Partial human Janus kinase 1 deficiency predominantly impairs responses to interferon gamma and intracellular control of mycobacteria

Purpose Janus kinase-1 (JAK1) tyrosine kinase mediates signaling from multiple cytokine receptors, including interferon alpha/beta and gamma (IFN-α/β and IFN-γ), which are important for viral and mycobacterial protection respectively. We previously reported autosomal recessive (AR) hypomorphic JAK1 mutations in a patient with recurrent atypical mycobacterial infections and relatively minor viral infections. This study tests the impact of partial JAK1 deficiency on cellular responses to IFNs and pathogen control. Methods We investigated the role of partial JAK1 deficiency using patient cells and cell models generated with lentiviral vectors expressing shRNA. Results Partial JAK1 deficiency impairs IFN-γ-dependent responses in multiple cell types including THP-1 macrophages, Epstein-Barr Virus (EBV)-transformed B cells and primary dermal fibroblasts. In THP-1 myeloid cells, partial JAK1 deficiency reduced phagosome acidification and apoptosis and resulted in defective control of mycobacterial infection with enhanced intracellular survival. Although both EBV-B cells and primary dermal fibroblasts with partial JAK1 deficiency demonstrate reduced IFN-α responses, control of viral infection was impaired only in patient EBV-B cells and surprisingly intact in patient primary dermal fibroblasts. Conclusion Our data suggests that partial JAK1 deficiency predominantly affects susceptibility to mycobacterial infection through impact on the IFN-γ responsive pathway in myeloid cells. Susceptibility to viral infections as a result of reduced IFN-α responses is variable depending on cell type. Description of additional patients with inherited JAK1 deficiency will further clarify the spectrum of bacterial and viral susceptibility in this condition. Our results have broader relevance for anticipating infectious complications from the increasing use of selective JAK1 inhibitors.

(FCS) and 1% penicillin-streptomycin (P/S). Primary dermal fibroblasts from patients and healthy controlsand 293T cells were cultured in DMEM medium and 10% heat-inactivated FCS and 1% P/S. For the different experiments fibroblasts were detached using Accutase® solution (A6964, Sigma Aldrich). Regular checks for mycoplasma contamination were performed. JAK1-complented stable clones were kept in the presence of puromycin (3 ug/ml).

Lentivirus preparation and transductions
JAK1 knock down (KD) and scrambled control (Sc) cell lines were generated using lentiviral vectors expressing short hairpin RNA (shRNA) sequences.pGIPZ vectors carrying the short hairpin RNA against JAK1(TAGTACACACATTTCCATG) or scrambled control (TGAACTCATTTTTCTGCTC) sequences as well as puromycin resistance cassette and turbo-GFP marker for selection were supplied by University College London Open Biosystems (London UK). Lentivirus stocks were prepared by transfection of 293T cells (80-90% confluence) cultured in DMEM medium and 10% heat-inactivated fetal bovine serum, with the envelope plasmid 17.5 µg pMD.G2 (VSV-G/envelop), 32.5 µg p8.74 plasmid (gagpol) and 25 µg vector construct with the transfection reagent PEI/Optimen following the manufacture instructions. Medium was replaced 5h post transfection and medium was harvested after 24 and 48 h, cleared by centrifugation (4,000 rpm, 5 min), filtered through 0.22 µm filters and left to spin for 2h 4ºC 50,000 g. Viruses were tittered on 293T cells by scoring GFP positive cells by flow cytometry 3 days post transduction. Virus stocks were stored at -80°C. Transductions of cells were carried out by infection at a multiplicity of infection of 1:10 for 6h, and then the virus containing media was replaced by fresh media.
Cells were selected in puromycin-containing medium (3 ug/ml) and the efficiency of transduction was assessed as percentage of GFP positive cells by flow cytometry. Lack of JAK1 expression in JAK1-deficient cells was verified by reverse transcription polymerase chain reaction (RT-PCR).
Western blot analysis 2x10 6 THP1 SC or THP1 KD cells were collected, washed with cold PBS, and lysed in RIPA (Sigma-Aldrich) lysis buffer containing 1X protease inhibitor cocktail (Roche) for 30min on ice. Cell lysates were centrifuged at 12000rpm for 20min at 4°C and supernatant was collected. Samples were subjected to SDS-polyacrylamide gel electrophoresis (10% Mini-Protean TGX Precast Protein Gels, Biorad) and transferred to nitrocellulose membranes (Biorad). After blocking with 5% BSA for 1h at room temperature, the membranes were probed with the following primary (overnight at 4°C) and HPR-conjugated secondary antibodies (1h at room temperature): mouse anti-JAK-1 (clone 73/JAK1, BD Biosciences), rabbit anti-GAPDH (clone D16H11, Cell Signalling Technology), anti-mouse-IgG-HRP (Cell Signalling Technology) and anti-rabbit-IgG-HPR (Cell Signalling Technology). The proteins were detected with SuperSignal West Pico Plus chemiluminescent substrate (ThemoFisherScientific) using the ChemiDoc Imaging System (Biorad). Mycobacteria were grown to mid-log phase (OD between 0.6-1) in Middlebrook 7H9 medium supplemented with 10% OADC enrichment medium (BD Biosciences), plus 50 μg/ml Hygromycin for BCG expressing-mCherry. Stock cultures were maintained in glycerol at -80° C until later use. Viable cell counts in thawed aliquots of BCG were determined by plating serial dilutions of cultures onto supplemented Middlebrook 7H11 agar plates followed by incubation at 37ºC for 14-21 days. Salmonella was grown to mid-log phase in LB broth overnight with agitation.
The MOI calculation was performed using the following conversion: OD of 1 = 1 x 10 8 CFU/ml for BCG and OD of 1 = 10 9 CFU/ml for Salmonella.Bacteria were washed and suspended in RPMI medium and 10% heat-inactivated FCS.
Microscopy 200,000 THP-1 cells were differentiated on 35mm glass bottom dishes (Fluorodish). After phagocytosis of BCG expressing-mCherry, cells were washed and incubated in complete medium in the presence or absence of IFN-γ (50 ng/ml) for 3 days. Subsequently, cells were incubated with 50 nMLysoTracker Deep Red (Life Technologies) for 30min, washed and then fixed in 4% paraformaldehyde (PFA) for 10 min. Nuclei were stained with 5 µg/ml DAPI for 10 min, cells were then washed and kept on PBS. Cells were acquired using Leica inverted fluorescent microscope equipped with 60x oil objective for quantification of infected cells or Nikon Eclipse Ti-E confocal microscope equipped with 40x objectivefor colocalization analysis. Infected cells were counted manually and at least 100 cells per experiment were analysed. Images were processed using ImageJ (National institute of health) and Imaris image analysis software.

Viral assays
For the plaque assays, control and patient fibroblasts were grown in six-well dishes and infected with different dilutions of PIV5VΔC and PIV5 for 1h. Subsequently, 0.1% Avicel (FMC Biopolymer) was included in the overlay medium and cells were incubated for 5 days at 37ºC in 0.5% CO 2 . Plaques were visualized by immunostaining using a pool of monoclonal virus-specific antibodies for the viruses as described previously (29,30), together with alkaline phosphatase-conjugated secondary antibody by using SIGMAFAST BCIP/NBT as the substrate.
Control and patient fibroblasts were grown on 13 mm diameter coverslips in individual wells of 24-well plates and then were left unstimulated or stimulated with different concentrations (10 3 -10 4 IU/ml) of IFN-α2b overnight. Cells were infected with PIV5 at a multiplicity of infection of 10 pfu/cell. The inoculum was adsorbed for 1 h and then cells were incubated in complete medium in the presence or absence of IFN for 24h. Monolayers were incubated in fixing solution (5 % formaldehyde and 2 % sucrose in PBS) for 15 min at room temperature, then permeabilized (0.5 % Nonidet-P40 and 10 % sucrose in PBS) for 5 min, and washed three times in PBS containing 1 % FCS and 0.1 % azide (PBS, 1 % FCS, 0.1 % azide). To detect the proteins of interest, cell monolayers were incubated with 10-15 μl of an antibody used to detect PIV5 as previously described (30). Cells were subsequently washed (PBS, 1 % FCS, 0.1 % azide). In addition, cells were stained with the DNA-binding fluorochrome DAPI (0.5 μg ml−1; Sigma-Aldrich) for nuclear staining. Following staining, monolayers were washed with PBS, mounted using Mowiol and examined using a Nikon Microphot-FXA immunofluorescence microscope.
EBV-B cells were either left untreated or were treated with 10 4 IU/ml IFN-α2b for 18 h. The kinetics of vesicular stomatitis virus (VSV) growth in EBV-B was determined by resuspending the cells in RPMI medium containing the virus inoculum and incubating for 1 h (VSV MOI = 1), washing with PBS and resuspending in fresh complete medium. Viruscontaining supernatants were then collected at the indicated time points. VSV titers were determined by calculating the 50% end point (TCID50), as previously described (31), after the inoculation of 96-well plates with Vero cell cultures.
Supplementary Tables   Table S1. Clinical diseases allelic with MSMD and viral susceptibility due to defects in type I and/or II IFN signalling pathways.