TLR-Signaling Induces Granulocytic MDSCs Generation by Directing Myeloid Lineage Commitment During Polymicrobial Sepsis

Elevated leukocyte frequency at early stage of polymicrobial sepsis is a common phenomenon, which is considered to be a result of rapid migration of neutrophils from the bone marrow (BM). However, a dramatic increase of granulocytic myeloid-derived suppressor cells (MDSCs) of sepsis usually occurs after sustained stimuli of the pathogens, which implies that emergency granulopoiesis is involved in negative regulation of immune response. In the present study, we try to reveal the mechanisms by which TLR signal regulates emergency myelopoiesis during infection.

:B Mice were subjected to cecal ligation and puncture to make polymicrobial sepsis model. Blood, spleens and bone marrow were harvested post operation at 1, 3, 7 and 12 day, and analyzed using flow cytometry. Hematopoietic stem cells and special progenitors were isolated using an FACS Aria sorter, and then total mRNA was extracted and reverse-transcripted. Some lineage commitment-related genes including Ikzf1,Gfi1, Cebp/a, PU.1, Csf1r, Csf2r, Csf3r, Notch1, IL-7r, Flt3 were analyzed by Real-time PCR. The methylcellulose colony assay of single-cell suspensions of bone marrow cells was used to identify myeloid progenitors in sepsis model.

We found that the elevated frequency of myeloid cells was associated with an expansion of hematopoietic stem cell (HSC) pool accompanied by augmented proportion of cells entering cell cycle. Furthermore, increased HSC numbers resulted in gradually elevated proportions of common myeloid progenitors (CMPs) and granulocyte/macrophage progenitors (GMPs), but a decrease of the frequency of common lymphoid progenitors (CLPs). In line with the decreased ratio of lymphoid/myeloid progenitors, real-time PCR revealed impaired expression levels of lymphoid differentiation-related genes in HSCs and CLPs, including Flt3, IL7R, Notch1, Ikaros and Rag1. By contrast, the expression levels of myeloid differentiation-related genes (eg., CEBP/a, Gfi1, PU.1, GM-CSFR) were up-regulated or remained unchanged in the stem cell and myeloid progenitor compartments. Moreover, consistent with elevated frequency of granulocytic MDSCs, an increase of Gfi-1 expression which made a granulocytic decision and a decrease of M-CSFR expression which played a crucial role in monopoiesis were noticed in CMPs and GMPs. More importantly, similar results were obtained in the mice challenged with LPS peritoneal injection.

Our study sheds light on its importance of TLR-signaling in generation and replenishment of granulocytic MDSCs by affecting early myeloid /lymphoid lineage decision and late granulocyte /monocyte lineage decision.

No relevant conflicts of interest to declare.