In Vivo Visualization of Stat3 Activation in Myeloid Cells during Inflammation and Tumor Growth.
Authors: Horna Pedro , Cheng Fengdong , Jove Richard , Mora Linda , Sotomayor Eduardo M. 
Published: October 2019

Abstract
Signal transducer and activator of transcription 3 (Stat3) is a key mediator of several cytokine and growth factor signaling pathways. On myeloid cells, activation of Stat3 to its phosphorylated form (pStat3) has been shown to negatively regulate inflammatory responses and play a central role in the decision leading to immune activation versus immune tolerance of antigen-specific T-cells1. Little is still known however, about the status of Stat3 signaling in myeloid cells in the steady state and during ongoing immune responses in vivo. To address this question we recently developed flow-cytometric and immuno-histochemistry assays that have allowed us to visualize the in vivo dynamics of Stat3 activation in myeloid cells during immune responses leading to divergent outcomes: productive inflammatory response to adjuvant immunization and tumor-induced unresponsiveness or tolerance. In the steady state we found that in peripheral blood only Ly6G+ polymorphonuclear cells display a positive nuclear staining for pStat3. Analysis of lymphoid organs revealed that although Stat3 protein was expressed almost ubiquitously on spleen sections of normal mice, only a small number of cells were positive for pStat3. Following immunization with complete Freund adjuvant (CFA) a dramatic increase in the number of cells expressing pStat3 was observed in the peripheral blood and spleen of treated animals. Ly6G+ pStat3+ were rapidly recruited from the blood to the inflammatory site where they now displayed significantly decreased levels of pStat3. During the growth of a subcutaneous tumor, a similar increase in the number of cells expressing pStat3 was observed in the blood and spleen of tumor-bearing mice. Further analysis by flow cytometry revealed that pStat3 expression was restricted to two sub-populations: a) CD11b+ myeloid cells expressing the lineage marker Gr-1 and b) Ly6G− mononuclear cells unable to down-regulate Stat3 activity following their migration from the blood into peripheral tissues. In vivo depletion of Gr-1+ cells eliminated most of the pStat3+ cells in tumor bearing mice. The immunoregulatory properties of these Gr-1+ cells was highlighted by the demonstration that in their absence, in vivo immunization with a peptide derived from influenza hemagglutinin (HA) in CFA markedly enhanced the priming of anti-HA specific CD4+ T-cells. More importantly, in animals depleted of Gr-1+ cells, the outcome in response to a tolerogenic stimuli was T-cell activation rather than tolerance induction. Taken together, although similar changes in the number of cells expressing pStat3 was observed in response to adjuvant immunization and during tumor progression, an important difference might relate to the extent of Stat3 activation in myeloid cells following their migration to the site of stimuli. While down-regulation of Stat3 in myeloid cells at the inflammatory site is an early event during productive inflammatory responses, a sustained Stat3 activation in myeloid cells such as that observed during tumor growth may provide an explanation for the state of immune unresponsiveness associated with malignancies.