Gentian Violet modulates cytokines levels in mice spleen toward an anti-inammatory prole

Introduction: Gentian Violet (GV) is a triphenylmethane industrial dye that is known for its antibacterial, antiviral, anti-helminthic and anti-tumor effects. Although many studies focused on determining the biological and pharmacological applications of GV, its exact effect on the immune response has not been elucidated yet. Methods: In this study, we investigate the immunomodulatory effects of GV in BALB/c mice after intraperitoneal injection of the dye by assessing cytokines levels in the spleen. Results: Our data show that GV-treated mice have decreased level of proinammatory cytokines (IL-1β and TNF-α) and increased level of anti-inammatory cytokines (IL-4) in their spleens. In addition, IFN-γ that can modulate pro-inammatory cytokine production was upregulated in GV-treated mice. Conclusion: Together, these ndings suggest an anti-inammatory activity of GV that warrant further studies investigating the potential of GV in immunotherapy.


Background
Gentian Violet (GV) is a triphenylmethane dye used in industry for ink, sanitary products, ceramics and photo-imaging system [1,2]. The dye, discovered by Charles Lauth [3], is also used by scientists in different biological applications including bactericidal, antifungal and anthelminthic activities [4,5]. In fact, GV was used in the treatment of skin burns, dermal and systemic Candidiasis. The molecule was recognized as an antiseptic of wounds and an inhibitor of mold in poultry. It is very bene cial against parasites such as cutaneous Leischmania and Chagas disease in the blood [6]. Gentian Violet was also known by its e ciency against gram positive organism [7] as well as Nipah and Hendra virus [8]. Despite of all its bene ts, GV is toxic at high doses, and it cannot be managed easily [9][10][11][12] as it can be carcinogenic, and cause gastrointestinal side effects and taste alteration in human body [13,14]. Recent study showed that GV reduces the effect of angiopoetin-2 (ang-2), a proin ammatory receptors in angiogenesis [7], which suggest an immunomodulatory effect of the molecule. In agreement with this nding, GV inhibited the proliferation of breast cancer cells by suppressing the activity of the proin ammatory molecule NF-κB [15][16][17]. Gentian violet was also shown to inhibits reactive oxygen species (ROS), leading to the decline of the in ammatory activity of NF-κB [18,19]. In this study we investigated the immunomodulatory potential of GV in vivo. We show that GV has anti-in ammatory potential by downregulating proin ammatory cytokines (IL-1β and TNF-α) and upregulating antiin ammatory cytokines (IL-4) and IFN-γ that modulates pro-in ammatory cytokine production.

Gentian Violet
Gentian Violet was supplied from Sigma Aldrich (G2039) in powder form and stored at room temperature.
Gentian Violet was dissolved in PBS prior to the experiment and ltered through 0.2 µm sterile syringe lters.

Mice handling and treatment
Eight to ten weeks old female BALB/c mice procured from the from the University of Balamand (UOB) animal house were used in this study. Animals were fed a standard diet and kept at 25 °C in 12 hours day/night cycle and handled according to the Guide for Care and Use of Laboratory Animals of the UOB Faculty of Sciences. Mice were divided into two groups that were injected intraperitoneally with either Gentian Violet (5 mg/kg) or PBS (control). Each mouse underwent three injections with 48 hours (hrs) of interval between each injection. Mice were sacri ced 24 hrs following the last injection by cervical dislocation. Spleens were removed, weighted, and then cut in half. For histology, spleen parts were reserved in chloroform at -20 °C; while those for ELISA were kept in eppendorf tubes at -80 °C. All animals were handled and experimental procedures were carried out according to the guidelines of the Institutional Animal Care and Use Committee at the University of Balamand, with strict adherence to the ethical guidelines for the study of experimental pain in conscious animals [20].

Histopathology
Tissue for histopathology was processed as described in [21,22]. Brie y, spleen samples were processed for dehydration, clearing, and impregnation by Leica TP1020 Tissue Processor. Para n blocks were prepared using ThermoFisher Histostar Tissue Embedding Station and serial sections of 3 µm thickness were cut using Leica RM2255 Fully Automated Rotary Microtome. Sections were placed on slides with 50 mM ethanol, then immerged in dissolved 0.1% gelatin. Dewaxing was performed by emerging the prepared slides 2 × 5 min in Xylol. Samples were then washed 3 × 2 min with 95% ethanol, then for 2 min with 75% ethanol, then 50% ethanol. Samples were drained for 3 min with water, treated with 0.37% HCL in 70% ethanol to remove hemotoxylin excess, and redrained for 2 min with water. Nucleus was stained using ammonia, and cytoplasm was stained using eosin. Slides were washed 5 × 10 min with 95% ethanol, emerged in Xylol and mounted with coverslips. Mounted tissue sections were observed under Swift M2250 Series Monocular lab light microscope for structural changes and abnormalities.

Tissue Homogenization and Protein Quanti cation
Frozen spleen samples were weighted, then homogenized at 4 °C in 1 ml RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS pH = 7.6) supplemented with protease inhibitors. The homogenates were incubated on ice for 30 minutes and then centrifuged at 10 000 g for 30 minutes at 4 °C. Supernatants were transferred to labeled Eppendorf tubes and protein concentration of each sample was quanti ed using Bio-Rad Protein Assay kit according to the manufacturer's recommendations. Samples were stored at -80 °C for cytokine measurement.

Cytokine Measurement
Quantitative evaluation of cytokines was performed using Enzyme-Linked Immuno-Sorbent Assay (ELISA) Development Kits (Cat. #900-K00, Peprotech) according to the manufacturer's recommendations. Brie y, 100 µl of prepared supernatants/standards were added into the 96-well plates in duplicates.
Plates were read with an ELISA plate reader at 405 nm with 650 nm as the correction wavelength. Concentrations of the cytokines TNF-α, IFN-γ, IL-4, IL-10, IL-1ß and IL-13 were estimated using standard curves established with the appropriate recombinant cytokines. The results were expressed as pg/mg of total proteins.

Statistical analysis
Differences among groups were analyzed using GraphPad Prism 6.00 software (GraphPad Software Inc., San Diego USA) by one-way analysis of variance (ANOVA). Results were expressed as means ± SEM. p < 0.05 was considered statistically signi cant.

Results
Histological disorganization of GV-injected mice spleen tissue First, we checked the histology of mice spleen, the site of innate and adaptive immune processes [23], after GV injection. Compared to controls (Fig. 1A, B), which did not show any signi cant changes in spleen tissue histology, we found that spleens from GV-injected mice (Fig. 1C) show indeed remarkable histological changes. In fact, we reported the presence of megacaryocytes (Fig. 1C 1 ) with polynuclear in ammatory in ltration (Fig. 1C 3 ) in GV-injected mice spleen tissue. In addition, slight extension in red pulps (Fig. 1C 4 ) and enlarged lymphoid follicles with aggregates of monocyte-like cells (Fig. 1C 2 ) were remarked. Together, these ndings show that GV injection induce spleen histological disorganization, which suggest an immunomodulatory effect of GV.
Proin ammatory cytokines levels are decreased in GVtreated mice Next, we dissected the immunomodulatory effect of GV by investigating changes in pro-and antiin ammatory cytokine levels in mice spleens after GV intraperitoneal injection. Compared to controls, results show that GV-injected mice show a signi cant decrease in interleukin-1β (IL-1β) levels, a key mediator of the in ammatory response [24] (Fig. 2A). Tumor necrosis factor alpha (TNF-α) levels showed also a signi cant decrease in GV-injected mice (Fig. 2B). TNF-α is a multifunctional cytokine secreted primarily by macrophages, natural killer (NK) cells, and lymphocytes; therefore, holds diverse proin ammatory actions [25]. Given that both downregulated cytokines (IL-1β and TNF-α) are key mediators of pro-in ammatory response, these results suggest that GV negatively affects proin ammatory cytokines levels.
IFN-γ concentration is upregulated upon GV injection GV-treated mice showed increased interferon gamma (IFN-γ) level that was ~ 4.5-fold higher in GVinjected than control mice (Fig. 2C). Interestingly, IFN-γ has dual role in in ammation. In fact, IFN-γ is not only associated with the pathogenesis of chronic in ammatory; but it can also induce anti-in ammatory molecules and modulates pro-in ammatory cytokine production [26]. Taken with proin ammatory cytokines decrease (IL-1β and TNF-α), our ndings suggest that upregulation of IFN-γ is more likely in favor of regulating pro-in ammatory cytokine levels and promotes anti-in ammatory cytokines secretion.

Discussion
Immunomodulation refers to the regulation of the immune response by suppressing or enhancing its components for therapeutic purposes [27][28][29]. Since cytokines are key components of immune response, altering released cytokines concentration will have immediate effects on the immune response. This strategy, termed immunotherapy, is used for the treatment of infections, autoimmune diseases, and cancer, and rely on chemical and biological molecules developed by pharmaceutical companies [30]. Among chemical compounds, triphenylmethane dyes have shown anti-in ammatory activities [31].
Gentian Violet (GV) is a triphenylmethane industrial dye that is known for its antibacterial, antiviral, antihelminthic and anti-tumor effects [4,5]. This work constitutes the rst in vivo study highlighting the immunomodulatory effect of gentian violet. Herein, we show that gentian violet modulates cytokines level in mice spleen. In fact, while pro-in ammatory cytokines (IL-1β, TNF-α) levels are decreased, antiin ammatory cytokine IL-4, and IFN-γ, an inducer of anti-in ammatory molecules and modulator of proin ammatory cytokine production, are upregulated. In addition, observed changes in GV-treated mice spleen histology that are characterized by the presence of in ammation, in ltration and hyperplastic lymphoid follicles emphasize the immunomodulatory role of gentian violet. This activity could be of interest in studies aiming to develop cancer treatment strategies. As such, studies on animal models described cytokines' roles in the pathogenesis of angiogenesis. IL-1β and TNF-α are potent proangiogenic cytokines, while IL-4 are anti-angiogenic cytokines [32,33]. The ability of gentian violet to increase IL-4 and decrease IL-1β and TNF-α levels warrant further studies investigating the antiangiogenic potential of gentian violet in cancer treatment. In agreement with this hypothesis, IL-4 can exhibit an anti-tumor activity, as it is involved in promoting immune response against tumor models including renal cancer, colorectal cancer, spontaneous adenocarcinoma, colon carcinoma, brosarcoma and melanoma [34,35].
Despite the fact that IL-13 shares many biologic activities with IL-4, the insensitivity of IL-13 levels to gentian violet treatment could be explained by the different T-cell subsets that produce IL-13, which may not be affected by gentian violet treatment. Alternatively, since IL-13 secretion requires CD4 + T cells differentiation into Th2 cells in the presence of IL-4 [36][37][38], IL-13 levels may increase later as a consequence of IL-4 upregulation. Contrarily to Humans, where IL-4 inhibits IFN-γ secretion [39,40], in mice, IL-4 is known to enhance IFN-γ secretion in response to a variety of stimuli [41]. These ndings are in agreement with ours showing an increase in IFN-γ levels in GV-treated mice. On the other hand, IL-4, IL-10, and IL-13 were previously shown to inhibit the production of IL-1β and tumor necrosis factor-α [42], which is comparable to our ndings showing an increase in IL-4 along with a decrease in IL-1β and TNFα. These ndings suggest that IL-1β and TNF-α levels decrease could be mediated by IL-4 level increase. In fact, it is known that IL-4 plays a crucial role in shaping the nature of immune responses [43] and that IL-4 production is triggered in response to receptor activation by TH2-type CD4 + T cells, basophils, and mast cells [44]. Since gentian violet signi cantly upregulates IL-4 levels, it is likely to suggest that this dye can activate IL-4-dependant adaptive immune system represented by naïve CD4 + T cells differentiation into Th2 cells [36]. Given the importance of IL-4 in shaping the immune system and disease emergence [43], our ndings highlight the importance of depicting the mechanism by which gentian violet triggers IL-4 upregulation and its possible bene cial applications.

Conclusion
The present study demonstrates that Gentian Violet can act as an immumomodulator in rodent model, which favors anti-in ammation by downregulating pro-in ammatory cytokines levels and upregulating anti-in ammatory cytokines and modulator of pro-in ammatory cytokine production. These ndings warrant further studies understanding the mechanism by which Gentian Violet modulate the immune system and investigating its potential in developing new therapies.

Declarations Ethical Approval and Consent to participate
The authors certify that all animals were handled and experimental procedures of this work were carried out according to the guidelines of the Institutional Animal Care and Use Committee at the University of Balamand, with strict adherence to the ethical guidelines for the study of experimental pain in conscious animals (Zimmermann, 1983).