Glycolytic reprogramming of macrophages activated by NOD1 and TLR4 agonists: No association with proinflammatory cytokine production in normoxia.

Upon activation with pathogen-associated molecular patterns, metabolism of macrophages and dendritic cells is shifted from oxidative phosphorylation to aerobic glycolysis, which is considered important for proinflammatory cytokine production. Fragments of bacterial peptidoglycan (muramyl peptides) activate innate immune cells through nucleotide-binding oligomerization domain (NOD) 1 and/or NOD2 receptors. Here, we show that NOD1 and NOD2 agonists induce early glycolytic reprogramming of human monocyte-derived macrophages (MDM), which is similar to that induced by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide. This glycolytic reprogramming depends on Akt kinases, independent of mTOR complex 1 and is efficiently inhibited by 2-deoxy-d-glucose (2-DG) or by glucose starvation. 2-DG inhibits proinflammatory cytokine production by MDM and monocyte-derived dendritic cells activated by NOD1 or TLR4 agonists, except for tumor necrosis factor production by MDM, which is inhibited initially, but augmented 4 h after addition of agonists and later. However, 2-DG exerts these effects by inducing unfolded protein response rather than by inhibiting glycolysis. By contrast, glucose starvation does not cause unfolded protein response and, in normoxic conditions, only marginally affects proinflammatory cytokine production triggered through NOD1 or TLR4. In hypoxia mimicked by treating MDM with oligomycin (a mitochondrial ATP synthase inhibitor), both 2-DG and glucose starvation strongly suppress tumor necrosis factor and interleukin-6 production and compromise cell viability. In summary, the requirement of glycolytic reprogramming for proinflammatory cytokine production in normoxia is not obvious, and effects of 2-DG on cytokine responses should be interpreted cautiously. In hypoxia, however, glycolysis becomes critical for cytokine production and cell survival.

A cell-based test system for the assessment of pharmacokinetics of NOD1 and NOD2 receptor agonists.

Agonists of nucleotide oligomerization domain (NOD) 1 and NOD2 receptors represent a promising class of immunostimulants and immunological adjuvants. Here, we describe a cell-based test system to assess their pharmacokinetics. In this system, NOD1 and NOD2 agonist concentrations in sera are determined using a reporter cell line, 293Luc, which contains an NF-κB-inducible luciferase reporter construct and naturally expresses NOD1 and NOD2. The 293Luc cells dose-dependently respond to different NOD1 and NOD2 agonists in the nanomolar to low-micromolar concentration range. To verify that the NF-κB-inducing activity of serum samples is due to the administered agonist and not to secondarily induced endogenous molecules, a 293Luc-derived NOD1/NOD2 double-knockout clone is used. Within-run and between-run precisions of the system are <15% and <20%, respectively. Applicability of the novel assay is illustrated by studying pharmacokinetics of two specific NOD2 agonists (N­acetyl­d­glucosaminyl­N­acetyl­d­muramyl­l­alanyl­d­isoglutamine and N­glycolyl­d­muramyl­l­alanyl­d­isoglutamine) and a specific NOD1 agonist (N­acetyl­d­glucosaminyl­N­acetyl­d­sorbitolamine­d­lactoyl­l­alanyl­d­isoglutamyl­meso­diaminopimelic acid). In summary, the test system described here can potentially be used to assess pharmacokinetics of NOD1 and NOD2 agonists in different animal species.