Taurine (chemical structure, C₂H₇NO₃S or NH₂CH₂CH₂SO₃H) was purchased from AppliChem ITW Companies (Taurine BioChemica, A1140,1000, Lot 3M004589). The purity was >99% and the molecular weight was 125.15 g/mol. BAT (chemical structure, C₇H₇BrNNaO₂S x 2H₂O or [CH₃-C₆H₄-SO₂-N-Br]-Na⁺ x 2H₂O) was kindly provided by the laboratories of Professor Dr Waldemar Gottardi and Professor Dr Markus Nagl at the Institute of Hygiene and Medical Microbiology of the Medical University of Innsbruck (Bromamine T, Lot no 29/06/2016). BAT (N-bromo-4-toluenesulfonamide sodium salt, BAT x 2H₂O) was prepared by the reaction of chloramine T with elemental bromine according to the method of Nair et al (39). The specifications were a potency of 95.8%, a bromine content of 24.83% and an effective molecular weight of 322.02 g/mol. As a result, Nair et al (39) demonstrated the synthesis of BAT and Walczewska et al (38) demonstrated that BAT was a stable active bromine compound.

The J774.A1 murine Mφs were obtained from the laboratories of Professor Dr Waldemar Gottardi and Professor Dr Markus Nagl at the Institute of Hygiene and Medical Microbiology of the Medical University of Innsbruck. All the cells were cultured in Dulbecco's modified Eagle's medium (DMEM, 41966-029; Gibco; Thermo Fisher Scientific, Inc.). All culture media were supplemented with 10% fetal bovine serum (FBS, 16000-044; Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin) (full DMEM). The cells were grown in a humidified incubator with 5% CO₂ at 37°C. The J774.A1 Mφs were pre-incubated with various concentrations of the tested agents (BAT or taurine), diluted in pure DMEM without FBS for 1.5 h at 37°C. Subsequently, 100 ng/ml of LPS were diluted in full DMEM and were administered to the Mφs for an additional 24 h at 37°C, as previously described (38). Data are presented as the means ± SEM of 3 independent experiments.

Total RNA isolation from the cultured Mφs, as well as exudates derived from the pouches of LPS-exposed mice (model described below), was carried out using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA quality was confirmed, through the presence of 28s, 18s and 5s RNA ribosomal subunits in agarose gel electrophoresis and taking into consideration the ratio of OD260/280 to be approximately 2 as well as the ratio of OD260/230 to be approximately 2.2 in all samples.

Reverse transcription was performed from 1.0 μg of purified RNA using the SuperScript Reverse Transcriptase II (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. Quantification at the mRNA level was conducted in 96-well polymerase chain reaction (PCR) plates using a Bio-Rad iCycler and the iQ5 Multicolor Real-Time polymerase chain reaction (RT-PCR) detection system (Bio-Rad Laboratories, Inc.). Each reaction contained 1X IQ SYBR-Green Supermix (Bio-Rad Laboratories, Inc.) and 150 nmol/l of each primer. All genes were tested in triplicate. The results were analyzed on the iCycler software. Values were normalized against β-actin. The relative quantification of complementary DNA (cDNA) was performed according to the ΔΔCq method (42). Selected primers are presented in Table SI.

To detect the putative suppressive effect of BAT or taurine on the nuclear translocation of phosphorylated NF-κB p65 protein (Ser 536), immunofluorescence experiments were performed. In detail, 24-well plates were seeded overnight with 4.5x10⁴ cells/well. The cells were pre-treated with 0.1-0.5 mM BAT or 100 mM taurine diluted in pure DMEM without FBS for 1.5 h at 37°C. Subsequently, LPS (100 ng/ml) diluted in full DMEM was added to each well, and the cells were incubated for 24 h at 37°C, as previously described (38). The Mφs were washed with 1X phosphate-buffered saline (PBS) for 5 min and fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min. The fixed cells were permeabilized with 1x PBS/0.5% Triton X-100 for 5 min at 4°C and blocked with 1% BSA/1X PBS, diluted in 1X PBS at room temperature. The Mφs were then incubated with the primary anti-phospho-NF-κB-p65 monoclonal antibody (mAb; #3033; Cell Signaling Technology, Inc.) diluted in 1% BSA/1X PBS at 4°C overnight. The following day, the cells were incubated with (4 μg/ml) Alexa Fluor 488-labeled secondary antibody (A11008; Thermo Fisher Scientific, Inc.) diluted in 1% BSA/1X PBS for 1 h at room temperature, after washing with 1X PBS. Hoechst dye No. 33342 (B2261; Sigma-Aldrich Merck KGaA; 0.5 μg/ml) was used for cellular chromatin staining. Finally, the coverslips were mounted in Prolong Gold antifade media (Molecular Probes, Inc.) and the cells were observed under a confocal microscope (Leica Microsystems GmbH) with an excitation wavelength of 355 nm for Hoechst and with an excitation wavelength of 488 nm for phosphorylated NF-κB (Ser 536). The LAS AF program was used to acquire the images. Experiments were repeated independently 3 times and representative images are presented.

Male black C57BL/6 mice (20 g in weight, 6 weeks of age) were obtained from the National Hellenic Research Foundation and housed under controlled temperature (22±2°C) and photoperiod (12 h light; 12 h dark) with free access to water and food. All experiments with mice were performed in the authorized animal house of the National Hellenic Research Foundation (License no. EL 25 BIObr 031 as a breeding facility; License no. EL 25 BIOsup 032 as a supply facility; and License no. for EL 25 BIOexp 033 as a research installation). The experiments complied with the Protocol on the Protection and Welfare of Animals, as obliged by the rules of the National Hellenic Research Foundation the regulations of the National Bioethics Committee and article 3 of the presidential decree 160/1991 (in line with 86/609/EEC directive) regarding the protection of experimental animals. A total of 3 mice/group were used. The minimum number of animals was achieved according to the 3Rs (replacement, reduction and refinement), to ensure scientific and statistical validity. The health and behaviour of the animals were monitored daily. The mice were gradually euthanized, using carbon dioxide (CO₂). The gradual flow rate of CO₂ was as per the guidelines. The death of the animals was confirmed through the verification of cardiac and respiratory arrest.

The present study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Bioethics Committee of the National Hellenic Research Foundation (date of approval: 29/3/2020). The ethic code is PN 2-3/29-3-2020.

Air-pouches were created according to a modified method described in the study by Sedgwick et al (43). An area of dorsal skin (4 cm²) was shaved, and 3 ml of sterile air were subcutaneously injected to establish a single air-pouch in 6-week-old black (C57BL/6) mice. The mice were anaesthetized by an intraperitoneal (i.p) injection of ketamine at 100 mg/kg together with xylazine at 10 mg/kg, prior to the subcutaneous injections during the LPS-induced air-pouch model of inflammation. The mice weighed approximately 20 g and were housed in filtered-air laminar-flow cabinets at a controlled temperature (22±2°C) with a 12-h light/dark cycle. A total of 3 ml of sterile air were administered on alternate days to maintain the pouch. At 10 days after air-pouch formation, the pouches were injected with a single dose of 1 ml (1 μg/ml) LPS alone (positive control) or a single dose of 1 ml (1 μg/ml) LPS plus a single dose of (3 or 6 or 9 mg/mouse) BAT concurrently or a single dose of 1 ml (1 μg/ml) LPS plus a single dose of (9 mg/mouse) taurine concurrently, inside the air pouches of the mice for 8 h, as previously described (44). Each group was composed of 3 mice. This air pouch model of inflammation has been similarly used in a wide range of studies (44-48). Inflammatory exudates were then harvested by collecting the lavage fluids after washing the air-pouch cavities with 2 ml PBS (1X, pH 7.4) followed by RNA extraction. The pouch membranes were fixed in 10% (v/v) buffered formalin for histological analysis.

Ten days after air-pouch formation, substances were administered and after 8 h, mice were euthanized and their pouches were dissected, sampled and processed for paraffin embedding. The protocol for paraffin embedding was as follows (total 16 h): 70% ethanol (2 h), 80% ethanol (1 h), 95% ethanol (1 h), 100% ethanol (4.5 h), xylene (4.5 h), paraffin (58-60°C) (4 h), embedding tissues into paraffin blocks and trimming into the suitable 6 μm. Sections were then stained with hematoxylin (8 min) and eosin (1 min) (H&E) at 30°C, using the following protocol: The sections were deparaffinized, and treated with absolute alcohol (10 min), 95% alcohol (2 min), 70% alcohol (2 min) and stained with Harris hematoxylin solution (Thermo Fisher Scientific, Inc.) (8 min) and saturated lithium carbonate (1 min) at 30°C. After rinsing, the sections were counterstained with eosin-phloxine solution (Thermo Fisher Scientific, Inc.) (1 min) and dehydrated using 95% alcohol. Microphotographs were captured using a Nikon Eclipse 80i upright microscope with CFI60 lenses, using a Leica DFC 450 C digital camera. The magnification utilized for their capture was x200. In each section, pouch wall thickness was measured at 6 regions randomly at the upper, at the back and at the middle side of the pouch wall. The mean of six different regions in each section was determined. The thickness of the pouch and the area of the corresponding region were calculated using ImageJ software (v1.52a). The total cell number (based on nucleus count) was calculated as cells/mm², using the 'analyze particles' feature of ImageJ software, in LPS-exposed mice bearing air-pouch with or without treatment, manually. The number of PMNs was calculated per mm², in LPS-exposed mice bearing air pouch with or without treatment, manually.

Data are presented as the means ± SEM. One-way ANOVA, followed by Tukey's multiple comparison test was used to evaluate the significance of all experiments between the LPS group with the LPS/BAT and LPS/taurine-treated groups. Two-way ANOVA was used to evaluate the statistical significance of weights between the LPS group with the LPS/BAT group and LPS/taurine group. Statistics were calculated with GraphPad Prism 6.0 (GraphPad Software, Inc.).

To investigate the putative inhibitory effects of BAT and taurine on the LPS-induced inflammatory response in vitro, the mRNA expression levels of various pro-inflammatory cytokines secreted by J774.A1 Mφs were assessed following 1.5 h of incubation with either BAT or taurine alone, prior to their stimulation with LPS for 24 h. In the present study, RT-qPCR was used to detect the mRNA expression levels of TNF-α, IL-1β, IL-18 and IL-23 following treatment of J774.A1 Mφs with either (0.1, 0.3, 0.5, 1 and 1.75 mM) BAT or (100 and 200 mM) taurine prior to their stimulation with LPS for 24 h. Of note, BAT and taurine exerted potent protective effects against inflammation, by reducing the mRNA expression levels of pro-inflammatory cytokines in the LPS-stimulated J774.A1 Mφs (Fig. 1 and Table SII). In particular, the IL-1β mRNA levels were decreased by (51, 59, 59, 60, 81, 59 and 71%), the IL-23 mRNA levels by (68, 70, 73, 75, 81, 72 and 77%), the IL-18 mRNA levels by (15, 25, 50, 57, 81, 15 and 55%) and the TNF-α mRNA levels by (56, 79%, 83, 89, 96, 88 and 89%) in the (0.1, 0.3, 0.5, 1 and 1.75 mM) BAT-, and in the (100 and 200 mM) taurine-treated LPS-stimulated J774.A1 Mφs compared to the LPS-stimulated Mφs. A dose-dependent transcriptional downregulation in the levels of pro-inflammatory cytokines was proved in the BAT-treated LPS-stimulated J774.A1 Mφς, with the highest inhibitory effect observed at the concentration of 1.75 mM BAT. Notably, the highest suppression of IL-1β, IL-23 and IL-18 mRNA expression (81%) and the greatest inhibition of TNF-α (96%) were evidenced in the 1.75 mM BAT-treated Mφs prior to the induction of LPS-mediated inflammation. In all cases, the 1.75 mM BAT-treated LPS-stimulated J774.A1 Mφς exhibited a greater transcriptional inhibition of all pro-inflammatory cytokines than those of the 100 and 200 mM taurine-treated LPS-stimulated J774.A1 Mφs. As a result, BAT potentially exerted a more significant protective action than taurine, by hindering the LPS inflammatory stimulus to activate cytokine secretion from macrophages. These data were consistent with the reduced protein expression levels of pro-inflammatory mediators (IL-6, IL12p40, TNF-α), as previously indicated by ELISA experiments in LPS-stimulated J774.A1 Mφs (38).

To elucidate the molecular mechanisms underlying the protective effects of BAT and taurine against LPS-mediated-inflammation, immunofluorescence experiments were employed to examine the exact localization of phosphorylated NF-κB p65 subunit (Ser536) in the J774A.1 Mφs. The dynamics (serine 536 phosphorylated p65 subunit of NF-κB) were measured in BATor taurine-treated J774.A1 Mφς, prior to their stimulation with LPS. Following treatment with BAT or taurine, the nuclear translocation of phospho-NF-κB p65 subunit (Ser 536) was hindered in a dose-dependent manner, since it was mainly found in the cell cytoplasm (Fig. 2). In particular, the inhibition of phospho-NF-κB p65 (Ser 536) nuclear translocation dynamics emerged in the 0.1 mM BAT-treated J774.A1 Mφς prior to their stimulation with LPS, while the most effective inhibition of NF-κB p65 phosphorylation (Ser 536) was observed in the 0.5 mM BAT-treated J774.A1 Mφς prior to their stimulation with LPS, as compared to the LPS-stimulated J774A.1 Mφs. Of note, the levels of phosphorylated NF-κB p65 in the NC J774.A1 Mφς were similar to those of LPS-stimulated J774A.1 Mφς following BAT or taurine treatment. As a result, BAT and taurine restored not only the translocation, but also the levels of phosphorylated NF-κB p65 in the LPS-stimulated J774A.1 Mφς, as observed in the NC Mφς. It was hypothesized that the sequestration of p-NF-κB p65 subunit (Ser 536) occurs in the cytoplasm of J774.A1 Mφς following BAT or taurine treatment, prior to their stimulation with LPS, probably due to other post-translational modifications or other mechanisms that inhibit phospho-NF-κB p65 nuclear shutting.

To confirm the protective effects of BAT and taurine against LPS-mediated inflammation in vivo, the LPS-induced air-pouch model of inflammation was used. This animal model has been widely used to evaluate acute inflammation due to its high sensitivity and cost-efficiency (41). In the present analysis, 4 groups of C57BL/6 mice were subjected to the following procedure: They were subjected to air-pouch formation subcutaneously; air injections were administered on specific days according to the timeline to maintain the air-pouch. At 10 days after air pouch formation, LPS inflammatory stimulus and optionally BAT or taurine were administered to the mice for 8 h. To elucidate the potential protective effect of BAT against LPS-induced inflammation in vivo, we compared its impact with that of a known anti-inflammatory agent, namely taurine. The 4 groups of animals were composed as follows: Group 1, mice with an air-pouch and treated with saline; group 2, LPS-exposed mice with an air pouch; group 3, BAT-treated LPS-exposed mice with an air pouch; and group 4, taurine-treated LPS-exposed mice with an air pouch. To ensure that the presence of inflammation formed inside the air-pouch, the results derived from the normal saline-negative control (NC) and the LPS-treated group (LPS) were compared. Following the induction of inflammation, the functional significance of either BAT or taurine in orchestrating the distribution of inflammatory cell populations and in determining the mRNA expression levels of pro-inflammatory cytokines was elucidated.

Air-pouch tissues were stained with H&E to evaluate the histological presence of various inflammatory cell populations. The injection of (1 μg/ml) LPS into the air-pouch on the backs of mice for 8 h resulted in increased inflammatory cell infiltration and elevated pouch wall thickness. All the examined parameters in the LPS group were significantly increased compared with those in the NC group (Fig. 3 and Table SIII). Representative images with histological changes in the pouch wall (synovial membrane and connective tissue) in the differently treated groups compared to the LPS group are presented in Fig. 3B. In particular, the LPS-exposed mice bearing an air pouch treated with BAT or taurine had a fewer number of infiltrating cells, as well as polymorphonuclear cells (PMNs) in the pouch wall (synovial membrane and connective tissue) and thinner pouch wall formation than those injected with LPS alone (Fig. 3 and Table SIII). Notably, pouch wall thickness was similar between the LPS and 9 mg BAT-treated mice bearing an air-pouch and the LPS and 9 mg taurine-treated mice bearing an air-pouch (Fig. 3G). The analysis of the exudates obtained from the pouches also revealed that the inhibitory effects of BAT and taurine treatment on the secretion of pro-inflammatory cytokines (Fig. 4 and Table SIV) were similar to those observed in vitro (Fig. 1 and Table SI). In particular, the LPS-exposed mice bearing an air pouch and treated with 3 or 6 or 9 mg of BAT or 9 mg of taurine exhibited decreased IL-1β mRNA levels by 48, 81, 91 and 84%, decreased IL-23 mRNA levels by 19, 93, 94 and 39%, and decreased IL-18 mRNA levels by 26, 63, 86 and 87% respectively, as compared to the untreated LPS-exposed mice bearing an air pouch. The IL-17 mRNA levels were reduced by 34, 51, 78 and 80% following treatment of the LPS-exposed mice bearing an air pouch with 3, 6 and 9 mg of BAT and 9 mg of taurine, respectively, compared to those of untreated LPS-exposed mice bearing an air pouch. Similarly, the TNF-α mRNA levels were reduced by 19, 53, 86 and 73% in the pouch exudates obtained from the LPS-exposed mice and treated with 3, 6 and 9 mg of BAT or 9 mg of taurine, respectively, as compared to those of untreated LPS-exposed mice bearing an air pouch. Last but not least, the mRNA levels of thymic stromal lymphopoietin (TSLP) were suppressed by 17, 61, 85 and 87% in the LPS-exposed mice with the air pouch treated with 3, 6 and 9 mg of BAT or 9 mg of taurine, respectively, as compared to those of untreated LPS-exposed mice bearing an air pouch. At the transcriptional level, IL-1β, IL-23 and TNF-α were produced at lower levels in the LPS plus 9 mg BAT-treated mice bearing an air pouch compared to those in the LPS plus 9 mg taurine-treated mice bearing an air pouch (Fig. 4A, B and E). Notably, the LPS plus 6 mg BAT-treated mice bearing an air pouch exhibited very low transcriptional levels of IL-23, as opposed to the LPS plus 9 mg taurine-treated mice bearing an air pouch (Fig. 4B). As regards the transcriptional levels of IL-18, IL-17 and TSLP, the 2 groups of LPS plus 9 mg of BAT and LPS plus 9 mg of taurine-treated mice bearing an air pouch presented similar values (Fig. 4C and F). Of note, the inhibitory effect of 9 mg BAT was the most effective on the IL-23 mRNA levels in the LPS-exposed mice bearing an air pouch (Fig. 4B). As a result, the LPS plus 9 mg BAT-treated mice bearing an air pouch exhibited a superior effect on the majority of the pro-inflammatory cytokines tested and on cellular infiltration, compared to the aforementioned parameters of the LPS plus 9 mg taurine-treated mice bearing an air pouch.