Imipramine hydrochloride (ImiP) (Sigma, I7379) was dissolved in deionized water to prepare a 0.1 M stock solution. The solution was sterilized by filtration through a 0.22 μm pore-size membrane to eliminate impurities and subsequently stored at −20°C. Working solutions were prepared by diluting the stock with sterilized phosphate-buffered saline (PBS).

RAW 264.7 cells (ATCC, TIB-71) were cultured in RPMI 1640 medium (Gibco, CA, USA, 11875119) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, 1600–044), with or without 1% of 100 × penicillin/streptomycin (Gibco, Invitrogen, NY, USA) at 37°C in a 5% CO₂ incubator. For specific assays, cells were seeded in 6-well and 96-well plates at densities of 1 × 10⁶ and 5 × 10⁴ cells per well, respectively, in RPMI medium with 10% FBS. The plates were incubated overnight before use.

The smooth, virulent, wild-type B. abortus 544 biovar 1 (ATCC 23448) was cultured on Brucella agar at 37°C in a 5% CO₂ atmosphere for 72 h. For cell infection and in vivo experiments, one colony was inoculated into Brucella broth (BBL BD, San Jose, CA, USA) and incubated at 37°C with continuous shaking for 48 h in a biosafety level 3 (BSL-3) containment facility.

RAW 264.7 cells were seeded in 96-well tissue culture plate were treated with different concentrations of ImiP (1,000; 500; 250; 125; 62.5; 31.25; 15.63; 7.81; and 3.91 μM) for 72 h. The cells were washed with PBS then fresh RPMI medium containing 10% FBS and 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well. The plate was wrapped in foil and incubated for 4 h under the same conditions. Thereafter, the medium was removed, 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the plate was incubated for 15 min. Cell viability was measured at 540 nm using a spectrophotometer (Thermo Labsystems Multiskan, Chantilly, VA, USA).

B. abortus 544 at a concentration of approximately 10⁴ colony-forming units (CFUs)/well was incubated with different concentrations of ImiP (37.5, 7.5, and 0.25 mM), with PBS as the control. The plate was incubated for 0, 4, 8, 24, and 48 h at 37°C in a 5% CO₂ atmosphere. At each time point, the mixture was serially diluted in RPMI, plated onto Brucella agar, and incubated for 72 h. CFUs were counted to evaluate bacterial survival.

For the bacterial internalization assay, 96-well tissue culture plate seeded with RAW 264.7 cells were pre-treated with ImiP at a concentration of 125 and 250 μM for 6 h before infection with B. abortus 544 at a multiplicity of infection (MOI) of 1,000. The plate was centrifuged at 200 × g for 5 min and then incubated at 37°C in a 5% CO₂ atmosphere. At 5, 20, and 60 min post-infection (pi), the cells were treated with gentamicin (50 μg/mL) per well for 30 min. The cells were washed with PBS, lysed in 100 μL distilled water, serially diluted and plated onto Brucella agar plates.

For the intracellular replication assay, cells were infected with B. abortus for 1 h and treated with ImiP (125 and 250 μM) and gentamicin (50 μg/mL) at 4, 24, and 48 h post-infection (pi). At each time point, the cells were washed, lysed and plated same as described in the internalization assay. CFUs were counted after 3 days to evaluate internalization and intracellular replication efficiency.

Following the protocol for intracellular growth assay, RAW 264.7 cells were infected with B. abortus and treated with ImiP. Cell supernatants were collected at 24 and 48 h pi and nitrite levels were measured using the Griess reagent kit (Promega, Cat# G2930) according to the manufacturer’s instructions.

Eight-week-old, specific-pathogen-free female BALB/c mice (Samtako Bio Co., Ltd., Korea) were randomly grouped into six groups consisting of six mice each. The mice were acclimatized for a week in a microisolator cage at 23 ± 1°C with a 12 h light/dark cycle, with ad libitum water and feed. Prior to infection, the mice were orally treated with ImiP (10 mg/kg/day or 20 mg/kg/day) or PBS (control) via gavage needle for 7 days. The selected ImiP doses were based on previous studies in mouse models examining its effects on neurological behavior, antidepressant activity, and antibacterial properties (17–20). Three groups of BALB/c mice were intraperitoneally infected with B. abortus 544 (5 × 10⁶ CFU in 100 μL PBS), while the remaining three groups received treatment without infection. The treatment continued for 14 days pi, during which all animal groups were monitored for clinical symptoms. On day 14 pi, peripheral blood samples were collected from the tail vein and the mice were sacrificed via cervical dislocation to collect the liver and spleen. The organs were individually weighed, a 1 cm sample was collected and fixed in 10% neutral-buffered formalin for histopathological examination, and a 0.05 g portion of each organ was collected for bacterial proliferation. The organs were homogenized in PBS then the homogenates were serially diluted and plated onto agar incubated at 37°C in a 5% CO₂ for 3 days. Bacterial CFUs were log₁₀-transformed to calculate protection units. All animal handling and sacrifice procedures were reviewed and approved by the Animal Ethical Committee of Chonbuk National University (Authorization Number CBNU-2021-037).

CD4⁺ and CD8⁺ T-cell populations were determined using two-color flow cytometry. Briefly, 100 μL of whole mouse blood was mixed with 75 μL of FITC-conjugated anti-mouse CD4⁺ and PE-conjugated anti-mouse CD8⁺ monoclonal antibodies (BD Pharmingen, USA) and incubated in the dark at room temperature for 30 min. Afterwards, 2 mL of red blood cell lysis buffer (Roche, Germany) was added and incubated for 10 min. The reaction was terminated by adding 3 mL of PBS and the samples were centrifuged at 380 × g for 5 min. The cell pellet was washed twice with PBS, resuspended in 0.5 mL PBS and the stained blood cells were analyzed using BD FACSLyric flow cytometer and BD FACSuite software (BD Biosciences, USA).

Peripheral blood samples were centrifuged at 2,000 rpm for 10 min at 4°C to obtain serum. Following the manufacturer’s instructions, 50 μL of mouse serum was analyzed using the Cytometric Bead Array (CBA) Mouse Inflammatory Kit (BD, Cat# 552364) to quantify cytokine levels, including IL-6, IL-12, IFN-γ, TNF-α, IL-10, and MCP-1. The samples were processed and analyzed using a BD FACSLyric flow cytometer with BD FACSuite software (BD Biosciences, USA).

Spleen and liver samples were fixed in 10% neutral-buffered formalin for 48 h. The fixed tissues were then dehydrated using a series of alcohol and xylene treatments before being embedded in paraffin. The paraffin-embedded tissues were sectioned into 3-μm-thick slices using a rotary microtome and mounted onto microscopic slides. The sections were subsequently stained with hematoxylin and eosin (H&E) for histological analysis. For each sample, ten randomly selected areas were examined microscopically at ×100 magnification. Tables 1, 2 present the histological grading criteria for brucellosis in the liver and spleen of mice, respectively. The severity of microgranulomas, periportal inflammation, and necrosis was assessed using a 0–4 scale, as described in previous studies (21–23). The mean total score for each group was then compared.

All experiments were performed in triplicate and repeated in at least three independent experiments. The data were analyzed using Student’s t-test or one-way ANOVA in GraphPad Instat. Results were expressed as means ± standard deviation (SD). Differences with p-values < 0.05 *, p < 0.01 **, p < 0.001 *** were considered statistically significant.

RAW 264.7 cells incubated with different concentrations of ImiP exhibited reduced cell viability after 48 h of incubation with 1,000 μM and 500 μM of ImiP. In contrast, cell viability significantly increased at 3.91 μM compared to the control group. Treatment with 125 μM and 250 μM maintained cell viability at 107 and 97%, respectively, representing the highest non-cytotoxic concentrations. Therefore, 125 μM and 250 μM were selected for subsequent experiments (Figure 1A).

B. abortus were treated with 37.5 mM, 7.5 mM, and 0.25 mM ImiP to evaluate its bactericidal effect. Bacterial survival was assessed using triplicate wells for each concentration. As shown in Figure 1B, ImiP demonstrated a time- and dose-dependent effect against B. abortus. A marked reduction in CFU was observed in 37.5 mM group after 4 h and in the 7.5 mM group at 24 h post-treatment. The 0.25 mM group showed a significant CFU reduction at 48 h post-treatment compared to PBS, but to a lesser extent than the higher concentrations.

Pre-treatment with 125 and 250 μM of ImiP did not affect the number of internalized bacteria in RAW 264.7 cells at 5, 20, and 60 min pi (Figure 2A). However, it significantly reduced the intracellular growth of B. abortus in cells treated with 125 μM ImiP at 4 (*p < 0.05), 24 (*p < 0.05) and 48 h (**p < 0.01) pi. Cells treated with 250 μM ImiP showed decreased intracellular bacterial growth at 48 h (**p < 0.01) pi (Figure 2B). Taken together, these findings suggest that ImiP treatment could interfere with the intracellular survival of B. abortus in macrophages.

RAW 264.7 cells were infected and treated using the same method as the intracellular growth assay. After infection the cells were cultured in fresh medium with ImiP and gentamicin for 24, and 48 h. Nitric oxide (NO) plays a multifaceted role, particularly in host defense against pathogens, impacting both the progression and persistence of infections (24). ImiP-treated murine macrophages infected with B. abortus exhibited a significant, dose-dependent reduction in nitrite levels compared to the control at both 24 and 48 h pi (Figure 3).

For the in vivo experiments, groups of six BALB/c mice were used (n = 6 per group). No clinical symptoms or mortality were observed throughout the treatment period. As shown in Figure 4A, BALB/c mice were orally treated with PBS (control), 10 mg/kg/day (low dose), or 20 mg/kg/day (high dose) of ImiP for 7 days, followed by intraperitoneal infection with B. abortus. The treatment was continued for 14 days then the mice were sacrificed to assess spleen and liver weights. In non-infected ImiP-treated mice, spleen weight significantly decreased in a dose-dependent manner compared to the control. However, infected mice treated with ImiP exhibited increased spleen weight at both doses (Figure 4B). A similar pattern was observed for liver weight, however only the non-infected mice treated with ImiP 20 showed a significant reduction (Figure 4C). Additionally, Imip treatment significantly reduced bacterial burden in the spleen, with log protection units of 0.25 for the low dose and 0.31 in the high dose group. Similarly, bacterial load in the liver decreased, with log protection units of 0.35 for the low dose and 0.57 in the high dose group (Figure 4D, Table 3).

To evaluate the effect of ImiP on the host immune response, CD4⁺ and CD8⁺ cell populations were analyzed, along with cytokine measurement using a CBA kit. At 14 days pi, CD4⁺ levels decreased in both non-infected and infected groups. In the non-infected group, no significant difference was observed between the treated and control groups. However, a significant reduction was noted in the group treated with the higher dose of ImiP compared to the low dose group. In the infected group, CD4⁺ levels significantly decreased at both treatment doses compared to the control. Similarly, CD8⁺ levels also declined, with a significant reduction in the non-infected mice treated with 250 μM ImiP and in infected mice treated with 125 μM ImiP (Figures 5A,B).

The CBA kit quantified the level of six cytokines which were IL-6 (Figure 5C), IL-12 (Figure 5D), IFN-γ (Figure 5E), TNF-α (Figure 5F) while IL-10 (Figure 5G) and MCP-1 (Figure 5H). The cytokine analysis of peripheral blood revealed that infected mice treated with ImiP significantly increased levels of IL-12 while IL-10 and MCP-1 levels decreased. These findings suggest that ImiP possesses immunomodulatory properties that may enhance host defenses.

Microscopic examination of stained liver and spleen sections revealed no significant lesions in the spleen. In the liver, the non-infected group treated with ImiP exhibited minimal microgranuloma formation and periportal inflammation. Conversely, these lesions were reduced in Brucella-infected groups receiving ImiP treatment, with microgranuloma formation lower in the high dose group compared to low dose and control group (Figure 6A). Notably, necrosis was absent in all treated liver samples. Representative histological liver samples from infected groups treated with PBS (Figure 6B), 10 mg/kg ImiP (Figure 6C), and 20 mg/kg ImiP (Figure 6D) further confirmed the reduction in microgranuloma formation and periportal inflammation, supporting the histological assessment.