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Poly-dispersed Acid-Functionalized Single-Walled Carbon Nanotubes (AF-SWCNTs) Are Potent Inhibitor of BCG Induced Inflammatory Response in Macrophages

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Abstract

The present study is focused on the modulation of Mycobacterium bovis BCG-induced inflammatory response by poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) in macrophages. Flow cytometric and confocal microscopy studies indicated that both BCG and AF-SWCNTs were efficiently internalized by RAW 264.7 and MH-S macrophage cell lines and were essentially localized in the cytoplasmic area. BCG-induced production of reactive oxygen species (ROS) and nitric oxide by the two cell lines was significantly inhibited by AF-SWCNTs. Using RT-PCR technique, a marked decline was observed in the expression of BCG-induced pro-inflammatory genes COX-2, iNOS, TNF-α, IL-6, and IL-1β upon treatment with AF-SWCNTs. Results of gelatin zymography indicated that the AF-SWCNTs treatment also induced a marked decline in BCG-induced release of matrix metalloproteinases MMP-2 and MMP-9 by the two macrophage cell lines. The anti-inflammatory effect of AF-SWCNTs in downregulating BCG-induced inflammatory response was further validated in murine peritoneal macrophages. Treatment with AF-SWCNTs led to a steep decline in BCG-induced NO production in murine peritoneal macrophages in vitro as well as in vivo. Peritoneal macrophages isolated from mice treated with BCG and AF-SWCNTs had a significantly lower intracellular expression of COX-2 as compared to the peritoneal macrophages derived from mice treated with BCG alone. Taken together, our results demonstrate a potent anti-inflammatory effect of AF-SWCNTs in alleviating BCG-induced inflammatory responses in macrophages in vitro and in vivo.

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All data is available in the manuscript. Raw data will be provided upon request.

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Acknowledgments

Research funding from the Department of Science and Technology, Government of India, and fellowship support to DB from the Department of Biotechnology are gratefully acknowledged.

Funding

This study was funded by a research grant to Professor Rajiv K. Saxena, from the Nano-science Mission, Department of Science and Technology, Government of India. Grant number SR/NM/NS-1219.

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Authors and Affiliations

  1. Faculty of Life Sciences and Biotechnology, South Asian University, Akbar Bhawan, Chanakyapuri, New Delhi, 110021, India

    Deepika Bhardwaj & Rajiv K. Saxena

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  1. Deepika Bhardwaj
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Contributions

Deepika Bhardwaj (DB) the first author is a PhD student of Professor Rajiv K. Saxena (RKS) the corresponding author. The idea of this study was that of RKS and was executed by DB with continuing consultations with RKS on a day to day basis. DB worked under the supervision of RKS and conducted all laboratory experiments after discussion with RKS. Experiments were planned jointly. DB also plotted all graphs and prepared all illustrations included in the paper that were in turn critically examined by RKS and modified where required. DB also wrote the first draft of the manuscript that was examined by RKS and suitable modifications made. Funding of the study was obtained by RKS in form of a research grant. DB was supported by a research fellowship from the Department of Biotechnology of the Government of India.

Corresponding author

Correspondence to Rajiv K. Saxena.

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Ethics Approval

In one part of the manuscript, mice were used to harvest peritoneal macrophages. Mice were used under approval from the Institutional Animal Ethical Committee (IAEC) of the South Asian University. IAEC project approval code: SAU/IAEC/2016/02. This information is provided in the Materials and Methods section of the manuscript.

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Both authors fully consented to participate in this study.

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Both the authors contributed physically and intellectually in this study and fully consent to the material in the manuscript.

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Supplementary Information

ESM 1

Supplementary Fig. 1: Characterization of AF-SWCNTs. Panel-a shows the Zeta potential and Zeta size of AF-SWCNTs measured using Malvern Zetasizer. Panel-b shows the mid-infrared and near-infrared spectroscopic results for AF-SWCNTs (Data from Sigma-Aldrich). Panel-c shows the TEM image of strands of AF- SWCNTs. Supplementary Fig. 2: Determination of percentage of cells that internalize DiI C-18 labeled BCG or FAF-SWCNTs by Flow cytometry. RAW 264.7 and MH-S cells [0.1 × 106/ml/well] were cultured with DiI C-18 labelled BCG MOI 20:1 or FAF-SWCNTs (10 μg/ml) for up to 24 hours. After incubation, cells were washed with PBS, detached using trypsin and analyzed on a flow cytometer. Representative results show the fluorescence distribution for control cells and cells that were incubated with fluorescent BCG or FAF-SWCNTs, and how the percentages of cells that internalized DiI C-18 labelled BCG or FAF-SWCNTs were determined. Top panel has data for RAW 264.7 and the bottom panel for MH-S cells. Vertical lines show the gates in all panels. Supplementary Fig. 3: Z sectioning images showing uptake of DiI C-18 labelled BCG and FAF-SWCNTs by macrophages using confocal microscopy. RAW 264.7 and MH-S cells [0.1 × 106/ml/well] were cultured on a coverslip with DiI C-18 labelled BCG (MOI 20:1) and FAF-SWCNTs (10 μg/ml) for 12 hours. After incubation, cells were washed with PBS, fixed using paraformaldehyde, stained nuclei using Hoechst 33342 dye and mounted on a glass slide to visualize the uptake of DiI C-18 labelled BCG and FAF-SWCNTs and performed Z sectioning using Confocal microscope. Confocal microscopy images showing internalization of labelled BCG (left panel) and FAF-SWCNTs (right panel) by RAW 264.7 and MH-S are shown. In each image, micrographs represent Z sectioning images at 3 μm showing DiI C-18 labelled BCG uptake/FAF-SWCNTs uptake (red fluorescence) and nuclei are defined by blue stain of Hoechst 33342 dye. Magnification 100X in all cases. Supplementary Fig. 4: Modulation of expression of BCG induced pro-inflammatory genes by AF-SWCNTs. RAW 264.7 and MH-S cells [0.5 × 106/ml/well] were cultured with AF-SWCNTs (100 μg/ml), BCG (MOI 100:1) and BCG + AF-SWCNTs for 12 hours. RNA was extracted using Trizol RNA extraction method, quantified using nanodrop and 2 μg of RNA was reverse transcribed to form cDNA. RT-PCR was performed to check relative gene expression of pro-inflammatory genes under different conditions as described in Methods. Fold change in gene expression was calculated using 2−∆∆Ct method. Histograms show result of 3 independent experiments (Mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001. (PDF 1.63 mb)

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Bhardwaj, D., Saxena, R.K. Poly-dispersed Acid-Functionalized Single-Walled Carbon Nanotubes (AF-SWCNTs) Are Potent Inhibitor of BCG Induced Inflammatory Response in Macrophages. Inflammation 44, 908–922 (2021). https://doi.org/10.1007/s10753-020-01386-8

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