Age-associated accumulation of B cells promotes macrophage inflammation and inhibits lipolysis in adipose tissue during sepsis. Carey et al.
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Full, raw western membrane images for all western blots shown in manuscript. Abstract: Non-canonical lipolysis induced by inflammatory cytokines or Toll-like receptor ligands is required for the regulation of inflammation during endotoxemia and sepsis. Canonical lipolysis induced by catecholamines declines during aging due to factors including an expansion of lymphocytes, pro-inflammatory macrophage polarization, and an increase in chronic low-grade inflammation; however, the extent to which the non-canonical pathway of lipolysis is active and impacted by immune cells during aging remains unclear. Therefore, we aimed to define the extent to which immune cells from old mice influence non-canonical lipolysis during sepsis. We identified age-associated impairments of non-canonical lipolysis and an accumulation of dysfunctional B1 B cells in the vWAT of old mice. Life-long deficiency of B cells resulted in restored non-canonical lipolysis and reductions in pro-inflammatory macrophage populations. Our study suggests that targeting the B cell-macrophage signaling axis may resolve metabolic dysfunction in aged vWAT and attenuate septic severity in older individuals.
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Western blotting- Whole vWAT tissue lysates were prepared from flash frozen tissues by homogenization in RIPA with phosphatase and protease inhibitors (Sigma-Aldrich; P0044, P5726, P8340). Lysates were left on ice for one hour, vortexing every 15 min. Lysates were then spun twice at 14,000 rpm for 10 minutes at 4°C, and the pellet and lipids were discarded after each spin. Total protein concentration was measured using a Bradford Assay (Fisher Sci; 23246) and protein amounts are standardized (15μg) for gel loading. Loading Buffer and Reducing Agent (Thermo Fisher; B0008, B0004) was added to samples, and protein was separated across a 4-12% Bis-Tris SDS Gel. After separation, protein was blotted onto a PVDF membrane (Invitrogen; PB5210) via semi-dry transfer, and blots were blocked with 5% non-fat milk in 0.5% TBST solution for 1 hour. Blots were incubated in primary antibody solution in 50 mL conical tubes on a rotator overnight at 4°C in either 2.5% BSA or nonfat milk in 0.5% TBST according to the antibody datasheets. Blots were then washed three times in a 0.5% TBST solution for 5 min each and incubated in goat anti-rabbit secondary antibody (Invitrogen; 31460) solution for 45 min at room temperature on a benchtop rocking table. Following three final washes in 0.5% TBST before, blots were imaged with ECL substrate (Thermo Fisher; PI32106) on a chemiluminescent imaging camera. Quantification of bands were performed with Thermo Fisher’s iBright Analysis software. More method details found in manuscript STAR Methods.