclin D1.

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Acorus gramineusand and Euodia ruticarpa Steam Distilled Essential Oils Exert Anti-Inflammatory Effects Through Decreasing Th1/Th2 and Pro-/Anti-Inflammatory Cytokine Secretion Ratios In Vitro

Biomolecules ◽

10.3390/biom10020338 ◽

2020 ◽

Vol 10 (2) ◽

pp. 338 ◽

Cited By ~ 4

Author(s):

Tzu-He Yeh ◽

Jin-Yuarn Lin

Keyword(s):

Essential Oils ◽

Immune Cells ◽

Cytokine Secretion ◽

Enzyme Linked Immunosorbent Assay ◽

Total Flavonoids ◽

Immune Functions ◽

Immune Balance ◽

Tnf Α ◽

Th2 Polarization ◽

Anti Inflammatory

To clarify the effects of steam distilled essential oils (SDEO) from herbs used in traditional Chinese medicine on immune functions, two potential herbs, Acorus gramineusand (AG) and Euodia ruticarpa (ER) cultivated in Taiwan, were selected to assess their immunomodulatory effects using mouse primary splenocytes and peritoneal macrophages. T helper type 1 lymphocytes (Th1) (IL-2), Th2 (IL-5), pro-inflammatory (TNF-α) and anti-inflammatory (IL-10) cytokines secreted by correspondent immune cells treated with SDEO samples were determined using enzyme-linked immunosorbent assay. The total amounts of potential phytochemicals, including total flavonoids, polyphenols and saponins, in these two selected SDEOs were measured and correlated with cytokine levels secreted by immune cells. Our results evidenced that ER SDEO is rich in total flavonoids, polyphenols and saponins. Treatments with AG and ER SDEO significantly (p < 0.05) increased IL-5/IL-2 (Th2/Th1) cytokine secretion ratios by splenocytes, suggesting that both AG and ER SDEO have the Th2-polarization property and anti-inflammatory potential. In addition, AG and ER SDEO, particularly ER SDEO, markedly decreased TNF-α/IL-10 secretion ratios by macrophages in the absence or presence of lipopolysaccharide (LPS), exhibiting substantial effects on spontaneous and LPS-induced inflammation. Significant correlations were found between the total polyphenols, flavonoids or saponins content in the two selected SDEOs and Th1/Th2 immune balance or anti-inflammatory ability in linear, non-linear or biphasic manners, respectively. In conclusion, our results suggest that AG and ER, particularly ER, SDEO have immunomodulatory potential in shifting the Th1/Th2 balance toward Th2 polarization in splenocytes and inhibiting inflammation in macrophages in the absence or presence of LPS.

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Involvement of natural killer cells in the pathogenesis of endometriosis in patients with pelvic pain

Journal of International Medical Research ◽

10.1177/0300060519871407 ◽

2020 ◽

Vol 48 (7) ◽

pp. 030006051987140

Author(s):

Jue He ◽

Yan Xu ◽

Minhui Yi ◽

Cancan Gu ◽

Yi Zhu ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Pelvic Pain ◽

Immune Cells ◽

Expression Profiles ◽

Gradient Centrifugation ◽

Enzyme Linked Immunosorbent Assay ◽

Sterile Alpha Motif Domain ◽

Factor Α ◽

Protein Expression Profiles

Objectives To detect the involvement of immune cells in the pathogenesis of endometriosis in patients with stable status or pelvic pain. Methods Blood was collected from patients with endometriosis with and without pelvic pain. Natural killer (NK) and Th17 cells were analyzed by flow cytometry, and secretion of inflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-7) was verified by enzyme-linked immunosorbent assay. We isolated immune cells from blood by density-gradient centrifugation to investigate the expression of functional molecules including sterile alpha motif domain-containing protein 9 (SAMD9), Ral guanine nucleotide dissociation stimulator-like 2 (RGL2), early growth response protein 1, and Akirin2. We also searched the BIOGPS database for protein expression profiles. Results SAMD9 and RGL2 expression levels were significantly upregulated in patients with pelvic pain. Furthermore, lysophosphatidic acid receptor 1 expression was higher in endometrial tissues from patients with pelvic pain, and was mainly localized in stromal and glandular epithelial cells in ectopic lesions. Conclusion NK cells play an important role in the pathogenesis of endometriosis in patients with pelvic pain. Suppressing the cytotoxic activity of NK cells may thus help to reduce the progression of pelvic pain in patients with endometriosis.

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Hsp60 and IL-8 axis promotes apoptosis resistance in cancer

British Journal of Cancer ◽

10.1038/s41416-019-0617-0 ◽

2019 ◽

Vol 121 (11) ◽

pp. 934-943 ◽

Cited By ~ 2

Author(s):

Sandeep Kumar ◽

Jordan O’Malley ◽

Ajay Kumar Chaudhary ◽

Joseph R. Inigo ◽

Neelu Yadav ◽

...

Keyword(s):

Cancer Cells ◽

Combined Treatment ◽

Annexin V ◽

Underlying Mechanism ◽

Competitive Inhibitor ◽

Enzyme Linked Immunosorbent Assay ◽

Dependent Manner ◽

Heat Shock Protein 60 ◽

Apoptosis Resistance ◽

Upstream Regulator

Abstract Background Interleukin-8 (IL-8) and heat shock protein 60 (Hsp60) play crucial roles in cell survival and maintenance of cellular homoeostasis. However, cross talks between these two proteins are not defined. Methods IL-8 expression in tumour tissue sections was analysed by immunohistochemistry. IL-8 expression and release in cancer cells was quantified using enzyme-linked immunosorbent assay (ELISA). Apoptosis was quantified using caspase activity and Annexin-V/PI staining. Results We observed IL-8 release from cancer cells in response to histone deacetylase inhibitor, apicidin (Api), and non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase, thapsigargin (TG). IL-8 release was increased upon TG-treatment. TG-induced IL-8 expression was reduced in the presence of Api in Bax-dependent manner. Increased apoptosis was associated with decreased IL-8 expression in response to combined treatment of TG and Api. TG and Api combination induced caspase-8 and caspase-9 dependent apoptosis. Hsp60 knockdown abrogated IL-8 expression induced by Api, TG, and their combination. The level of TGF-β, an upstream regulator of IL-8, was decreased upon Hsp60-silencing. Knocking down Hsp60 decreased IL-8 expression and its release in prostate cancer cell xenograft tumours in SCID mice. Conclusion This study describes the underlying mechanism associated with apoptosis resistance mediated via Hsp60-IL-8 axis in cancer.

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Glucocorticoids mobilize macrophages by transcriptionally up-regulating the exopeptidase DPP4

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010894 ◽

2020 ◽

Vol 295 (10) ◽

pp. 3213-3227 ◽

Cited By ~ 5

Author(s):

David Diaz-Jimenez ◽

Maria Grazia Petrillo ◽

Jonathan T. Busada ◽

Marcela A. Hermoso ◽

John A. Cidlowski

Keyword(s):

Gene Expression ◽

Immune Cells ◽

Inflammatory Diseases ◽

Expression Patterns ◽

Peritoneal Macrophages ◽

Macrophage Migration ◽

Dipeptidyl Peptidase 4 ◽

Ru 486 ◽

Conserved Gene ◽

Inflammatory Molecules

Glucocorticoids are potent endogenous anti-inflammatory molecules, and their cognate receptor, glucocorticoid receptor (GR), is expressed in nearly all immune cells. Macrophages are heterogeneous immune cells having a central role in both tissue homeostasis and inflammation and also play a role in the pathogenesis of some inflammatory diseases. Paradoxically, glucocorticoids have only a limited efficacy in controlling the resolution of these macrophage-related diseases. Here, we report that the transcriptomes of monocyte-like THP-1 cells and macrophage-like THP-1 cells (THP1-MΦ) have largely conserved gene expression patterns. In contrast, the differentiation to THP1-MΦ significantly altered the sensitivity of gene transcription to glucocorticoids. Among glucocorticoid-regulated genes, we identified the exopeptidase dipeptidyl peptidase-4 (DPP4) as a critical glucocorticoid-responsive gene in THP1-MΦ. We found that GR directly induces DPP4 gene expression by binding to two glucocorticoid-responsive elements (GREs) within the DPP4 promoter. Additionally, we show that glucocorticoid-induced DPP4 expression is blocked by the GR antagonist RU-486 and by GR siRNA transfection and that DPP4 enzyme activity is reduced by DPP4 inhibitors. Of note, glucocorticoids highly stimulated macrophage mobility; unexpectedly, DPP4 mediated the glucocorticoid-induced macrophage migration, and siRNA-mediated knockdowns of GR and DPP4 blocked dexamethasone-induced THP1-MΦ migration. Moreover, glucocorticoid-induced DPP4 activation was also observed in proinflammatory M1-polarized murine macrophages, as well as peritoneal macrophages, and was associated with increased macrophage migration. Our results indicate that glucocorticoids directly up-regulate DPP4 expression and thereby induce migration in macrophages, potentially explaining why glucocorticoid therapy is less effective in controlling macrophage-dominated inflammatory disorders.

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Targeting Negative and Positive Immune Checkpoints with Monoclonal Antibodies in Therapy of Cancer

Cancers ◽

10.3390/cancers11111756 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1756 ◽

Cited By ~ 20

Author(s):

Katsiaryna Marhelava ◽

Zofia Pilch ◽

Malgorzata Bajor ◽

Agnieszka Graczyk-Jarzynka ◽

Radoslaw Zagozdzon

Keyword(s):

Monoclonal Antibodies ◽

Immune Cells ◽

Combined Treatment ◽

Adaptive Immune Response ◽

Bispecific Antibodies ◽

Immune Checkpoints ◽

Immune Homeostasis ◽

Physiological Conditions ◽

Adaptive Immune ◽

Current State

The immune checkpoints are regulatory molecules that maintain immune homeostasis in physiological conditions. By sending T cells a series of co-stimulatory or co-inhibitory signals via receptors, immune checkpoints can both protect healthy tissues from adaptive immune response and activate lymphocytes to remove pathogens effectively. However, due to their mode of action, suppressive immune checkpoints may serve as unwanted protection for cancer cells. To restore the functioning of the immune system and make the patient’s immune cells able to recognize and destroy tumors, monoclonal antibodies are broadly used in cancer immunotherapy to block the suppressive or to stimulate the positive immune checkpoints. In this review, we aim to present the current state of application of monoclonal antibodies in clinics, used either as single agents or in a combined treatment. We discuss the limitations of these therapies and possible problem-solving with combined treatment approaches involving both non-biological and biological agents. We also highlight the most promising strategies based on the use of monoclonal or bispecific antibodies targeted on immune checkpoints other than currently implemented in clinics.

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Flow cytometric analysis of the cell-cycle distribution of spleen lymphocytes isolated from Fischer 344 rats exposed to ethyl nitrosourea

Cell Biology and Toxicology ◽

10.1007/bf00755141 ◽

1993 ◽

Vol 9 (1) ◽

pp. 77-83 ◽

Cited By ~ 1

Author(s):

Suzanne M. Morris ◽

Olen E. Domon ◽

Lynda J. McGarrity ◽

Anane Aidoo ◽

Ralph L. Kodell ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometric Analysis ◽

Cell Cycle Distribution ◽

Fischer 344 Rats ◽

Fischer 344 ◽

Flow Cytometric ◽

Spleen Lymphocytes

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Aspirin Blocks Orthodontic Relapse via Inhibition of CD4+ T Lymphocytes

Journal of Dental Research ◽

10.1177/0022034516685527 ◽

2017 ◽

Vol 96 (5) ◽

pp. 586-594 ◽

Cited By ~ 13

Author(s):

Y. Liu ◽

T. Zhang ◽

C. Zhang ◽

S.S. Jin ◽

R.L. Yang ◽

...

Keyword(s):

T Lymphocytes ◽

Relapse Rate ◽

Immune Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Th1 Cells ◽

Tnf Α ◽

Inflammatory Drugs ◽

Ifn Γ ◽

Anti Inflammatory

Immunologic response plays an important role in orthodontic tooth movement (OTM) and relapse. Nonsteroidal anti-inflammatory drugs, such as aspirin, affect immune cells and clinical orthodontic treatment. However, the mechanisms by which nonsteroidal anti-inflammatory drugs regulate immune cells to affect orthodontic relapse are unclear. In this study, male Sprague-Dawley rats were grouped as relapse and relapse + aspirin for 10 d after 14 d of OTM. Silicone impressions of the rats’ maxillary dentitions were obtained to record the distance of OTM at the indicated time point. CD4+ T lymphocytes in spleen were examined by flow cytometry. Serum levels of type 1 T-helper (Th1) cell–associated cytokines tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) were determined through enzyme-linked immunosorbent assay. The effects of aspirin on CD4+ T and Th1 cells were also analyzed in vitro. Aspirin treatment significantly reduced the relapse rate. More interestingly, injection of CD25 neutralizing antibody basiliximab or TNF-α inhibitor etanercept can significantly reduce the relapse rate as well. Correspondingly, aspirin treatment significantly accelerated the decrease of orthodontic force–induced secretion of TNF-α and IFN-γ in serum and the expression of TNF-α and IFN-γ in periodontal ligament during relapse. Furthermore, aspirin treatment in vitro significantly repressed the differentiation of CD4+ T and Th1 cells. Overall, results indicated that aspirin treatment can block orthodontic relapse by regulating Th1 cells.

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