Metabolic activation and colitis pathogenesis is prevented by lymphotoxin β receptor expression in neutrophils

Inflammatory bowel disease is characterized by an exacerbated intestinal immune response, but the critical mechanisms regulating immune activation remain incompletely understood. We previously reported that the TNF-superfamily molecule TNFSF14 (LIGHT) is required for preventing severe disease in mouse models of colitis. In addition, deletion of lymphotoxin beta receptor (LTβR), which binds LIGHT, also led to aggravated colitis pathogenesis. Here, we aimed to determine the cell type(s) requiring LTβR and the mechanism critical for exacerbation of colitis. Specific deletion of LTβR in neutrophils (LTβRΔN), but not in several other cell types, was sufficient to induce aggravated colitis and colonic neutrophil accumulation. Mechanistically, RNA-Seq analysis revealed LIGHT-induced suppression of cellular metabolism, and mitochondrial function, that was dependent on LTβR. Functional studies confirmed increased mitochondrial mass and activity, associated with excessive mitochondrial ROS production and elevated glycolysis at steady-state and during colitis. Targeting these metabolic changes rescued exacerbated disease severity. Our results demonstrate that LIGHT signals to LTβR on neutrophils to suppress metabolic activation and thereby prevents exacerbated immune pathogenesis during colitis.


Percent Starting Body Weight
Ltbr fl/fl x Cx3cr1 Cre -Ltbr fl/fl x Cx3cr1 Cre + n.s.     , g-h), or are combined from three experiments (f).   Glutathione Peroxidase 4 Redox Table 1 List of genes that are among top50 differentially regulated by LIGHT and with redox or mitochondrial Gene Ontology

Cell isolation
Bone marrow neutrophils were obtained by crushing tibia and femur with a mortar and pistil in RPMI1640 supplemented with 5% FBS. The suspension was filtered through a 70um mesh washed and pelleted. Following red blood cell lysis, non-granulocytic cells were immunolabelled with a mixture of antibodies according to the Neutrophil magnetic bead negative selection kit (Stem Cell Technologies), or labelled with CD11b and Ly6G antibodies for direct, positive sorting via flow cytometry. Purity of obtained neutrophils was controlled post-sort and was typically >90% (negative selection) or >95% (positive selection).

RNA-Seq data acquisition and analysis
Neutrophils were isolated from the blood of 8-week-old LTβR ΔN mice, and littermate controls, Data were acquired using Fortessa or LSR II flow cytometers (BD Biosciences) and analyzed with FlowJo software. Metabolic marker fluorescence intensity depends on the instrument type and laser intensity, and therefore does not allow inter-experiment comparisons.

Metabolic flux analysis
3 × 10 5 freshly isolated Ly6G + BM neutrophils were obtained by flow-cytometric sorting using CD11b and Ly6G. Post-sort purity was >95%. Neutrophils were washed twice in the assay medium consisting of bicarbonate-free DMEM base (Sigma, D-5030-1L), 1.85 g/L NaCl and 6 mg/ml Phenol Red supplemented with 5 mM HEPES-Na, pH7.6, 10 mM glucose, 10 mM pyruvate and 4 mM glutamine, then attached in the assay medium to a XF plate coated with Cell-Tak (Sigma) by spinning at 500 g for 5min. Cells were rested for 20min in a non-CO2 incubator at 37°C before analysis. Mitochondrial stress test was performed with all rates read twice, with 30 sec of mixing followed by 2min data acquisition. The initial reading of the basal rates was followed by injection of 2 µg/ml oligomycin to stop mitochondrial ATP synthesis and force compensatory stimulation of glycolysis. Subsequently, two sequential injections of 300 µM uncoupler 2,4-dinitrophenol (DNP) were made to ensure attaining the maximal OCR 3 . The run was terminated with the injection of 2 µM myxothiazol, a respiratory inhibitor targeting Complex III to control for non-mitochondrial background rate 4 . The basal rates reflect cellular ATP turnover and, thus, served as a measure of cellular energy demand; maximal uncoupler-stimulated OCR and oligomycin-induced ECAR served as the measures of cellular respiratory and glycolytic capacities, respectively.

Electron Microscopy
Incubation of the cell pellet with 1 % OsO4 in buffer for 30 min was followed by three washing steps with dH2O. The sample was incubated with 2 % uranyl acetate, rinsed, and dehydrated in EtOH. Embedded cells were then placed in a 60°C oven overnight. Sections were cut on a Leica Ultracut microtome and collected on copper grids. Sections were stained with 2 % uranyl acetate in dH2O and Sato Lead. The TEM evaluation was carried out on the JEOL-1200 EX microscope.