Conclusion
Our results indicate a novel role for the Sts enzymes in regulating monocyte antibacterial responses downstream of IFNγ.
Methods
Monocyte bacterial clearance was assayed using ex vivo coculture infections followed by colony-forming unit analysis of intracellular bacteria. Levels of gene expression were quantified by quantitative reverse-transcription polymerase chain reaction, levels of Nos2 protein levels were quantified by Western blot analysis, and levels of nitric oxide (NO) were quantified directly using the Griess reagent. We characterized monocyte cytokine production via enzyme-linked immunosorbent assay.
Results
We demonstrate that Sts-/- monocyte cultures produce elevated levels of interferon-γ (IFNγ) after infection, relative to wild type cultures. Sts-/- monocytes also demonstrate heightened responsiveness to IFNγ. Specifically, Sts-/- monocytes produce elevated levels of antimicrobial NO following IFNγ stimulation, and this NO plays an important role in LVS restriction. Additional IFNγ-stimulated genes, including Ip10 and members of the Gbp gene family, also display heightened upregulation in Sts-/- cells. Both Sts-1 and Sts-2 contribute to the regulation of NO production, as evidenced by the responses of monocytes lacking each phosphatase individually. Finally, we demonstrate that the elevated production of IFNγ-induced NO in Sts-/- monocytes is abrogated following chemical inhibition of Syk kinase.