University of Calgary
UofC Navigation

Injury

 Injury 

Tissue injury results in acute inflammation and activation of the innate immune system. However how the innate immune system detects and orchestrates a response to non-infectious stimuli has not been well defined.  The Kubes lab is actively studying multiple forms of sterile tissue injury in the liver and the brain and have discovered novel inflammatory pathways.

 

Intravascular danger signals guide neutrophils to sites of sterile inflammation 

To understand how PMN arrive to sites of sterile inflammation, we have developed a novel thermal injury model of the liver parenchyma and visualized the sinusoidal recruitment of vascular PMN using spinning disk confocal intravital microscopy (Fig1A, Video).  PMN rapidly responded to intravascular danger signals (adenosine triphosphate) and used a hierarchy of distinct directional cues to lead them through healthy liver tissue (chemokine gradient) and then through dead injured liver (formyl-peptide gradient)(Fig 1B and Fig2).

 Figure 1.
Injury Figure 1A: (A) Time-lapse images from SD-IVM demonstrating the response of neutrophils (green) to focal hepatic necrosis (red, propidium iodide). Scale bar indicates 200 μm

Injury Figure 1A: (A) Time-lapse images from SD-IVM demonstrating the response of neutrophils (green) to focal hepatic necrosis (red, propidium iodide). Scale bar indicates 200 μm

   Injury Figure 1B: (B) Representative SD-IVM images at 2 and 3 hours after injury, demonstrating the intravascular (blue, Alexa-647-BSA) route taken by neutrophils (green) to reach necrotic foci (red). Arrows show path of travel of selected neutrophils. Scale bars, 100 μm

Injury Figure 1B: (B) Representative SD-IVM images at 2 and 3 hours after injury, demonstrating the intravascular (blue, Alexa-647-BSA) route taken by neutrophils (green) to reach necrotic foci (red). Arrows show path of travel of selected neutrophils. Scale bars, 100 μm 

 Injury Figure 2Injury Figure 2

 

 

 

Braedon 1

Response of circulating neutrophils to focal sterile injury in vivo. The response of eGFP-expressing neutrophils (green) to a focal necrotic lesion in the liver (red, propidium iodide) in an untreated lysM-eGFP mouse was visualized by SD-IVM (x4 objective) for four hours after injury. Elapsed time shown in top right corner. Scale bar: 200 µm.

 

 

Braedon 2 

Rapid chemotaxis of neutrophils to sites of sterile injury through the intravascular channels. Migratory behavior of eGFP-expressing neutrophils (green) in a lysM-eGFP mouse visualized by SD-IVM (x10 objective) between 2 and 3 hours after induction of a focal necrotic lesion in the liver (red, propidium iodide). Hepatic microvasculature is visualized by circulating Alexa-fluor-647 labeled BSA (blue). Elapsed time shown in top right corner. Scale bar: 100 µm.

 

 Braedon 3

Chemotaxing neutrophils remain intravascular as they migrate to sites of sterile injury. Representative 3-dimentional volume rendering of the liver microvasculature in a lysM-eGFP mouse 2 hours after induction of a focal necrotic lesion. eGFP-expressing neutrophils (green) remain within the vascular channels (blue; Alexa-fluor-647 labeled BSA) en route to foci of sterile tissue injury (red, propidium iodide).

 

 Intravascular chemotaxis of neutrophils to sites of sterile injury requires functional Mac1. Migratory behavior of eGFP-expressing neutrophils (green) in a lysMeGFP mouse treated with a Mac1-blocking antibody visualized by SD-IVM (x10 objective) after induction of a focal necrotic lesion in the liver (red, propidium iodide). Hepatic microvasculature is visualized by circulating Alexa-fluor-647 labeled BSA (blue). Elapsed time shown in top right corner. Scale bar: 100 µm.

 

Braedon 5 
Apyrase administration reduces neutrophil infiltration to sites of sterile injury in the liver. The response of eGFP-expressing neutrophils (green) in a lysMeGFP mouse treated with apyrase was visualized by SD-IVM (x4 objective) for four hours after induction of a focal necrotic lesion in the liver (red, propidium iodide). Elapsed time shown in top right corner. Scale bar: 240 µm.

 

A novel link between vascular brain injury and infection

 Patients suffering a non-infectious injury to the brain, either due to blunt trauma or a vascular insult such as a stroke, develop bacterial infections immediately following the injury.  This common clinical observation has perplexed physicians and researchers for years.  We have investigated how injury to the brain leads to immunosuppression resulting in infection.  Using a mouse stroke model we found that rapid and profound immunosuppression occurred because of changes in the behavior of liver invariant NKT cells.  Sympathetic innervation linked the central nervous system to the immune system through its effects on liver iNKT cells.  Stimulation of liver iNKT cells promoted a pro-inflammatory response that was protected the host from infection.

 

 

Crawling behavior of hepatic iNKT cells in vivo. Spinning disk microscopy was used to visualize iNKT cells within the livers of sham-operated Cxcr6gfp/+ mice. Elapsed time is shown at the top right. The time lapse was recorded at 10 frames per minute (fpm) and exported to video at 20 frames per second (fps). Scale bar indicates 50 μm.

 

Cessation of crawling behavior of post-MCAO hepatic iNKT cells in vivo. Spinning disk microscopy was used to visualize iNKT cells within the livers of post-MCAO Cxcr6gfp/+ mice at 24 h. Elapsed time is shown at the top right. The time lapse was recorded at 10 fpm and exported to video at 20 fps. Scale bar indicates 50 μm.

 

Pirouetting behavior of post-MCAO hepatic iNKT cells in vivo. Spinning disk microscopy was used to visualize the "pirouetting" behavior of iNKT cells within the livers of post-ischemic Cxcr6gfp/+ mice at 24 h. Elapsed time is shown at the top right. The time lapse was recorded at 10 fpm and exported to video at 20 fps. Scale bar indicates 25 μm.

 

Propranolol reversed crawling cessation of post-MCAO hepatic iNKT cells in vivo. Spinning disk microscopy was used to visualize iNKT cells within the livers of Cxcr6gfp/+ mice. Elapsed time is shown at the top right. The time lapse was recorded at 10 fpm and exported to video at 20 fps. Scale bar indicates 50 μm

  

References: 

  • McDonald B, Kubes P.  Neutrophils and intravascular immunity in the liver during infection and sterile inflammation.  Toxicol Pathol. 2012;40(2):157-65. 
  • McDonald B, Kubes P.  Cellular and molecular choreography of neutrophil recruitment to sites of sterile inflammation.  J Mol Med (Berl). 2011 Nov;89(11):1079-88. Epub 2011 Jul 13. Review. 
  • McDonald B, Pittman K, Menezes GB, Hirota SA, Slaba I, Waterhouse CC, Beck PL, Muruve DA, Kubes P.  Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science. 2010 Oct 15;330(6002):362-6. Erratum in: Science. 2011 Mar 25;331(6024):1517. 
  • Wong CH, Jenne CN, Lee WY, Léger C, Kubes P.  Functional innervation of hepatic iNKT cells is immunosuppressive following stroke.  Science. 2011 Oct 7;334(6052):101-5.