Activation of human macrophages for the killing of intracellular Trypanosoma cruzi by TNF-α and IFN-γ through a nitric oxide-dependent mechanism

Trypanosoma cruziis the causative agent of chronic chagasic cardiomyopathy. Why macrophages (mφs), the early responders to infection, fail to achieve parasite clearance is not known. Mouse (RAW 264.7) and human (THP-1 and primary) mφs were infected for 3 h and 18 h withT. cruziTcI isolates, SylvioX10/4 (SYL, virulent) and TCC (nonpathogenic), which represent mφ stimulation and infection states, respectively. Mφs incubated with lipopolysaccharide and gamma interferon (LPS/IFN-γ) and with interleukin-4 (IL-4) were used as controls. We monitored the cytokine profile (using enzyme-linked immunosorbent assay [ELISA]), reactive oxygen species (ROS; fluorescent probes), nitric oxide (·NO; Griess assay), and metabolic state using a custom-designed mitoxosome array and Seahorse XF24 Analyzer. LPS/IFN-γ treatment of mφs elicited a potent increase in production of tumor necrosis alpha (TNF-α) at 3 h and of ROS and ·NO by 18 h. Upon SYL infection, murine mφs elicited an inflammatory cytokine profile (TNF-α ≫ TGF-β + IL-10) and low levels of ·NO and ROS production. LPS/IFN-γ treatment resulted in the inhibition of oxidative metabolism at the gene expression and functional levels and a switch to the glycolytic pathway in mφs, while IL-4-treated mφs utilized oxidative metabolism to meet energy demands. SYL infection resulted in an intermediate functional metabolic state with increased mitoxosome gene expression and glycolysis, and IFN-γ addition shut down the oxidative metabolism in SYL-infected mφs. Further, TCC- and SYL-stimulated mφs exhibited similar levels of cell proliferation and production of TNF-α and ROS, while TCC-stimulated mφs exhibited up to 2-fold-higher levels of oxidative metabolism and ·NO production than SYL-infected mφs. Inhibiting ATP-coupled O2consumption suppressed the ·NO generation in SYL-infected mφs. Mitochondrial oxygen consumption constitutes a mechanism for stimulating ·NO production in mφs duringT. cruziinfection. Enhancing the oxidative metabolism provides an opportunity for increased ·NO production and pathogen clearance by mφs to limit disease progression.

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Intracellular Growth of Trypanosoma cruzi in Cardiac Myocytes Is Inhibited by Cytokine-Induced Nitric Oxide Release

Infection and Immunity ◽

10.1128/iai.72.1.359-363.2004 ◽

2004 ◽

Vol 72 (1) ◽

pp. 359-363 ◽

Cited By ~ 27

Author(s):

Laura Edith Fichera ◽

Maria Cecilia Albareda ◽

Susana Adriana Laucella ◽

Miriam Postan

Keyword(s):

Nitric Oxide ◽

Trypanosoma Cruzi ◽

Cardiac Myocytes ◽

Cardiac Myocyte ◽

Parasite Growth ◽

Inos Expression ◽

No Production ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Ifn Γ

ABSTRACT The effect of interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) on Trypanosoma cruzi multiplication and nitric oxide (NO) production in cardiac myocytes was investigated. Cardiac myocyte cultures were obtained from neonatal Wistar rat hearts, infected with T. cruzi, and treated with IL-1β, TNF-α, IFN-γ, or N-monomethyl-l-arginine (l-NAME) for 72 h. Parasite growth was calculated from the number of infected cells in Giemsa-stained smears. Nitric oxide production was determined with the Griess reagent. Inducible nitric oxide synthase (iNOS) expression by cardiac myocytes was detected by Western blot. The results showed that the percentages of cardiac myocytes containing T. cruzi amastigotes in cytokine-treated cultures were significantly lower than in nontreated cultures. The addition of l-NAME reversed the inhibitory effect on parasite growth of IL-1β and TNF-α but not of IFN-γ. Nitrite levels released by T. cruzi-infected and noninfected cardiac myocyte cultures after 72 h of stimulation with IL-1β were significantly higher than those produced upon treatment with TNF-α, IFN-γ, or medium alone, regardless of the infection status. Nitrite levels in TNF-α-stimulated infected cultures were significantly higher than in untreated infected cultures and TNF-α-treated noninfected cultures. l-NAME inhibited IL-1β- but not TNF-α-induced NO production, indicating the presence of iNOS-dependent and iNOS-independent mechanisms for NO formation in this experimental system. iNOS expression was detected in infected and noninfected cardiac myocytes stimulated with IL-1 β and TNF-α but not with IFN-γ. These results suggest an important role for cardiac myocytes and locally secreted cytokines in the control of parasite multiplication in T. cruzi-induced myocarditis.

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Leukotriene B4 Induces Nitric Oxide Synthesis in Trypanosoma cruzi-Infected Murine Macrophages and Mediates Resistance to Infection

Infection and Immunity ◽

10.1128/iai.70.8.4247-4253.2002 ◽

2002 ◽

Vol 70 (8) ◽

pp. 4247-4253 ◽

Cited By ~ 55

Author(s):

A. Talvani ◽

F. S. Machado ◽

G. C. Santana ◽

A. Klein ◽

L. Barcelos ◽

...

Keyword(s):

Nitric Oxide ◽

Trypanosoma Cruzi ◽

Leukotriene B4 ◽

No Production ◽

Dependent Manner ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Ifn Γ

ABSTRACT The production of nitric oxide (NO) by gamma interferon (IFN-γ)-activated macrophages is a major effector mechanism during experimental Trypanosoma cruzi infection. In addition to IFN-γ, chemoattractant molecules, such as platelet-activating factor (PAF) and CC chemokines, may also activate macrophages to induce NO and mediate the killing of T. cruzi in an NO-dependent manner. Here we investigated the ability of leukotriene B4 (LTB4) to induce the production of NO by macrophages infected with T. cruzi in vitro and whether NO mediated LTB4-induced parasite killing. The activation of T. cruzi-infected but not naive murine peritoneal macrophages with LTB4 induced the time- and concentration-dependent production of NO. In addition, low concentrations of LTB4 acted in synergy with IFN-γ to induce NO production. The NO produced mediated LTB4-induced microbicidal activity in macrophages, as demonstrated by the inhibitory effects of an inducible NO synthase inhibitor. LTB4-induced NO production and parasite killing were LTB4 receptor dependent and were partially blocked by a PAF receptor antagonist. LTB4 also induced significant tumor necrosis factor alpha (TNF-α) production, and blockade of TNF-α suppressed LTB4-induced NO release and parasite killing. A blockade of LTB4 or PAF receptors partially inhibited IFN-γ-induced NO and TNF-α production but not parasite killing. Finally, daily treatment of infected mice with CP-105,696 was accompanied by a significantly higher level of blood parasitemia, but not lethality, than that seen in vehicle-treated animals. In conclusion, our results suggest a role for LTB4 during experimental T. cruzi infection. Chemoattractant molecules such as LTB4 not only may play a major role in leukocyte migration into sites of inflammation in vivo but also, in the event of an infection, may play a relevant role in the activation of recruited leukocytes to kill the invading microorganism in an NO-dependent manner.

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Comparing the protective effects of resveratrol, curcumin and sulforaphane against LPS/IFN-γ-mediated inflammation in doxorubicin-treated macrophages

Scientific Reports ◽

10.1038/s41598-020-80804-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Haidy A. Saleh ◽

Eman Ramdan ◽

Mohey M. Elmazar ◽

Hassan M. E. Azzazy ◽

Anwar Abdelnaser

Keyword(s):

Nitric Oxide ◽

Inflammatory Response ◽

Raw 264.7 Cells ◽

Inos Mrna ◽

No Production ◽

Raw 264.7 ◽

Mrna Levels ◽

Protective Effects ◽

Tnf Α ◽

Ifn Γ

AbstractDoxorubicin (DOX) chemotherapy is associated with the release of inflammatory cytokines from macrophages. This has been suggested to be, in part, due to DOX-mediated leakage of endotoxins from gut microflora, which activate Toll-like receptor 4 (TLR4) signaling in macrophages, causing severe inflammation. However, the direct function of DOX on macrophages is still unknown. In the present study, we tested the hypothesis that DOX alone is incapable of stimulating inflammatory response in macrophages. Then, we compared the anti-inflammatory effects of curcumin (CUR), resveratrol (RES) and sulforaphane (SFN) against lipopolysaccharide/interferon-gamma (LPS/IFN-γ)-mediated inflammation in the absence or presence of DOX. For this purpose, RAW 264.7 cells were stimulated with LPS/IFN-γ (10 ng/mL/10 U/mL) in the absence or presence of DOX (0.1 µM). Our results showed that DOX alone is incapable of stimulating an inflammatory response in RAW 264.7 macrophages. Furthermore, after 24 h of incubation with LPS/IFN-γ, a significant increase in tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) mRNA levels was observed. Similarly, nitric oxide (NO) production and TNF-α and IL-6 protein levels were significantly upregulated. Moreover, in LPS/IFN-γ-treated macrophages, the microRNAs (miRNAs) miR-146a, miR-155, and miR-21 were significantly overexpressed. Interestingly, upon testing CUR, RES, and SFN against LPS/IFN-γ-mediated inflammation, only SFN was able to significantly reverse the LPS/IFN-γ-mediated induction of iNOS, TNF-α and IL-6 and attenuate miR-146a and miR-155 levels. In conclusion, SFN, at the transcriptional and posttranscriptional levels, exhibits potent immunomodulatory action against LPS/IFN-γ-stimulated macrophages, which may indicate SFN as a potential treatment for DOX-associated inflammation.

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Cytokine and inflammatory mediators are associated with cytotoxic, anti-inflammatory and apoptotic activity of honeybee venom

Journal of Complementary and Integrative Medicine ◽

10.1515/jcim-2019-0182 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Mohamed A. Salama ◽

Mohamed A. Younis ◽

Roba M. Talaat

Keyword(s):

Nitric Oxide ◽

Cell Lines ◽

Cancer Cell ◽

Hela Cells ◽

No Production ◽

Hep G2 ◽

Normal Cells ◽

Tnf Α ◽

Ifn Γ ◽

Mcf 7

AbstractObjectiveThe present study aimed to evaluate cytotoxic, apoptotic, and anti-inflammatory properties of bee venom (BV) as well as changes in cytokine secretion levels and nitric oxide (NO) production using three different cancer cell lines [liver (Hep-G2), breast (MCF-7), and cervical (HPV-18 infected HeLa cells)] and two normal cells (splenocytes and macrophages (MQ).MethodsCytotoxic activity of BV against tumor cell lines and normal splenocytes/MQ was tested by MTT assay. By ELISA (ELISA); Tumor necrosis factor (TNF-α), Interleukine (IL-10) and interferon (IFN-γ) were measured. Caspase three expressions was evaluated using reverse transcription-polymerase chain reaction (RT-PCR). Nitric oxide (NO) was estimated using a colorimetric assay.ResultsBV has a significant cytotoxic effect on all cell lines in a dose- and time-dependent manner; none of them was toxic for normal cells. Treating Hep-G2 cells with BV showed a reduction in IL-10, elevation in TNF-α with no change in IFN-γ level. MCF-7 cells have low IL-10 and TNF-α and high IFN-γ production level. Elevation of IL-10 and IFN-γ coincides with a reduction in TNF-α level was demonstrated in HeLa cells. The expression of Caspase three was dramatically increased with elevation in BV concentration in all tested cancer cell lines. A gradual decrease in NO production by MQ with increasing BV dose was observed.ConclusionTaken together, our results stressed on the importance of BV as a potent anti-tumor agent against various types of cancers (Liver, Breast, and Cervix). Further steps towards the use of BV for pharmacological purposes must be done.

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CD154 Activates Macrophage Antimicrobial Activity in the Absence of IFN-γ through a TNF-α-Dependent Mechanism

The Journal of Immunology ◽

10.4049/jimmunol.171.12.6750 ◽

2003 ◽

Vol 171 (12) ◽

pp. 6750-6756 ◽

Cited By ~ 27

Author(s):

Rosa M. Andrade ◽

Matthew Wessendarp ◽

Carlos S. Subauste

Keyword(s):

Antimicrobial Activity ◽

Tnf Α ◽

Ifn Γ ◽

Dependent Mechanism

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IFN-γ inhibits AP-1 binding activity in human brain-derived cells through a nitric oxide dependent mechanism

Journal of Neuroimmunology ◽

10.1016/s0165-5728(98)00069-1 ◽

1998 ◽

Vol 88 (1-2) ◽

pp. 39-44 ◽

Cited By ~ 7

Author(s):

Katherine Conant ◽

Umbereen Ahmed ◽

Joan P Schwartz ◽

Eugene O Major

Keyword(s):

Nitric Oxide ◽

Human Brain ◽

Binding Activity ◽

Ifn Γ ◽

Dependent Mechanism

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Nitric oxide interacts with cholinoceptors to modulate insulin secretion by pancreatic β cells

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-020-02443-9 ◽

2020 ◽

Vol 472 (10) ◽

pp. 1469-1480

Author(s):

Bashair M. Mussa ◽

Ankita Srivastava ◽

Abdul Khader Mohammed ◽

Anthony J. M. Verberne

Keyword(s):

Nitric Oxide ◽

Insulin Secretion ◽

Cell Viability ◽

Combined Treatment ◽

No Production ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

Tnf Α ◽

Ifn Γ

Abstract Dysfunction of the pancreatic β cells leads to several chronic disorders including diabetes mellitus. Several mediators and mechanisms are known to be involved in the regulation of β cell secretory function. In this study, we propose that cytokine-induced nitric oxide (NO) production interacts with cholinergic mechanisms to modulate insulin secretion from pancreatic β cells. Using a rat insulinoma cell line INS-1, we demonstrated that β cell viability decreases significantly in the presence of SNAP (NO donor) in a concentration- and time-dependent manner. Cell viability was also found to be decreased in the presence of a combined treatment of SNAP with SMN (muscarinic receptor antagonist). We then investigated the impact of these findings on insulin secretion and found a significant reduction in glucose uptake by INS-1 cells in the presence of SNAP and SMN as compared with control. Nitric oxide synthase 3 gene expression was found to be significantly reduced in response to combined treatment with SNAP and SMN suggesting an interaction between the cholinergic and nitrergic systems. The analysis of gene and protein expression further pin-pointed the involvement of M3 muscarinic receptors in the cholinergic pathway. Upon treatment with cytokines, reduced cell viability was observed in the presence of TNF-α and IFN-γ. A significant reduction in insulin secretion was also noted after treatment with TNF-α and IFN-γ and IL1-β. The findings of the present study have shown for the first time that the inhibition of the excitatory effects of cholinergic pathways on glucose-induced insulin secretion may cause β cell injury and dysfunction of insulin secretion in response to cytokine-induced NO production.

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