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Scientific Reports
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10.1038/s41598-024-51319-w
Article
RETRACTED ARTICLE: Arsenic album 30C exhibits crystalline nano structure of arsenic trioxide and modulates innate immune markers in murine macrophage cell lines
Rath Suvasmita 12
Jema Jyoti Prava 2
Kesavan Kamali 3
Mallick Sagar 3
Pradhan Jyotsnarani 2
Chainy Gagan Bihari Nityananda 2
Nayak Debadatta 4
Kaushik Subhash 4
Dandapat Jagneshwar jd.biotech@utkaluniversity.ac.in
jdandapat.nou@gmail.com

25
1 https://ror.org/0034eez47 grid.412779.e 0000 0001 2334 6133 Centre of Environment, Climate Change and Public Health, Utkal University, Vani Vihar, Bhubaneswar, Odisha India
2 https://ror.org/0034eez47 grid.412779.e 0000 0001 2334 6133 Post Graduate Department of Biotechnology, Utkal University, Bhubaneswar, Odisha India
3 https://ror.org/01g7qth32 grid.418808.d 0000 0004 1792 1607 CSIR-Institute of Minerals and Materials Technology, Bhubaneswar, Odisha India
4 https://ror.org/04qawwn42 grid.464749.c 0000 0001 1482 3022 Central Council for Research in Homeopathy, New Delhi, India
5 https://ror.org/0034eez47 grid.412779.e 0000 0001 2334 6133 Centre of Excellence in Integrated Omics and Computational Biology, Utkal University, Bhubaneswar, Odisha 751004 India
7 1 2024
7 1 2024
2024
14 7451 6 2023
3 1 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Macrophages are associated with innate immune response and M1-polarized macrophages exhibit pro-inflammatory functions. Nanoparticles of natural or synthetic compounds are potential triggers of innate immunity. As2O3 is the major component of the homeopathic drug, Arsenic album 30C.This has been claimed to have immune-boosting activities, however, has not been validated experimentally. Here we elucidated the underlying mechanism of Ars. alb 30C-mediated immune priming in murine macrophage cell line. Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD) used for the structural analysis of the drug reveals the presence of crystalline As2O3 nanoparticles of cubic structure. Similarly, signatures of M1-macrophage polarization were observed by surface enhanced Raman scattering (SERS) in RAW 264.7 cells with concomitant over expression of M1 cell surface marker, CD80 and transcription factor, NF-κB, respectively. We also observed a significant increase in pro-inflammatory cytokines like iNOS, TNF-α, IL-6, and COX-2 expression with unaltered ROS and apoptosis in drug-treated cells. Enhanced expression of Toll-like receptors 3 and 7 were observed both in transcriptional and translational levels after the drug treatment. In sum, our findings for the first time indicated the presence of crystalline As2O3 cubic nanostructure in Ars. alb 30C which facilitates modulation of innate immunity by activating macrophage polarization.

Subject terms

Biological techniques
Biophysics
Biotechnology
Cell biology
Immunology
Molecular biology
Materials science
Central Council for Research in Homeopathy (CCRH), Ministry of AYUSH, and Government of India17-6/2015-16/CCRH/Tech/Coll/Utkal University/1762 issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Innate immunity is the body’s first line of defence against any pathogenic attack1. Microbes and microbes-derived pathogenic factors are recognized and cleared by the action of innate immune effector molecules2. Various cellular components of innate immune system include neutrophils, dendritic cells, eosinophils, Langerhans cells, macrophages, and Natural killer (NK) cells3. An array of cell surface receptors on these innate immune cells, also known as pattern recognition receptors (PRRs), enables them to detect pathogens or pathogen-associated molecular patterns (PAMPs)4. Toll-like receptors (TLRs) are major PRRs responsible for the detection of microbial signature molecules5. Among all TLRs, TLR-3 and TLR-7 are predominantly involved in antiviral immunity6,7. TLR-3 recognizes double stranded RNA (dsRNA) molecules and exhibits antiviral host response by the production of type I interferons (IFNs), pro-inflammatory cytokines, and chemokines8. Toll-interleukin-1 receptor (TIR)-domain containing adaptor-inducing IFN-‍β (TRIF) is the adaptor molecule essential for TLR-3 function9. Similarly, TLR-7 recognizes single stranded RNA (ssRNA) molecules and executes its function through Myeloid differentiation primary response 88 (MyD88) adaptor protein10. TLR signalling induces the transcription factor NF-κB, which further activates pro-inflammatory genes associated with M1 macrophage polarization11. Elevated expressions of both TLR-3 and TLR-7 are evident in M1 polarized macrophages, which are associated with induced pro-inflammatory cytokine production12,13.

The TLR signalling cascade is coupled with macrophage polarization and its phenotypic expression14,15. Macrophages are active members of the innate immune system with impressive plasticity and potential for “differentiation into distinct phenotypes and functional attributes,” commonly referred to as macrophage polarization. Such cellular responses to ambient stimuli under various pathophysiological conditions result in optimum and differential macrophage function16,17. The tissue‐resident macrophages (M0) are undifferentiated macrophages and precursors of polarized macrophages M1 and M218. M1, or classically activated macrophages have pro-inflammatory activities, while M2, or alternatively activated macrophages have anti-inflammatory activities19. Activated macrophages are primed against pathogenic attacks; therefore, their activation plays a protective role in the pathogenesis of several inflammatory conditions20. M1-polarized macrophages produce effector molecules such as reactive nitrogen intermediates and inflammatory cytokines, including IL-1β, TNF-α, and IL-6, which act against pathogens by promoting immune responses20,21.

TLR stimulation has also been linked to activation of adaptive immune response. Recent studies indicate the use of various TLR agonists like imiquimod, and CpG oligodeoxynucleotide (CpG ODN) as potential adjuvants during the design and development of COVID-19 vaccine. Further, some of the adjutants have also been reported to induce a synergized effect comprising both adaptive and innate immunity22,23. Several drugs and natural compounds are reported to have the ability to induce macrophage polarization, hence have significant therapeutic potential as immune modulators24. Various preclinical studies demonstrate immunomodulatory, anti-oxidative, and therapeutic applications of naturally derived homeopathic drugs in a dose response manner25,26. Arsenic album (Ars. alb) is a homeopathic drug prepared from arsenic trioxide and has a significant anticancer properties25. It has been reported that Ars. alb ameliorates the toxic effect of arsenic trioxide in ultra-diluted form27. Recently, Ars. alb 30C has also been reported to have preventive role against COVID-19 infection as evident from community-based and parallel cohort study in a contaminant zone of the Delhi region in India28,29. Ye et al., demonstrated the immunomodulatory effect of trivalent arsenic, As (III) at a non-toxic concentration30. Several other studies demonstrated the anticancer properties of As2O3 nanoparticles and their application in cancer immunotherapy31,32. Although priming of innate immunity has been hypothesized behind the protective role of Ars. alb 30C described above, its function as an immune booster and detailed mechanism of its action is not been experimentally validated to date.

With this backdrop, the present study aims to underpin the possible causal mechanism of immunomodulation by Ars. alb 30C in RAW 264.7 macrophage cell line. Hypothesis of the study is that the immune priming potential of Ars. alb 30C is associated with the induction of innate immunity. Through our integrated experimental approaches, we have validated that Ars. alb 30C enhances innate immune signatures through upregulated expression of pro-inflammatory cytokines and concomitant polarization of M1 macrophages, as a part of its immune function. To gain insight into the mode of action of Ars. alb 30C, we have analysed its structure in Transmission Electron Microscope (TEM), X-ray diffraction (XRD) and M1 macrophage phenotype by Surface Enhanced Raman Scattering (SERS). For the first time, we have reported the presence of arsenic trioxide nanoforms in Ars. alb 30C and correlated such nano structure behind its mechanism of immunomodulation. Our findings are in line with the previous reports which clearly supports nanostructure-driven innate immunity through macrophage polarization33,34.

Therefore, the present study, with novel findings opens a new window to revisit Ars. alb 30C with different perspectives. Furthermore, this will provide hitherto unexplored opportunities and contribute to gaining its translational potential as a prospective alternative strategy for immune priming linked to several health consequences.

Results

Structural analysis with transmission electron microscopy (Fig. 1a–d) and X-ray diffraction (XRD) (supplementary Fig. S2)

Figure 1 TEM analysis of Ars. alb 30C. (a) Low magnification bright field TEM image of the ultra-high-diluted Ars. alb 30C sample. (b) Selected area electron diffraction (SAED) taken along [111] zone-axis shows various planes corresponds to the d-spacings of 0.466 nm, 0.268 nm, and 0.176 nm of cubic As2O3 structure [JCPDS 00-002-1451]. (c) High-Resolution TEM (HRTEM) image of the sample indicates the formation of nanoparticles of average size ~ 15 nm. (d) HRTEM image of a single nano particle and corresponding fast Fourier transform (FFT) image (inset) clearly shows that the nanoparticles are highly crystalline in nature and various planes (along [100] zone-axis) confirms the presence of cubic As2O3 structure [JCPDS 00-002-1451].

Structural analysis of Ars. alb 30C was performed by transmission electron microscopy (TEM) and X-ray diffraction (XRD). Figure 1a, b showed the bright field low magnification TEM image of the ultra-high-diluted Ars. alb 30C sample and corresponding Selected Area Electron Diffraction (SAED) taken along [111] zone-axis. It clearly shows various planes such as (2 2¯ 0), (20 2¯) and (02 2¯) planes that corresponds d-spacings of 0.466 nm of cubic As2O3 structure. Similarly, (42 2¯)and (22 4¯) planes correspond to 0.268 nm, while (40 4¯)and (04 2¯) planes correspond to 0.235 nm. Lastly, (42 6¯) and (24 6¯) planes correspond to 0.176 nm of cubic As2O3 structure [JCPDS 00-002-1451]. Figure 1c shows the High-Resolution TEM (HRTEM) image of the samples having nanoparticles. It indicates the formation of highly crystalline nanoparticles of an average size ~ of 15 nm. In Fig. 1d, the HRTEM image of a single nanoparticles and corresponding Fast Fourier Transform (FFT) image (inset) clearly shows that the nanoparticles are highly crystalline in nature and (040) (440), (004) planes (along [100] zone-axis) confirm the presence of cubic As2O3 structure [JCPDS 00-002-1451]. To further characterize, XRD scans were acquired from the drop-cast sample of Ars. alb 30C on 300 nm SiO2/Silicon substrate to investigate the crystallinity of As2O3 nanoparticles. In supplementary figure (Fig. S2), XRD pattern were analyzed, indexed and the peak position at 63.63° (2θ value) coincide with the (731) plane of As2O3 structure [Database: JCPDS 00-002-1451] Both TEM and XRD results confirmed the formation of highly crystalline nanoparticles of cubic As2O3 structure and their aggregates in the sample.

Macrophage polarization study by SERS (Fig. 2)

Figure 2 SERS fingerprint of M0 and M1-phenotype macrophages. SERS spectra represent the averages obtained from 20 cells for each group in the spectral range of 700–1800 cm−1. The peaks positions with red fonts indicate the positions where the largest spectral variations are found between M0 and M1 phenotypes.

SERS is a phenomenon of enhancing Raman signal intensity using surface plasmonic nanoparticle substrate. Macrophage polarization states were evaluated by the SERS technique using gold plasmonic nanoparticles. RAW 264.7 cells were treated with Ars. alb 30C for 24 h or left untreated. 90% alcohol (10–4 dilution) was used as vehicle control. The spectral assignments35 of Fig. 2 are depicted in Table 1. Macrophages were stimulated with Lipopolysaccharide (LPS) which acts as a positive control for M1 macrophage phenotype and the Raman spectra were evaluated in order to verify whether Raman spectroscopic analysis might distinguish M1-polarized macrophages from resting M0-macrophages upon activation with Ars. alb 30C. Comparing M0 and M1 spectra, the Raman bands around 910 cm−1 due to C–C stretching of amino acids of proteins and 1100 cm−1 due to lipid signatures undergoing major changes in peak intensity. Apart from these changes, there was neither the appearance nor disappearance of peaks. The other peaks exhibit small variations in the peak intensities. The spectrum corresponding to Ars. alb 30C matches with LPS-simulated macrophage spectrum. This confirms the M1 macrophage phenotype in Ars. alb 30C-treated cells.Table 1 Sequences of forward primer and reverse primer used for real-time PCR.

Primers	Forward primer	Reverse primer	Annealing temperature (°C)	
TLR-3	5′-TCACTTGCTCATTCTCCCTT-3’	5’-GCCTGGCTAAGTTATTGTGC-3’	58	
TLR-7	5’-TTCCTTCCGTAGGCTGAACC-3’	5’-GTAAGCTGGATGGCAGATCC-3’	58	
TRIF	5’-CACGATCCTGCTCCTGACTG-3’	5’-CTGTGGAGCAGTCTGGTTGT-3’	56	
MyD88	5’-CATACCCTTGGTCGCGCTTA-3’	5’-CCAGGCATCCAACAAACTGC-3’	56	
NF-κB	5’-CAGACCGCAGTATCCATAGC-3’	5’-CGTGAAAGGGGTTATTGTTGG-3’	56	
COX-2	5’-ACAACATCCCCTTCCTGCGA-3’	5’-TGGGCAGTCATCTGCTACGG-3’	58	
iNOS	5’-TGAAACTTCTCAGCCACCTTGG-3’	5’-TGAAGAGAAACTTCCAGGGGCA-3’	58	
TNF-α	5’-TGCCTATGTCTCAGCCTCTT-3’	5’-GAGGCCATTTGGGAACTTCT-3’	58	
CD 80	5’-CCAAAGCATCTGAAGCTATGGC-3’	5’-TTCCCAGCAATGACAGACAGC-3’	56	
IRF3	5’-CGGAAAGAAGTGTTGCGGTTAGC-3’	5’-CAGGCTGCTTTTGCCATTGGTG-3’	58	
GAPDH	5’-CAATGAATACGGCTACAGCAAC-3’	5’-AGGGAGATGCTCAGTGTTGG-3’	58	

Ars. alb 30C treatment is associated with unaltered ROS level and cell viability (Fig. 3a, b)

Figure 3 Measurement of total ROS level and microscopic detection of cell viability. (a) Total ROS level showing no significant change in Ars. alb 30C-treated cells as compared to the vehicle control and media control. Bars shown in panels represent normalized data (mean ± SEM, n = 3), *P ≤ 0.05. (b) Microscopic detection (magnification: ×10) of apoptosis using AO/PI double staining in untreated, alcohol and Ars. alb 30C-treated RAW 264.7 cells.

Total ROS level was measured by using DCFH-DA and no significant change in total ROS level was observed after Ars. alb 30C treatment as compared to vehicle control and media control cells (Fig. 3a). Further AO/PI double staining was performed to distinguish viable cells from dead cells by using fluorescence microscope. AO staining emits green fluorescence as it penetrates plasma membrane of viable cells. However, PI stains non-viable cells and emits red fluorescence. Our data showed uniform emission of green fluorescence with a healthy nucleus in control, alcohol, and Ars. alb 30C treated cells (Fig. 3b).

Induced expression of innate immune markers TLR-3, TLR-7, and their adaptor molecules in Ars. alb 30C-treated cells (Fig. 4a–h)

Figure 4 Expression of innate immune markers and downstream molecules in Ars. alb 30C-treated cells. (a–e) Real-time PCR showed induced TLR-3, TLR-7, TRIF, MyD88, and CD80 mRNA expression in Ars. alb 30C-treated cells as compared to the control samples. Bars shown in (a–e) represent normalized data (mean ± SEM, n = 3), *P ≤ 0.05. (f–h) Western blot analysis showed upregulation of TLR-3, TLR-7 & MyD88 in Ars. alb 30C-treated cell. Bars shown in (f–h) represent normalized data (mean ± SEM, n = 3), *P ≤ 0.05.

The present study aimed to validate the expression of innate immune markers TLR-3 and TLR-7 in Ars. alb 30C-treated cells both in transcript and translate levels. For this, RAW 264.7 cells were treated with Ars. alb 30C at a dilution of 10–4 along with 90% alcohol at a dilution of 10–4 as vehicle control and another group of cells left untreated for 24 h. Total RNA was isolated by TRIZOL method, and cDNA synthesis was performed using reverse transcriptase kit followed by real-time PCR. The RT-PCR data (n = 3) confirmed induced expression of TLR-3 and TLR-7 (P ≤ 0.05) in transcript level after Ars. alb 30C treatment (Fig. 4a, b). We also observed induced expression of TLR-3 and TLR-7 adaptor molecules TRIF and MyD88, respectively in Ars. alb 30C-treated cells (Fig. 4c, d). As CD80 is used as M1 macrophage marker, we further investigated its expression in the transcript level by RT-PCR. Our data revealed induced expression of CD80 in Ars. alb 30C- treated cells as compared to vehicle control and media control cells (Fig. 4e).

RAW 264.7 cells were treated with Ars. alb 30C for 24 h. Protein samples were isolated and immunoblotting was performed to study the expression pattern of TLR-3 and TLR-7 protein levels. Representative western blot results (n = 3) showed that there is induced expression of TLR-3 and TLR-7 in Ars. alb 30C-treated cells as compared to the vehicle control and untreated cells (Fig. 4f, g). Induced MyD88 expression was validated in Ars. alb 30C-treated cells (Fig. 4h).

Confocal analysis for TLR-3 and TLR-7 expression (Fig. 5a, b)

Figure 5 Confocal image analysis of Ars. alb 30C-treated RAW 264.7 cells. (a) Induced expression of TLR-3 was observed in Ars. alb 30C-treated cells as compared to control sample. Bars shown in panels represent normalized data (mean ± SEM, n = 3), *P ≤ 0.05. (b) Enhanced expression of TLR-7 observed in Ars. alb 30C-treated cells as compared to control samples. Bars shown in panels represent normalized data (mean ± SEM, n = 3), *P ≤ 0.05.

To study the expression pattern of innate immune markers TLR-3 and TLR-7, we also performed confocal experiment by staining macrophage cells with TLR-3 and TRL-7 specific antibodies tagged with Alexa fluor 488. We observed induced expression of both TLR-3 and 7 in Ars. alb 30 C-treated cells (Fig. 5a, b).

Up regulation of NF-κB in Ars. alb 30C-treated RAW 264.7 cells (Fig. 6a, b)

Figure 6 NF-κB expression in Ars. alb. 30C-treated cell. (a,b) Induced of expression NF-κB both at translational and transcript level was observed after Ars. alb 30C treatment. Bars shown in panels represent normalized data (mean ± SEM, n = 3).

As TLR signalling pathways activate downstream transcription factor NF-κB, so we further validated expression of NF-κB in transcript and translate levels. Our findings showed induced expression of NF-κB in Ars. alb 30 C-treated cells (Fig. 6a, b).

Induced expression of pro-inflammatory cytokines in Ars. alb 30C-treated cells (Fig. 7a–e)

Figure 7 Expression of pro-inflammatory markers in Ars. alb 30C-treated cell. (a–c) Pro-inflammatory molecules like iNOS, COX-2, and TNF-α induced at transcript level after Ars. alb 30C treatment. Bars shown in panels represent normalized data (mean ± SEM, n = 3), *P ≤ 0.05. (d,e) Induced expression of IL-6 & TNF-α was observed in Ars. alb 30C-treated cells by ELISA. Bars shown in panels represent normalized data (mean ± SEM, n = 3), *P ≤ 0.05.

Further, expression of pro-inflammatory cytokines TNF-α, COX-2, and iNOS at the transcript level was evaluated after Ars. alb 30C treatment. Our results confirmed the up regulation of these pro-inflammatory effector molecules in Ars. alb 30C-treated cells as compared to the untreated and alcohol-treated samples (Fig. 7a–c). The up-regulation of pro-inflammatory effector molecules like IL-6 and TNF-α after 30C treatment was confirmed by ELISA results (Fig. 7d. e).

Discussion

Research on nanoparticles (NPs) is a rapidly developing area of biomedicine and has made a significant contribution to health and the cure of diseases36. The size, shape, and surface charge of NPs affect their cellular uptake and effectiveness37. Due to larger surface area, a higher degree of surface charge, and proton exchangeability, nanoparticles show significant immunomodulatory activity38,39. Various engineered and naturally-derived nanoparticles like cell membrane-derived nanoparticles (CMDNs) and silver nanoparticles (AgNPs), are reported to have immunomodulatory activity40,41. Although, homeopathic drugs are prepared by ultrahigh dilutions; the possibility of formation of nanoparticles cannot be avoided42,43. For the first time, we have reported the existence of crystalline As2O3 nanoparticles of cubic structure with an average size of ~ 15 nm and their aggregates in Ars. alb 30C by TEM & XRD. Our results are in line with previous findings of Ganeshchandra et al., who demonstrated widespread organ distribution of 15 nm gold NPs that signifies the effect of size on nanoparticle function44. Based on the physicochemical properties and type of target cells, aggregates of NPs qualitatively influence their cellular uptake. Higher surface curvature, irregular shape, and asymmetric structure of the aggregates significantly affect their characteristics. This is the reason for the higher uptake of NPs aggregates rather than single NP45. Nanoparticles can modulate the activity of innate immune cells by inducing inflammatory response46. Several studies reported the ability of NPs to induce M0 to M1 macrophage polarization and reprogramming based on different physicochemical features, such as chemical composition, size, and surface modifications47. Our SERS data indicated M1 macrophage polarization with a change in lipid and protein signatures like LPS-induced M1 macrophages48. M1 macrophages are associated with TLR signalling pathways and induce the expression of pro-inflammatory effectors like TNF-α, COX-2, IL-6, and iNOS49. TLR-3 and TLR-7 are markers of innate immune response expressed in the endosome of macrophage cells and are associated with pro-inflammatory pathways50,51. TLR-3 responds to dsRNA and TLR-7 does the same against ssRNA52,53. In this context recent reports confirmed the activation of TLR-3 and TLR-7 during SARS-CoV-2 infection54. In a related study, Menezes et al., also demonstrated lower TLR-3 expression in patients having severe COVID-19 infections55. Nayak et al., in their recent study demonstrated the significance of Ars. alb 30C as a preventive against SARS-CoV-228,29, but the underlying mechanism was not resolved. Our findings indicate induced expression of TLR-3 and TLR-7 in murine macrophages after Ars. alb 30C treatment along with their adaptor molecules TRIF and MyD88, respectively. This strongly supports the immunomodulatory potential of Ars. alb 30C and substantiates its preventive role against COVID-19 as proposed in previous studies28,29. As TLR-3 and TLR-7 are mostly double-stranded and single-stranded RNA sensors, respectively, their induced expression might provide further antiviral responses in Ars. alb 30C-treated cells. It is essential to mention that several vaccines have been designed and developed taking TLR-3 and 7 agonists as adjuvants to improve the efficacy of the vaccines22,23. We have further reported an unaltered total ROS level in Ars. alb 30C-treated cells, which suggests that the drug does not contribute to additional ROS production. No change in cell viability was observed after Ars. alb 30C treatment which indicated no significant cell death after drug treatment. Our findings are consistent with previous studies which also reported that Ars. alb 30C could promote antioxidant defence and upregulation of glutathione (GSH) with a concomitant reduction of total ROS level in yeast and mammals, which supports its therapeutic potential against oxidative stress-associated pathogenesis25,56,57.

Taken together, this study, for the first time, reports the existence of a crystalline nanostructure of As2O3 in Ars. alb 30C dilution, with a clear evidence of drug-dependent macrophage polarization, upregulation of TLR pathways, and a consequent immune effector function with distinct signature molecules. Figure 8 summarizes Ars. alb 30C-mediated immune priming of macrophage and stimulation of “TLR-3-TRIF” & “TLR-7-MyD88”- signalling events leading to pro-inflammatory cytokine production as a part of its immune effector function.Figure 8 Proposed mechanism for Ars. alb 30C-induced M1-macrophage polarization and innate immune response. Ars. alb 30C crystalline nanostructure induces TLR-3 and TLR-7 expression in endosome of M1-polarized macrophage cells along with the adaptor molecules TRIF and MyD88 respectively, and surface marker CD80. Further, this activates transcription factor NF-κB and downstream pro-inflammatory cytokines COX-2, TNF-α, IL-6, and iNOS.

Immunomodulation which promotes the innate protective mechanism or surveillance in the host, is increasingly receiving attention as an alternative to vaccination. This is due to the rapid and unpredictable mutation of microbial pathogens which can evade the immune barrier58. An emerging strategy to combat infectious disease is the synergistic approach of vaccination and innate immune modulator (against TLR modulation). Mifsud et al., have opined that innate immune modulator-mediated long-term immunity is particularly important in cases of epidemic with recurrent infections. TLR stimulation by suitable agonist is crucial in sensing or detecting the pathogen/pathogen-derived signature molecule for their self-elimination58. This work, using in vitro model, established the mechanism of action of Ars. alb 30C as an immunomodulator. For the first-time our findings revealed the presence of As2O3 crystalline nanostructures in Ars. alb 30C, providing compelling evidence to explain the causal mechanism. Furthermore, the expression of innate immune markers and pro-inflammatory cytokines related to M1 macrophages is evident in Ars. alb 30C-treated murine macrophage cells.

As homeopathic drugs are less costly and have minimal side-effects, Ars. alb 30C can be a prospective alternative medicine to boost innate immunity, with significant clinical applications. However, a major challenge to the acceptance of homeopathic medications is the lack of pharmacological validation. In this context, our findings provide considerable evidence in support of its efficacy and reveal a novel mechanism of Ars. alb 30C-mediated M1 macrophage polarization and induction of innate immune response. Ars. alb 30C can be used as innate immune modulator, particularly as TLR-3 and TLR-7 agonist, which can provide host-dependent long lasting innate immunity in general, and against the invading pathogens or infectious diseases, in particular. Nevertheless, further study with animal models and human trials needs to be carried out before translational intervention.

Materials and methods

Cell culture and Ars. Alb 30C treatment

Murine macrophage cell lines RAW 264.7 were obtained from National Centre for Cell Science (NCCS), Pune, India and cultured in DMEM complete medium under standard cell culture conditions59. Ars. alb 30C was procured from Dr. Reckeweg.co, Germany and was used at a dilution of 10–4 with complete media by serial dilution. Treatment was done for 24 h as per the standardised protocol. Since the drug was prepared in alcohol (90% ethanol), we included a solvent control (SC) as 90% ethanol which was also diluted to 10–4 with complete media60.

Total RNA extraction and cDNA synthesis

1 × 106 RAW 264.7 cells were treated with Ars. alb 30C for 24 h and total RNA was extracted from treated, vehicle control and media control samples by using TRIZOL reagent (Invitrogen) according to the manufacturer’s manual. Then the total RNA was reverse transcribed to synthesize cDNA using reverse transcriptase Revert Aid H minus (Thermo Scientific, USA). cDNA templates were diluted to 100 ng of working concentration.

Quantitative real-time PCR analysis

Real-time PCR was performed by using specific primers for TLR-3, TLR-7 and their adaptor molecules TRIF and MyD88, respectively. M1-associated transcription factor NF-κB was also evaluated by real-time PCR. Expressions of pro-inflammatory effector molecules like COX-2, iNOS, and TNF-α were also checked for validation of M1-macrophage polarization. GAPDH was used as housekeeping gene (Table 1 for primer sequence). SYBR Green (ABI, USA) was used to amplify and detect DNA in qPCR by Quant studio 5 system (Applied Biosystems, CA, and USA). Real-time PCR amplification reactions were carried out in a final volume of 12.5 μl, which contained 8.5 μl dH2O, 2.5 μl SYBR Green, 0.5 μl of each primer and 0.5 μl diluted cDNA template. PCR conditions were as follows Hold stage: 50 °C for 2 min, 95 °C for 10 min, PCR stage: 95 °C for 15 s followed by suitable annealing for 1 min and 72 °C for 30 s.

SDS PAGE and western blotting

1 × 106 cells were seeded in each well of 60 mm cell culture plate and treated with Ars. alb 30C and 90% ethanol (10–4 diluted) as vehicle control. After 24 h of post-treatment, total proteins were isolated and estimated by Bradford’s method61. 30 µg of proteins were loaded in each well and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with a stacking gel concentration of 5% and a resolving gel concentration of 10%. The gel was run for 90 min at 45 milliamps, till the dye front moved out of the gel. Then, Western blotting was performed and the proteins were transferred (through wet transfer) to a polyvinylidene fluoride (PVDF) membrane using at 40 V for 4 h. After completion of transfer, membranes were incubated with blocking solution of 5% Non-Fat Dry milk (NFDM) in Tris-buffered saline with 0.1% Tween 20 detergent (TBST) buffer for 1 h. Further, membranes were probed with specific antibodies against target molecules like TLR-7, TLR-3, MyD88, and NF-κB (Santacruz, USA) at a dilution of 1:1000 in TBST for overnight at 4 °C. GAPDH (Imgenex India Pvt. Ltd., India) was used as a loading control at a dilution of 1:1000. Next day, blots were washed three times with 1× TBST followed by incubation with secondary antibodies (anti-rabbit and anti-mouse) at a dilution of 1:5000 in TBST for 1 h at room temperature, followed by another round of washing with 1× TBST and final wash with 1× TBS. ImmunoCruz western blotting luminol reagent (Santacruz, USA) was used as a detection reagent and developed using X-ray film. Images were scanned with a gel documentation system (VILBER, USA). Band intensity was quantified by using Image J densitometry software. GAPDH was used as an internal loading control to normalize the expression of the proteins. All the original blots referenced in the document are provided in the supplementary file.

Enzyme-linked immunosorbent assay (ELISA)

25 × 103 cells were seeded in each well of 96 well plates and Ars. alb 30C treatment was done along with vehicle control and media control. After 24 h of post-treatment cell supernatant were collected from all three groups, centrifuged at 1000×g at 4 °C for 20 min and the concentrations of TNF-α and IL-6 were measured with ELISA kits (Elabscience, USA) as per manufacturer’s instruction. All samples were assayed in triplicate and measured at 450 nm wavelength.

Confocal microscopy

1 × 103 RAW 264.7 cells were seeded on 18 mm cover slips and treated with Ars. alb 30C for 24 h. Another group of cells were treated with 90% alcohol (10–4 diluted) and the third group was left untreated. After the incubation period was over, cells were fixed with 4% paraformaldehyde at 37 °C for 15 min followed by permeabilization with 0.1% Triton-X-100 for 15 min. Cells were washed with 1X Phosphate Buffered Saline with Tween 20 (PBST) for 3 times followed by blocking with 3% BSA for 1 h at room temperature. Then, primary antibodies were added specific for TLR-3 and TLR-7 at a dilution of 1:200 in 3% BSA for overnight at 4 °C. The next day, cells were washed with 1X PBST and incubated with fluorescently conjugated (Alexa fluor 488) secondary antibodies at a dilution of 1:1000 in blocking solution for 1 h in dark. Subsequently 3 times washing was performed with 1X PBST followed by treatment with nuclear stain DAPI (4', 6-Diamidino-2-Phenylindole, Dilactate) (Invitrogen) for 20 min at a dilution of 1:2000 in 1X PBS. Finally, coverslips were washed with 1× PBS for three times and mounted over slides by using Fluoromount aqueous mounting medium (Sigma Aldrich). Once the slides were dried, expression of TLR-3 and TLR-7 were assessed by a laser scanning confocal microscope (Confocal Microscope Platforms, Leica, STELLARIS). All images were acquired at room temperature and digitally processed along with intensity measurement by image J software.

Total reactive oxygen species (ROS) measurement

Total reactive oxygen species measurement was evaluated by Dichlorodihydrofluorescein diacetate (DCFH-DA) treatment as per standardized protocol62. Ars. alb 30C treated cells, 90% alcohol (10–4 diluted)-treated cells and control cells were treated with DCFH-DA for 1 h followed by PBS wash three times. Then, cells were sonicated for 2–3 min followed by centrifugation at 10,000×g for 5 min. Then supernatant was collected and fluorescence reading (Cary Eclipse Fluorescence Spectrophotometer, Agilent Technologies, USA) was taken at 488 nm excitation and 522 nm emission.

Acridine orange (AO) and propidium iodide (PI) double staining for cell viability

Cell viability and apoptosis induction in Ars. Alb 30C-treated cells was evaluated by PI and AO double staining assay63by using a fluorescent microscope (BX53, Olympus,Japan). RAW 264.7 cells were seeded at a density of 5 × 103 cells/ml on cover slips in a six well plate and treated with Ars. alb 30C for 24 h. Another group of cells were treated with 90% alcohol (10–4 diluted) and the third group was left untreated. After the incubation period cells were washed with 1× PBS and treated with 10 µM of AO and 5 µM of PI for 30 min. The stained cells were visualized under fluorescence microscope.

Surface-enhanced Raman scattering (SERS)

Macrophage polarization was detected using surface enhanced Raman spectroscopy (SERS) technique. 1 × 103 RAW 264.7 cells were seeded on cover slips and treated with Ars. alb 30C for 24 h. Lippopolysaccharide (LPS) (100 ng/ml) treated cells were used as a positive control for macrophage polarization from M0 to M1. The rest of the two groups were treated with 90% alcohol (10–4 dilution) and left untreated. Plasmonic gold nanoparticles of 15 nm size were used for surface enhancement. After incubation for 24 h, cover slips with cells were fixed using cold methanol (to reduce the fluorescence background)64. Then gold nanoparticles were added to the cells and kept in the open air for 24 h. SERS spectra were obtained using a Renishaw Raman spectrometer coupled to an Olympus microscope and equipped with a 532 nm laser-focused on the cells' nucleus through a 100× objective (NA = 0.9). The same objective lens was used to collect Raman scattered light after interaction with the sample in backscattering geometry. The Raman peak positions were calibrated by the 520 cm−1 vibrational band of a silicon wafer. Under similar conditions, 20 cell spectra for each stimulated group (M0, and M1) were measured in the spectral range of 700–1800 cm−1. In order to minimize laser-induced heating of the specimens, low-power irradiation at the sample surface was used, approximately 3 mW, over a short exposure time (5 s laser exposure for 5 accumulations). The diffraction grating used had 2400 lines/mm, which yielded a spectral resolution of 1.5 cm−1.

Data pre-processing and spectral analysis

Before conducting the spectral analysis, the spectra were smoothed, background-corrected, and normalized by the area to suppress external noise and enhance useful information about the biochemical composition of cells. All procedures were performed using Origin Lab software. The background spectra are subtracted from cellular spectra, ensuring that the measured signal is assigned only to the cells.

Transmission electron microscope

Transmission electron microscopy (TEM) was performed to evaluate the structural arrangement of Ars. alb 30C. The sample was transferred to a carbon-coated Cu-TEM grids and left to dry before observation. TEM investigation was performed on a JEOL-JEM-F-200 TEM operated at 200 kV.

X-ray diffraction (XRD)

To characterize the crystal structure, powder XRD diffraction (θ − 2θ) scans were recorded for the drop-coated films of Ars. alb 30C sample on 300 nm SiO2/Silicon substrate, using Bruker DA VINCI D8 ADVANCE diffractometer equipped with a Cu-Kα source (λ = 0.154 nm).

Statistics

Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, California, USA). The statistical significance among groups were determined using analysis of variance (ANOVA). All data are presented as mean ± standard error of the mean (SEM) from three or more independent experiments. The level of significance was set at p ≤ 0.05.

Supplementary Information

Supplementary Figures.

Abbreviations

TLR Toll like receptors

NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)

IL-6 Interleukin 6

HIF-1α Hypoxia-inducible factor 1α

MyD88 Myeloid differentiation primary response 88

TRIF TIR-domain-containing adaptor-inducing interferon-β

LPS Lipopolysaccharides

SERS Surface-enhanced Raman scattering

TEM Transmission electron microscopy

PRRs Pattern recognition receptors

TIR Toll-interleukin-1 receptor

DMEM Dulbecco's modified eagle medium

TNF-α Tumor necrotic factor α

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

PVDF Polyvinylidene fluoride

DCFDA Dichlorodihydrofluorescein diacetate

ROS Reactive oxygen species

iNOS Inducible nitric oxide synthase

SAED Selected area electron diffraction

HR High resolution

FFT Fast Fourier transform

As2O3 Arsenic trioxide

PAMPs Pathogen-associated molecular patterns

NK Natural killer

CMDNs Cell-membrane derived nanoparticles

ANOVA Analysis of variance

ssRNA Single stranded ribonucleic acid

NP Nanoparticle

TBST Tris-buffered saline with 0.1% tween 20 detergent

NFDM Non-fat dry milk

PBST Phosphate buffered saline with tween 20

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-51319-w.

Acknowledgements

Authors would like to thank Central Council for Research in Homoeopathy, CCRH, Ministry of AYUSH, Government of India for a collaborative research grant and supporting open access publication. SR thankful to the RUSA 2.0 Centre of Excellence (CoE) in Environment, Climate Change and Public Health (ECCPH), Utkal University supported by MHRD, Govt of India for the provisional fellowship. Support from DBT, Government of India, New Delhi to Department of Biotechnology, Utkal University and Center of Excellence in Integrated Omics and Computational Biology (under WB-OHEPEE), Utkal University, Bhubaneswar are also gratefully acknowledged for infrastructure support. We are thankful to Dr. Ashutosh Rath, senior scientist, CSIR-IMMT, Bhubaneswar for analysis of TEM data and fruitful discussions. Our sincere gratitude to Dr. Soma Chattopadhyay, Scientist F, Institute of Life science, Bhubaneswar, for providing us Phospo-IRF and IRF antibodies. We are sincerely grateful to Dr. S. Deepika, Assistant Professor, Dept. of English, Utkal University, Bhubaneswar, for language correction and copy editing of this manuscript.

Author contributions

J.D., conceptualized, discussed, drafted, and revised the manuscript. S.R., conceptualized, discussed, performed experiments, drafted, and revised the manuscript. J.P.J., discussed, performed experiments, analysed data, and participated in manuscript writing. K.K., performed SERS and participated in manuscript writing. S.M., performed TEM. J.P., conducted experiments and analysing data, G.B.N.C., discussed and participated in manuscript writing. D.N., and S.K., revised the manuscript. All authors have read and agreed to the published version of the manuscript. All authors read and approved the final manuscript.

Funding

This work is supported by Central Council for Research in Homeopathy (CCRH), Ministry of AYUSH, Government of India (17-6/2015-16/CCRH/Tech/Coll/Utkal University/1762).

Data availability

The raw scanned blot images used in the figures are provided in Supplementary Data as supplementary Fig. 1. Other datasets used and/ or analysed in the current study are available from the corresponding author upon request.

Competing interests

The authors declare no competing interests.

This article has been retracted. Please see the retraction notice for more detail:https://doi.org/10.1038/s41598-024-70287-9

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Suvasmita Rath and Jyoti Prava Jema.

Change history

8/21/2024

This article has been retracted. Please see the Retraction Notice for more detail: 10.1038/s41598-024-70287-9
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References

1. Marshall JS Warrington R Watson W Kim HL An introduction to immunology and immunopathology Allergy Asthma Clin. Immunol. 2018 14 1 10 10.1186/s13223-018-0278-1 29339956
Marshall, J. S., Warrington, R., Watson, W. & Kim, H. L. An introduction to immunology and immunopathology. Allergy Asthma Clin. Immunol. 14, 1–10 (2018).29339956 10.1186/s13223-018-0278-1
2. Bikard D Marraffini LA Innate and adaptive immunity in bacteria: mechanisms of programmed genetic variation to fight bacteriophages Curr. Opin. Immunol. 2012 24 15 20 10.1016/j.coi.2011.10.005 22079134
Bikard, D. & Marraffini, L. A. Innate and adaptive immunity in bacteria: mechanisms of programmed genetic variation to fight bacteriophages. Curr. Opin. Immunol. 24, 15–20 (2012).22079134 10.1016/j.coi.2011.10.005
3. Li MO Innate immune cells in the tumor microenvironment Cancer Cell 2021 39 725 729 10.1016/j.ccell.2021.05.016 34129817
Li, M. O. et al. Innate immune cells in the tumor microenvironment. Cancer Cell 39, 725–729 (2021).34129817 10.1016/j.ccell.2021.05.016
4. Uthaisangsook S Day NK Bahna SL Good RA Haraguchi S Innate immunity and its role against infections Ann. Allergy Asthma Immunol. 2002 88 253 265 10.1016/S1081-1206(10)62005-4 11926618
Uthaisangsook, S., Day, N. K., Bahna, S. L., Good, R. A. & Haraguchi, S. Innate immunity and its role against infections. Ann. Allergy Asthma Immunol. 88, 253–265 (2002).11926618 10.1016/S1081-1206(10)62005-4
5. Fitzgerald KA Kagan JC Toll-like receptors and the control of immunity Cell 2020 180 1044 1066 10.1016/j.cell.2020.02.041 32164908
Fitzgerald, K. A. & Kagan, J. C. Toll-like receptors and the control of immunity. Cell 180, 1044–1066 (2020).32164908 10.1016/j.cell.2020.02.041
6. Matsumoto M Oshiumi H Seya T Antiviral responses induced by the TLR3 pathway Rev. Med. Virol. 2011 21 67 77 10.1002/rmv.680 21312311
Matsumoto, M., Oshiumi, H. & Seya, T. Antiviral responses induced by the TLR3 pathway. Rev. Med. Virol. 21, 67–77 (2011).21312311 10.1002/rmv.680
7. O’Neill LA TLR-7 and antiviral immunity Trends Immunol. 2002 23 234 10.1016/S1471-4906(02)02199-3
O’Neill, L. A. TLR-7 and antiviral immunity. Trends Immunol. 23, 234 (2002).10.1016/S1471-4906(02)02199-3
8. Su R The TLR3/IRF1/type III IFN axis facilitates antiviral responses against enterovirus infections in the intestine MBio 2020 11 22 10.1128/mBio.02540-20
Su, R. et al. The TLR3/IRF1/type III IFN axis facilitates antiviral responses against enterovirus infections in the intestine. MBio 11, 22 (2020).10.1128/mBio.02540-20
9. Chen Y Lin J Zhao Y Ma X Yi H Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses J. Zhejiang Univ. B 2021 22 609 632 10.1631/jzus.B2000808
Chen, Y., Lin, J., Zhao, Y., Ma, X. & Yi, H. Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses. J. Zhejiang Univ. B 22, 609–632 (2021).10.1631/jzus.B2000808
10. Pal A Pal A Baviskar P RIGI, TLR7, and TLR3 genes were predicted to have immune response against avian influenza in indigenous ducks Front. Mol. Biosci. 2021 8 633283 10.3389/fmolb.2021.633283 34970593
Pal, A., Pal, A. & Baviskar, P. RIGI, TLR7, and TLR3 genes were predicted to have immune response against avian influenza in indigenous ducks. Front. Mol. Biosci. 8, 633283 (2021).34970593 10.3389/fmolb.2021.633283
11. Ma B Modular bioinformatics analysis demonstrates that a Toll-like receptor signaling pathway is involved in the regulation of macrophage polarization Mol. Med. Rep. 2018 30592264
Ma, B. et al. Modular bioinformatics analysis demonstrates that a Toll-like receptor signaling pathway is involved in the regulation of macrophage polarization. Mol. Med. Rep.10.3892/mmr.2018.9486 (2018).30592264
12. Lu CH Involvement of M1 macrophage polarization in endosomal Toll-like receptors activated psoriatic inflammation Mediators Inflamm. 2018 2018 3523 10.1155/2018/3523642
Lu, C. H. et al. Involvement of M1 macrophage polarization in endosomal Toll-like receptors activated psoriatic inflammation. Mediators Inflamm. 2018, 3523 (2018).10.1155/2018/3523642
13. Vidyarthi A TLR-3 stimulation skews M2 macrophages to M1 through IFN-αβ signaling and restricts tumor progression Front. Immunol. 2018 9 1650 10.3389/fimmu.2018.01650 30072995
Vidyarthi, A. et al. TLR-3 stimulation skews M2 macrophages to M1 through IFN-αβ signaling and restricts tumor progression. Front. Immunol. 9, 1650 (2018).30072995 10.3389/fimmu.2018.01650
14. Koo J Hayashi M Verneris MR Lee-Sherick AB Targeting tumor-associated macrophages in the pediatric sarcoma tumor microenvironment Front. Oncol. 2020 10 1 19 10.3389/fonc.2020.581107 32076595
Koo, J., Hayashi, M., Verneris, M. R. & Lee-Sherick, A. B. Targeting tumor-associated macrophages in the pediatric sarcoma tumor microenvironment. Front. Oncol. 10, 1–19 (2020).32076595 10.3389/fonc.2020.581107
15. Meng L Toll-like receptor 3 upregulation in macrophages participates in the initiation and maintenance of pristane-induced arthritis in rats Arthritis Res. Ther. 2010 12 103 10.1186/ar3034 20156318
Meng, L. et al. Toll-like receptor 3 upregulation in macrophages participates in the initiation and maintenance of pristane-induced arthritis in rats. Arthritis Res. Ther. 12, 103 (2010).20156318 10.1186/ar3034
16. Kishore A Petrek M Roles of macrophage polarization and macrophage-derived miRNAs in pulmonary fibrosis Front. Immunol. 2021 12 6784 10.3389/fimmu.2021.678457
Kishore, A. & Petrek, M. Roles of macrophage polarization and macrophage-derived miRNAs in pulmonary fibrosis. Front. Immunol. 12, 6784 (2021).10.3389/fimmu.2021.678457
17. Twigg H Macrophages in innate and acquired immunity Semin. Respir. Crit. Care Med. 2004 25 21 31 10.1055/s-2004-822302 16088446
Twigg, H. Macrophages in innate and acquired immunity. Semin. Respir. Crit. Care Med. 25, 21–31 (2004).16088446 10.1055/s-2004-822302
18. Zhang Y Zou J Chen R An M0 macrophage-related prognostic model for hepatocellular carcinoma BMC Cancer 2022 22 791 10.1186/s12885-022-09872-y 35854246
Zhang, Y., Zou, J. & Chen, R. An M0 macrophage-related prognostic model for hepatocellular carcinoma. BMC Cancer 22, 791 (2022).35854246 10.1186/s12885-022-09872-y
19. Cendrowicz E Sas Z Bremer E Rygiel TP The role of macrophages in cancer development and therapy Cancers (Basel) 2021 13 1946 10.3390/cancers13081946 33919517
Cendrowicz, E., Sas, Z., Bremer, E. & Rygiel, T. P. The role of macrophages in cancer development and therapy. Cancers (Basel) 13, 1946 (2021).33919517 10.3390/cancers13081946
20. Glass CK Natoli G Molecular control of activation and priming in macrophages Nat. Immunol. 2016 17 26 33 10.1038/ni.3306 26681459
Glass, C. K. & Natoli, G. Molecular control of activation and priming in macrophages. Nat. Immunol. 17, 26–33 (2016).26681459 10.1038/ni.3306
21. Mosser DM Edwards JP Exploring the full spectrum of macrophage activation Nat. Rev. Immunol. 2008 8 958 969 10.1038/nri2448 19029990
Mosser, D. M. & Edwards, J. P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958–969 (2008).19029990 10.1038/nri2448
22. Angelopoulou A Imiquimod—A toll like receptor 7 agonist—Is an ideal option for management of COVID 19 Environ. Res. 2020 188 109858 10.1016/j.envres.2020.109858 32846644
Angelopoulou, A. et al. Imiquimod—A toll like receptor 7 agonist—Is an ideal option for management of COVID 19. Environ. Res. 188, 109858 (2020).32846644 10.1016/j.envres.2020.109858
23. Liang Z Adjuvants for coronavirus vaccines Front. Immunol. 2020 11 589833 10.3389/fimmu.2020.589833 33240278
Liang, Z. et al. Adjuvants for coronavirus vaccines. Front. Immunol. 11, 589833 (2020).33240278 10.3389/fimmu.2020.589833
24. Wang Y Smith W Hao D He B Kong L M1 and M2 macrophage polarization and potentially therapeutic naturally occurring compounds Int. Immunopharmacol. 2019 70 459 466 10.1016/j.intimp.2019.02.050 30861466
Wang, Y., Smith, W., Hao, D., He, B. & Kong, L. M1 and M2 macrophage polarization and potentially therapeutic naturally occurring compounds. Int. Immunopharmacol. 70, 459–466 (2019).30861466 10.1016/j.intimp.2019.02.050
25. Paumier A Verre J Tribolo S Boujedaini N Anti-oxidant effect of high dilutions of Arnica montana, Arsenicum Album, and Lachesis Mutus in microglial cells in vitro Dose-Response 2022 20 155932582211036 10.1177/15593258221103698
Paumier, A., Verre, J., Tribolo, S. & Boujedaini, N. Anti-oxidant effect of high dilutions of Arnica montana, Arsenicum Album, and Lachesis Mutus in microglial cells in vitro. Dose-Response 20, 155932582211036 (2022).10.1177/15593258221103698
26. Gupta V Mathur M Immunomodulatory effects of homoeopathic medicines: A review of pre-clinical studies Indian J. Res. Homoeopath. 2018 12 90 10.4103/ijrh.ijrh_26_18
Gupta, V. & Mathur, M. Immunomodulatory effects of homoeopathic medicines: A review of pre-clinical studies. Indian J. Res. Homoeopath. 12, 90 (2018).10.4103/ijrh.ijrh_26_18
27. Ive EC Couchman IMS Reddy L Therapeutic effect of arsenicum album on leukocytes Int. J. Mol. Sci. 2012 13 3979 3987 10.3390/ijms13033979 22489193
Ive, E. C., Couchman, I. M. S. & Reddy, L. Therapeutic effect of arsenicum album on leukocytes. Int. J. Mol. Sci. 13, 3979–3987 (2012).22489193 10.3390/ijms13033979
28. Nayak D Efficacy of Arsenicum album 30C in the prevention of COVID-19 in individuals residing in containment areas—A prospective, multicentre, cluster-randomized, parallel arm, community based, open-label study Complement. Med. Res. 2022 36195077
Nayak, D. et al. Efficacy of Arsenicum album 30C in the prevention of COVID-19 in individuals residing in containment areas—A prospective, multicentre, cluster-randomized, parallel arm, community based, open-label study. Complement. Med. Res.10.1159/000526897 (2022).36195077
29. Nayak D Effectiveness of Arsenicum album 30C in prevention of COVID-19 in individuals residing in containment zones of Delhi—A prospective, community-based, parallel cohort study Homeopathy 2022 35768003
Nayak, D. et al. Effectiveness of Arsenicum album 30C in prevention of COVID-19 in individuals residing in containment zones of Delhi—A prospective, community-based, parallel cohort study. Homeopathy10.1055/s-0042-1745755 (2022).35768003
30. Ye Y Gaugler B Mohty M Malard F Old dog, new trick: Trivalent arsenic as an immunomodulatory drug Br. J. Pharmacol. 2020 177 2199 2214 10.1111/bph.15011 32022256
Ye, Y., Gaugler, B., Mohty, M. & Malard, F. Old dog, new trick: Trivalent arsenic as an immunomodulatory drug. Br. J. Pharmacol. 177, 2199–2214 (2020).32022256 10.1111/bph.15011
31. Hu J Local delivery of arsenic trioxide nanoparticles for hepatocellular carcinoma treatment Signal Transduct. Target. Ther. 2019 4 1 7 30652029
Hu, J. et al. Local delivery of arsenic trioxide nanoparticles for hepatocellular carcinoma treatment. Signal Transduct. Target. Ther. 4, 1–7 (2019).30652029
32. Jadhav V Ray P Sachdeva G Bhatt P Biocompatible arsenic trioxide nanoparticles induce cell cycle arrest by p21WAF1/CIP1 expression via epigenetic remodeling in LNCaP and PC3 cell lines Life Sci. 2016 148 41 52 10.1016/j.lfs.2016.02.042 26883975
Jadhav, V., Ray, P., Sachdeva, G. & Bhatt, P. Biocompatible arsenic trioxide nanoparticles induce cell cycle arrest by p21WAF1/CIP1 expression via epigenetic remodeling in LNCaP and PC3 cell lines. Life Sci. 148, 41–52 (2016).26883975 10.1016/j.lfs.2016.02.042
33. Zanganeh S Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues Nat. Nanotechnol. 2016 11 986 994 10.1038/nnano.2016.168 27668795
Zanganeh, S. et al. Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues. Nat. Nanotechnol. 11, 986–994 (2016).27668795 10.1038/nnano.2016.168
34. Italiani P Della Camera G Boraschi D Induction of innate immune memory by engineered nanoparticles in monocytes/macrophages: From hypothesis to reality Front. Immunol. 2020 11 566309 10.3389/fimmu.2020.566309 33123137
Italiani, P., Della Camera, G. & Boraschi, D. Induction of innate immune memory by engineered nanoparticles in monocytes/macrophages: From hypothesis to reality. Front. Immunol. 11, 566309 (2020).33123137 10.3389/fimmu.2020.566309
35. Ribeiro ARB Application of Raman spectroscopy for characterization of the functional polarization of macrophages into M1 and M2 cells Spectrochim. Acta Part Mol. Biomol. Spectrosc. 2022 265 120328 10.1016/j.saa.2021.120328
Ribeiro, A. R. B. et al. Application of Raman spectroscopy for characterization of the functional polarization of macrophages into M1 and M2 cells. Spectrochim. Acta Part Mol. Biomol. Spectrosc. 265, 120328 (2022).10.1016/j.saa.2021.120328
36. Salata OV Applications of nanoparticles in biology and medicine J. Nanobiotechnol. 2004 6 1 6
Salata, O. V. Applications of nanoparticles in biology and medicine. J. Nanobiotechnol. 6, 1–6 (2004).
37. Kiio TM Park S Physical properties of nanoparticles do matter J. Pharm. Invest. 2021 51 35 51 10.1007/s40005-020-00504-w
Kiio, T. M. & Park, S. Physical properties of nanoparticles do matter. J. Pharm. Invest. 51, 35–51 (2021).10.1007/s40005-020-00504-w
38. Li Z Liu Y Fang X Shu Z Nanomaterials enhance the immunomodulatory effect of molecular targeted therapy Int. J. Nanomed. 2021 16 1631 1661 10.2147/IJN.S290346
Li, Z., Liu, Y., Fang, X. & Shu, Z. Nanomaterials enhance the immunomodulatory effect of molecular targeted therapy. Int. J. Nanomed. 16, 1631–1661 (2021).10.2147/IJN.S290346
39. Jang J Lim D-H Choi I-H The impact of nanomaterials in immune system Immune Netw. 2010 10 85 10.4110/in.2010.10.3.85 20631878
Jang, J., Lim, D.-H. & Choi, I.-H. The impact of nanomaterials in immune system. Immune Netw. 10, 85 (2010).20631878 10.4110/in.2010.10.3.85
40. Yang Y Wang K Pan Y Rao L Luo G Engineered cell membrane-derived nanoparticles in immune modulation Adv. Sci. 2021 8 1 20 10.1002/advs.202102330
Yang, Y., Wang, K., Pan, Y., Rao, L. & Luo, G. Engineered cell membrane-derived nanoparticles in immune modulation. Adv. Sci. 8, 1–20 (2021).10.1002/advs.202102330
41. Alsaleh NB Silver nanoparticle immunomodulatory potential in absence of direct cytotoxicity in RAW 264.7 macrophages and MPRO 2.1 neutrophils J. Immunotoxicol. 2019 16 63 73 10.1080/1547691X.2019.1588928 31282784
Alsaleh, N. B. et al. Silver nanoparticle immunomodulatory potential in absence of direct cytotoxicity in RAW 264.7 macrophages and MPRO 2.1 neutrophils. J. Immunotoxicol. 16, 63–73 (2019).31282784 10.1080/1547691X.2019.1588928
42. Chikramane PS Suresh AK Bellare JR Kane SG Extreme homeopathic dilutions retain starting materials: A nanoparticulate perspective Homeopathy 2010 99 231 242 10.1016/j.homp.2010.05.006 20970092
Chikramane, P. S., Suresh, A. K., Bellare, J. R. & Kane, S. G. Extreme homeopathic dilutions retain starting materials: A nanoparticulate perspective. Homeopathy 99, 231–242 (2010).20970092 10.1016/j.homp.2010.05.006
43. Vargheese, W. V. Characterization of Arsenic Trioxide Nanoparticles in Ultra-Dilutions of Arsenicum Album at 12c, 30c and 200c Strengths Using Raman Spectroscopy Technique Introduction (2021).
44. Sonavane G Tomoda K Makino K Biodistribution of colloidal gold nanoparticles after intravenous administration: Effect of particle size Colloids Surf. B Biointerfaces 2008 66 274 280 10.1016/j.colsurfb.2008.07.004 18722754
Sonavane, G., Tomoda, K. & Makino, K. Biodistribution of colloidal gold nanoparticles after intravenous administration: Effect of particle size. Colloids Surf. B Biointerfaces 66, 274–280 (2008).18722754 10.1016/j.colsurfb.2008.07.004
45. Behzadi S Cellular uptake of nanoparticles: Journey inside the cell Chem. Soc. Rev. 2017 46 4218 4244 10.1039/C6CS00636A 28585944
Behzadi, S. et al. Cellular uptake of nanoparticles: Journey inside the cell. Chem. Soc. Rev. 46, 4218–4244 (2017).28585944 10.1039/C6CS00636A
46. Swartzwelter BJ The impact of nanoparticles on innate immune activation by live bacteria Int. J. Mol. Sci. 2020 21 9695 10.3390/ijms21249695 33353206
Swartzwelter, B. J. et al. The impact of nanoparticles on innate immune activation by live bacteria. Int. J. Mol. Sci. 21, 9695 (2020).33353206 10.3390/ijms21249695
47. Boraschi D Nanoparticles and innate immunity: New perspectives on host defence Semin. Immunol. 2017 34 33 51 10.1016/j.smim.2017.08.013 28869063
Boraschi, D. et al. Nanoparticles and innate immunity: New perspectives on host defence. Semin. Immunol. 34, 33–51 (2017).28869063 10.1016/j.smim.2017.08.013
48. Orecchioni M Ghosheh Y Pramod AB Ley K Macrophage polarization: Different gene signatures in M1(LPS+) vs. classically and M2(LPS–) vs alternatively activated macrophages Front. Immunol. 2019 10 1084 10.3389/fimmu.2019.01084 31178859
Orecchioni, M., Ghosheh, Y., Pramod, A. B. & Ley, K. Macrophage polarization: Different gene signatures in M1(LPS+) vs. classically and M2(LPS–) vs alternatively activated macrophages. Front. Immunol. 10, 1084 (2019).31178859 10.3389/fimmu.2019.01084
49. Yunna C Mengru H Lei W Weidong C Macrophage M1/M2 polarization Eur. J. Pharmacol. 2020 877 173090 10.1016/j.ejphar.2020.173090 32234529
Yunna, C., Mengru, H., Lei, W. & Weidong, C. Macrophage M1/M2 polarization. Eur. J. Pharmacol. 877, 173090 (2020).32234529 10.1016/j.ejphar.2020.173090
50. Krieg AM Vollmer J Toll-like receptors 7, 8, and 9: linking innate immunity to autoimmunity Immunol. Rev. 2007 220 251 269 10.1111/j.1600-065X.2007.00572.x 17979852
Krieg, A. M. & Vollmer, J. Toll-like receptors 7, 8, and 9: linking innate immunity to autoimmunity. Immunol. Rev. 220, 251–269 (2007).17979852 10.1111/j.1600-065X.2007.00572.x
51. Hu Y Synergy of TLR3 and 7 ligands significantly enhances function of DCs to present inactivated PRRSV antigen through TRIF/MyD88-NF-κB signaling pathway Sci. Rep. 2016 6 1 15 28442746
Hu, Y. et al. Synergy of TLR3 and 7 ligands significantly enhances function of DCs to present inactivated PRRSV antigen through TRIF/MyD88-NF-κB signaling pathway. Sci. Rep. 6, 1–15 (2016).28442746
52. Crozat K Beutler B TLR7: A new sensor of viral infection Proc. Natl. Acad. Sci. USA 2004 101 6835 6836 10.1073/pnas.0401347101 15123819
Crozat, K. & Beutler, B. TLR7: A new sensor of viral infection. Proc. Natl. Acad. Sci. USA 101, 6835–6836 (2004).15123819 10.1073/pnas.0401347101
53. Tengroth L Functional effects of Toll-like receptor (TLR)3, 7, 9, RIG-I and MDA-5 stimulation in nasal epithelial cells PLoS One 2014 9 3 10 10.1371/journal.pone.0098239
Tengroth, L. et al. Functional effects of Toll-like receptor (TLR)3, 7, 9, RIG-I and MDA-5 stimulation in nasal epithelial cells. PLoS One 9, 3–10 (2014).10.1371/journal.pone.0098239
54. Bortolotti D TLR3 and TLR7 RNA sensor activation during SARS-CoV-2 infection Microorganisms 2021 9 1820 10.3390/microorganisms9091820 34576716
Bortolotti, D. et al. TLR3 and TLR7 RNA sensor activation during SARS-CoV-2 infection. Microorganisms 9, 1820 (2021).34576716 10.3390/microorganisms9091820
55. Menezes MCS Lower peripheral blood Toll-like receptor 3 expression is associated with an unfavorable outcome in severe COVID-19 patients Sci. Rep. 2021 11 15223 10.1038/s41598-021-94624-4 34315957
Menezes, M. C. S. et al. Lower peripheral blood Toll-like receptor 3 expression is associated with an unfavorable outcome in severe COVID-19 patients. Sci. Rep. 11, 15223 (2021).34315957 10.1038/s41598-021-94624-4
56. De A Potentized homeopathic drug Arsenicum Album 30C inhibits intracellular reactive oxygen species generation and up-regulates expression of arsenic resistance gene in arsenite-exposed bacteria Escherichia coli J. Chinese Integr. Med. 2012 10 210 227 10.3736/jcim20120212
De, A. Potentized homeopathic drug Arsenicum Album 30C inhibits intracellular reactive oxygen species generation and up-regulates expression of arsenic resistance gene in arsenite-exposed bacteria Escherichia coli. J. Chinese Integr. Med. 10, 210–227 (2012).10.3736/jcim20120212
57. Das D Potentized homeopathic drug Arsenicum Album 30C positively modulates protein biomarkers and gene expressions in Saccharomyces cerevisae exposed to arsenate J. Chinese Integr. Med. 2011 9 752 760 10.3736/jcim20110709
Das, D. Potentized homeopathic drug Arsenicum Album 30C positively modulates protein biomarkers and gene expressions in Saccharomyces cerevisae exposed to arsenate. J. Chinese Integr. Med. 9, 752–760 (2011).10.3736/jcim20110709
58. Mifsud EJ Tan ACL Jackson DC TLR agonists as modulators of the innate immune response and their potential as agents against infectious disease Front. Immunol. 2014 5 79 10.3389/fimmu.2014.00079 24624130
Mifsud, E. J., Tan, A. C. L. & Jackson, D. C. TLR agonists as modulators of the innate immune response and their potential as agents against infectious disease. Front. Immunol. 5, 79 (2014).24624130 10.3389/fimmu.2014.00079
59. Taciak B Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages PLoS One 2018 13 1 13 10.1371/journal.pone.0198943
Taciak, B. et al. Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages. PLoS One 13, 1–13 (2018).10.1371/journal.pone.0198943
60. Arora S Aggarwal A Singla P Jyoti S Tandon S Anti-proliferative effects of homeopathic medicines on human kidney, colon and breast cancer cells Homeopathy 2013 102 274 282 10.1016/j.homp.2013.06.001 24050774
Arora, S., Aggarwal, A., Singla, P., Jyoti, S. & Tandon, S. Anti-proliferative effects of homeopathic medicines on human kidney, colon and breast cancer cells. Homeopathy 102, 274–282 (2013).24050774 10.1016/j.homp.2013.06.001
61. Bradford M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal. Biochem. 1976 72 248 254 10.1016/0003-2697(76)90527-3 942051
Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).942051 10.1016/0003-2697(76)90527-3
62. Kim H Xue X Detection of total reactive oxygen species in adherent cells by 2’,7’-dichlorodihydrofluorescein diacetate staining J. Vis. Exp. 2020 2020 1 5
Kim, H. & Xue, X. Detection of total reactive oxygen species in adherent cells by 2’,7’-dichlorodihydrofluorescein diacetate staining. J. Vis. Exp. 2020, 1–5 (2020).
63. Bank HL Assessment of islet cell viability using fluorescent dyes Diabetologia 1987 30 812 10.1007/BF00275748 3322907
Bank, H. L. Assessment of islet cell viability using fluorescent dyes. Diabetologia 30, 812 (1987).3322907 10.1007/BF00275748
64. Pissuwan D Hattori Y Detection of adhesion molecules on inflamed macrophages at early-stage using SERS probe gold nanorods Nano-Micro Lett. 2017 9 1 9 10.1007/s40820-016-0111-7
Pissuwan, D. & Hattori, Y. Detection of adhesion molecules on inflamed macrophages at early-stage using SERS probe gold nanorods. Nano-Micro Lett. 9, 1–9 (2017).10.1007/s40820-016-0111-7
