Autoimmune susceptibility gene PTPN2 is required for clearance of adherent-invasive Escherichia coli by integrating bacterial uptake and lysosomal defence

Objectives Alterations in the intestinal microbiota are linked with a wide range of autoimmune and inflammatory conditions, including inflammatory bowel diseases (IBD), where pathobionts penetrate the intestinal barrier and promote inflammatory reactions. In patients with IBD, the ability of intestinal macrophages to efficiently clear invading pathogens is compromised resulting in increased bacterial translocation and excessive immune reactions. Here, we investigated how an IBD-associated loss-of-function variant in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene, or loss of PTPN2 expression affected the ability of macrophages to respond to invading bacteria. Design IBD patient-derived macrophages with wild-type (WT) PTPN2 or carrying the IBD-associated PTPN2 SNP, peritoneal macrophages from WT and constitutive PTPN2-knockout mice, as well as mice specifically lacking PTPN2 in macrophages were infected with non-invasive K12 Escherichia coli, the human adherent-invasive E. coli (AIEC) LF82, or a novel mouse AIEC (mAIEC) strain. Results Loss of PTPN2 severely compromises the ability of macrophages to clear invading bacteria. Specifically, loss of functional PTPN2 promoted pathobiont invasion/uptake into macrophages and intracellular survival/proliferation by three distinct mechanisms: Increased bacterial uptake was mediated by enhanced expression of carcinoembryonic antigen cellular adhesion molecule (CEACAM)1 and CEACAM6 in PTPN2-deficient cells, while reduced bacterial clearance resulted from defects in autophagy coupled with compromised lysosomal acidification. In vivo, mice lacking PTPN2 in macrophages were more susceptible to mAIEC infection and mAIEC-induced disease. Conclusions Our findings reveal a tripartite regulatory mechanism by which PTPN2 preserves macrophage antibacterial function, thus crucially contributing to host defence against invading bacteria.


Supplementary Methods
Macrophages. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls and IBD patients by density gradient centrifugation on a Ficoll layer (20,000rpm for 20 min. at room temperature), washed twice in ice cold PBS and frozen in FCS containing 10% DMSO. PBMCs were then thawed, washed twice with RPMI (Life Technologies) 10% FCS and CD14+ cells isolated using the Miltenyi CD14 + cell isolation kit according to the manufacturer's instructions.
For differentiation of THP-1 cells into macrophages, 10 6 cells were pulsed for 3 h with 50 ng/ml PMA, washed in serum-free RPMI and incubated for 48 h as described previously [1].
Bone marrow macrophages were prepared as described [1]. In brief, bone marrow was isolated from femori and tibiae, strained through a 70µm nylon mesh and cells incubated in differentiation medium (RPMI containing 1% pen/strep, 1% glutamine, 1% Na-pyruvate, 10 % FCS and 20 % L929 supernatant) for 7 days. On day 4, half of the culture medium was replaced by fresh differentiation medium.
Immunofluorescence staining. For immunofluorescence staining, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed in PBS and fixed with methanol for 10 min at -20°C. After washing three times with PBS, unspecific antibody binding was blocked by incubation with 10% normal goat serum in Tris-buffered saline with 0.01% Tween-20 (TBS-T) for 2 h at room temperature prior to incubation with anti-LAMP-1 (1:200) or anti- LC3B (1:200) were used as primary antibodies. After washing 3 x in PS-Tween, secondary antibody was applied for 1 h at room temperature, cells washed three times in PBS-Tween and slides mounted with DAPI containing ProlongGold anti-fade mounting medium (Thermo Fisher Scientific). Images were taken on a Leica DM5500B microscope with a DFC450C camera (Leica) or a SP5 confocal microscope (Leica) and processed using the Leica Application Suite AF3. Tris-Cl, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) for protein isolation. RNA lysates were processed using the RNeasy mini kit from Qiagen according to the manufacturer's instructions and RNA concentration estimated measuring absorbance at 260 and 280 nm. Protein lysates were sonicated on ice for 30 seconds, centrifuged at 13,000g for 10 min and protein containing supernatants transferred to fresh tubes and protein concentrations measured using a BCA kit.
Western blotting. For Western blot analyses, equal amount of protein were loaded onto polyacrylamide gels and separated by SDS-PAGE. Proteins were blotted onto PVDF membranes, blocked in 3% milk, 1% BSA in TBS-T (Trisbuffered saline with 0.01% Tween20) prior to incubation with primary antibody overnight. Membranes were then washed three times in TBS-T, incubated with HRP-labeled secondary antibody for 1 h at room temperature, washed 3x with TBS-T and immunoreactive proteins visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific) and x-ray films (GE Healthcare Systems).

Quantitative PCR.
Complementary DNA (cDNA) synthesis was performed using the qScript cDNA synthesis kit from Quantabio (Beverly, MA) following the manufacturer's instructions. Real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) on a C1000 Thermal cycler equipped with a CFX96 Real-Time PCR system using BioRad CFX Manager 3.1 Software.
Measurements were performed in triplicates, mouse GAPDH was used as endogenous control, and results were analyzed by the ΔΔCT method. The realtime PCR contained an initial enzyme activation step (3 min, 95 °C) followed by 45 cycles consisting of a denaturing (95 °C, 10 seconds), an annealing (53°-60°C, 10 seconds) and an extending (72 °C, 10 seconds) step. The used primers are listed in the Key Resources Table. BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)