Ifn β

Human PBMCs were trained as described before. In short, 500.000 PBMCs were added into 96-well flat bottom plates. Cells were allowed to adhere for 1h at 37°C. Cells were washed three times with PBS prior to stimulations. After washing cells were incubated with culture medium only as negative control, or treated with 100 μM chloroquine (Sigma Aldrich), 100 μM hydroxychloroquine (Sigma Aldrich) or 0.01 μM rapamycin (Selckchem) for 1 hour at 37°C. The chloroquine and hydroxychloroquine dose were based on a study by French et al.⁴⁹ (link) who showed that in vitro 100 uM is necessary to generate intracellular levels similar to those in patients receiving therapy with hydroxychloroquine 400 mg daily.⁴⁹ (link) Subsequently cells were incubated with 10⁵ cells/ml HKCA (Invivogen) for 24 hours together with the respective treatment for 24 hours at 37°C. Subsequently, cells were washed and cells were rested for five days in RPMI culture medium containing 10% FBS. After the resting period cells were stimulated with either RPMI as negative control, 10ng/ml LPS (Sigma Aldrich) 1ug/ml Pam3CSK4 (Invivogen), 10ug/ml polyI:C (Invivogen), 10ng/ml IFNα (Invivogen), 10ng/ml IFNβ (R&D systems) or 100ng/ml IFNγ (Invivogen). Where indicated hydroxychloroquine and chloroquine were added 1 hour prior to restimulation and 24 hours during restimulation.

Bone marrow derived cells were plated to 1 × 10⁶ cells/mL and cells in 1 mL of media in 24 well tissue culture-treated plates, rested overnight at 37°C in 5% CO₂, then treated with IFN-β (10 units/mL) or IFN-γ (10 ng/mL) (R&D Systems, MN). Twenty-four hours later, cell culture supernatants were harvested and cells were lysed in RLT buffer and frozen at −80°C for RNA extraction using the RNAeasy mini kit (QIAGEN, Germantown, MD. Gene expression was analyzed as described above.

Cell culture supernatants were harvested from control, noninfected, and M. tuberculosis-infected THP-1 or THP-1-derived macrophages, and cytokine expression was measured at 24 h postinfection. Human enzyme-linked immunosorbent assay (ELISA) kits were used as per the manufacturer’s protocol to measure the expression of TNF-α, IL-1β, IL-10 (Invitrogen, Carlsbad, CA), and IFN-β (R&D Systems). The absorbance of the samples was read at 450 nm using the Synergy H1 microplate reader.

Human interferons used were commercially available recombinant proteins i.e. IFN-α (PBL Assay Science, Piscataway, NJ, USA), IFN-β (R&D System, MN, USA) and IFN-λ (PBL). Confluent normal and tumor cell lines were prepared in a 12-well plates. Cells were washed and medium was replaced with fresh maintenance medium supplemented with IFN-α, -β and -γ (1000 IU each) or IFN-α, -β (1500 IU each) or IFN-γ (3000 IU) in 1 mL medium. Plates were then incubated for 24 hours at 37 °C and cell lysate was subjected to caspase-3 activity (Promega).