Intravital Imaging Reveals Differential Roles Of Neutrophilic and Stromal Toll-Like Receptor 4 In Hyper-Acute Bone Marrow Response To Systemic Lipopolysaccharide Challenge
Authors: Yee-Chen Huang Alex , Myers Jay 
Published: October 2019

Abstract

Introduction
Neutrophils are the first defenders of host innate immune response against a variety of pathogens. Produced in large quantities from myeloid progenitor cells in the bone marrow, neutrophils are rapidly mobilized and released from the marrow cavity into the general circulation in response to inflammation and invading pathogens. While how neutrophils are released from the bone marrow as a result of interplay among CXCL12 (osteoblastic) and CXCL1/CXCL2 (perivascular) gradients have been studied at a time scale of hours to days post systemic lipopolysaccharide (LPS) challenge, little is known about how the neutrophil and marrow stroma react on a single-cell level in vivo during the hyper-acute (minutes to hours) phase following LPS challenge.

Methods
We performed intravital two-photon microscopy (TPM) on intact cranium of live, anesthetized mice, in which the green fluorescent protein (GFP) is expressed under the Lysozyme M (LysM) promoter expressed predominantly in neutrophils and monocytes, before and immediately after systemic LPS challenge. The behavior of neutrophils and surrounding stroma was analyzed and correlated with systemic neutrophilic responses. Bone marrow chimeras were generated in order to differentiate TLR4-mediated responses in hematopoietic and radio-resistant stromal elements. Neutralizing antibodies to relevant chemokines responsible for neutrophil mobilizations were used to observe changes in intra-marrow neutrophil response following LPS challenge.

Results
We observed a 2.5-fold increase in the average instantaneous speed, as well as the formation of dynamic GFP+ cellular clusters in 50% of the marrow-resident neutrophils between 30 minutes to 60 minutes following i.v. administration of 100ng of LPS, with the GFP+ neutrophils achieving a peak average speed of 8.4um/min. This was accompanied by release of neutrophils into the systemic circulation as evidenced by a 40% reduction in the overall GFP fluorescence intensity 90 minutes following LPS challenge. Bone marrow chimera experiments revealed that while intra-marrow migration and cellular clustering are dependent on TLR4 signaling in neutrophils, the release of neutrophils into systemic circulation is facilitated by TLR4 signaling on stromal elements. Blocking CXCR2 signaling on neutrophils resulted in the same phenotype as chimeras expressing TLR4-defective stroma, further revealing that marrow stromal elements modulate TLR4-mediated neutrophil mobilization and release via CXCL1/CXCL2 chemokine signaling during this hyper-acute phase of marrow response to LPS.

Conclusions
Intravital imaging provides real-time insights to the mechanisms of dynamic regulation of marrow responses to hyper-acute systemic LPS challenge in vivo at a single-cell level within live hosts, and reveals differential contribution of TLR4 signaling on neutrophils and stroma in the bone marrow's response to systemic LPS challenge.

Disclosures:
No relevant conflicts of interest to declare.