Mice

Female wild-type (WT), 6 to 8 weeks old C57BL/6 mice, and mice deficient for the leptin receptor (db/db) and their respective control (db/+), were obtained from the breeding facility of the Ribeirao Preto Medical School, University of São Paulo (Ribeirao Preto, SP, Brazil). Although type 2 diabetes is associated with tuberculosis recurrence in men (1.23, 95% confidence interval 1.15–1.32) compared to women (0.96, 95% confidence interval 0.85–1.09) [29], we used female animals because the experimental design demands more than 10 weeks and male animals frequently hurt each other. All animals were maintained in sterile environmental cages and received sterile food and water ad libitum. All animals were euthanized with ketamine (300 mg/kg) (Agener, Embu-Guaçu, São Paulo, Brazil) and xylazine (30 mg/kg) (Syntec, Santana de Parnaiba, Bazil). All animal procedures were carried out in accordance with the Ribeirao Preto Medical School Ethic Committee in Animal Use (protocol numbers 116/2018, 226/2019, 8/2019, 72/2019).

Diet-induced Obesity

C57BL/6 mice were fed a high fat diet (HFD: 60% of lipid, 20% of protein and 20% of carbohydrate) or low-fat diet (LFD: 10% of lipid, 70% of carbohydrate and 20% of protein) for 12 weeks (Pragsoluções Biociências, Jaú, SP, Brazil). Body weight was measured weekly until the end of the experiment.

Obese and Diabetic Mice

db/db (homozygous) and db/+ (heterozygous) mice were fed a regular diet for 21 weeks. At 21 weeks, db/db mice developed obesity and type 2 diabetes, while db/+ mice - normoglycemic and lean - were used as controls.

Blood Glucose Measurement

Fasting blood glucose was measured in LFD and HFD groups at 18 weeks of age. Fasting blood glucose was determined in db/db and db/+ groups at 21 weeks (150 days) of age.

Bone Marrow-derived Macrophages (BMDM) Differentiation

Femurs and tibias harvested from WT on LFD or HFD, db/db, and db/+ mice were flushed with RPMI 1640 (Gibco, Waltham, MA, USA) medium. Precursor cells were cultured in RPMI supplemented with 20% L929 cell-conditioned medium and 20% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA). Seven days after culture, differentiated BMDM were harvested. Adherent cells were plated on 24-well culture plate (Costar®, Corning #3524) in RPMI medium with 10% FBS and mannitol (25 mM, normoglycemic condition) or glucose (25 mM, high glucose or hyperglycemic condition). Cells were cultured for 24 h at 37 °C and 5% CO2.

In vitro M. tuberculosis Infection

BMDM (1 × 106 cells) were infected with M. tuberculosis strain H37Rv (American Type Culture Collection 27294, Rockville, MD, USA) in a multiplicity of infection (MOI) 1. After 4 h, BMDM cultures were washed once with phosphate buffered saline (PBS 1x) to remove extracellular, non-phagocytized bacteria. Infected BMDM were cultured for 20 h in RPMI medium supplemented with 10% FBS and mannitol or glucose.

In vitro Treatment

BMDM were infected or non-infected with M. tuberculosis (MOI 1) and 4 h post infection, washed once and treated with CCX832 (1 µM, a gift from ChemoCentryx, Mountain View, CA). Chemerin (100 ng/mL, R&D Systems, Minneapolis, MN, USA) was added to cultures with normoglycemic or hyperglycemic medium 1 h after CCX832 treatment. The concentrations of CCX832 and chemerin were defined as described [30].

In vivo Infection

Mice were anesthetized by inhalation using isoflurane in an atmosphere containing 5% isoflurane, with a flow of 1 L per minute (LPM) by the infusion inhalation anesthesia equipment (Bonther, Ribeirao Preto, Sao Paulo, Brazil). After anesthetizing, db/db or db/+ were infected by the oropharyngeal route with 1 × 103 or 1 × 104 Colony Forming Unit (CFU) M. tuberculosis (Mtb). Mice were followed up until the final effect of anesthesia. Mice were euthanized 20 days post-infection.

Treatment

Ten days post-infection, M. tuberculosis-infected db/db or db/+ mice were treated with CCX832 (55 mg/kg), by oral gavage for 7 consecutive days.

Colony-Forming Unit (CFU) Assay

In the in vitro CFU assay, which quantifies bacterial load or CFU recovery, BMDM were lysed 24 h post-infection and plated on supplemented 7H11 agar medium (Sigma-Aldrich). In the in vivo CFU assay, the lower and middle right lobes of the lungs were digested with collagenase (2.2 mg/mL) and DNase (0.055 mg/mL) and lung cell suspensions were serially diluted and plated in supplemented 7H11 agar medium. The plates were cultured at 37 °C and 5% CO2 for 28 days.

ELISA

IL-6, TNF, IL-10, IFN-γ and chemerin concentrations were determined in lung homogenates and culture supernatants by ELISA kits, following the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). The limit of detection was 31.2 pg/mL for TNF, IFN-γ and IL-10 and 15.6 pg/mL for IL-6.

Western Blot Analysis

BMDM infected with M. tuberculosis and/or stimulated with chemerin were homogenized in a lysis buffer (50 mM Tris/HCl, 150 mM NaCl, 1% Nonidet P40, 1 mM EDTA, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 µg/mL aprotinin, 1 mM sodium orthovanadate, 1 mM PMSF, and 1 mM sodium fluoride) and centrifuged at 16,000 x g for 15 min. The supernatant was collected for protein quantification by the Bradford method. Proteins (15 µg) were separated by electrophoresis on 12% polyacrylamide gel and transferred onto nitrocellulose membranes. Non-specific binding sites were blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature. Membranes were incubated with rabbit anti-mouse IgG anti-ChemR23/CMKLR1 (1:1000, Abcam, Cambridge, MA, EUA) or rabbit anti-mouse IgG anti-β-actin (1:5000, Abcam, Cambridge, MA, EUA) overnight at 4 °C. The antibodies were diluted in TBS-T. The membranes were incubated with a secondary antibody goat anti-rabbit IgG (1:5000, Sigma Aldrich) for 1 h at room temperature. The signals were obtained by chemiluminescence and quantified by densitometric analysis using the software ImageJ (National Institute of Health, Bethesda, Maryland).

Histopathology Analysis and Inflammation Score

The upper right lobes of the lungs were fixed in 10% formalin, embedded in paraffin blocks and cut into 5 μm thick sections. Lungs were stained with hematoxylin & eosin (H&E). Pulmonary inflammation score was determined as previously described [31]. Briefly, score 0: without inflammation; 1: mild inflammation; 2: moderate inflammation; 3: severe inflammation.

Flow Cytometry

The phenotype of BMDM and lung cells populations was determined by flow cytometry as described in Table 1. Cultured BMDM were removed with PBS 1x plus EDTA 0.05% and stained with viability stain (Live/Dead, Thermo Fisher, Waltham, MA, USA). Anti-arginase 1 (Arg-1) and anti-inducible nitric oxide synthase enzyme (iNOS) monoclonal antibodies (mAbs) were used to characterize M1 and M2 macrophages subsets, respectively. Arg-1 and iNOS expression was evaluated on gated Live/Dead−CD11b+CD64+ cells. For vivo experiments, lung cell suspensions (1 × 106 cells) were obtained as previously described (Palma Albornoz et al. 2023; Palma Albornoz et al. 2021), stained with viability stain (FVS780, BD Biosciences, Franklin Lakes, NJ, USA) and mAbs to characterize alveolar (FVS−CD64+CD11b−SiglecF+ cells) or interstitial macrophages (FVS−CD64+CD11b+SiglecF− cells), M1 (iNOS+ cells) or M2 macrophages (Arg1+ cells) and Th1 cells (CD45+CD3+CD4+IFN-γ+ cells). Samples were fixed with 1% paraformaldehyde. Acquisition was performed in BD FACSCanto cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Total events per sample were collected and analyzed according to size, granularity and fluorescence intensity using FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA).

Table 1 Flow cytometry panels: antibodies, fluorochrome, clones and source. Statistical Analysis

GraphPad Prism 8 (GraphPad Software, Inc., San Diego CA, USA) was used for data analysis and preparation of all graphs. Data normality was evaluated by Shapiro-Wilk normality test. An unpaired Student’s t-test was used to detect significant differences between two groups for parametric data. Mann-Whitney U test was used to detect significant differences between two groups for non-parametric data. For inflammation score analysis, relative numbers of mice with different histological scores were calculated and Fisher’s exact test was used to compare overall scores between different groups. Data were shown as the mean ± standard deviation (SD). Results were considered statistically significant when p < 0.05.