Infection
Innate Immunity to Infection
The host immune system must remain vigilant and proactive to prevent pathogens from disseminating throughout the blood. Although it is becoming clear that a diversity of cells of the immune response reside in the vasculature, the mechanisms to prevent pathogen dissemination have not been fully elucidated, mainly because of the previous limitations of conventional imaging techniques required to study this dynamic environment.
Alveolar macrophages are mobile and capture inhaled bacteria
For the first time, we show that alveolar macrophages are dynamic immune cells that move around and through alveoli in search of harmful pathogens! But during viral infection, their migration is impaired. (Neupane et al., Cell, 2020)
Video_2.mp4Video 1. In Vivo Behavior of Alveolar macrophages
Video_7.mp4Video 2. Capture of Inhaled Bacteria by Crawling Alveolar Macrophages
The liver acts as an immune bottleneck to bloodstream pathogens
Kupffer cells, the tissue-resident macrophage of the liver, are housed within the sinusoids ready to capture any bloodstream pathogen. Using intravital microscopy, we have shown that most bacterial pathogens (Gram+ and Gram-) are caught by Kupffer cells within minutes of systemic infection (video 3). This immune bottleneck is critical to prevent bacterial dissemination and sepsis.
However, some pathogens such as Staphylococcus aureus are able to survive and replicate within Kupffer cells (video 4), which leads to Kupffer cell lysis and dissemination through the peritoneal cavity. In the peritoneal cavity, resident GATA6+ macrophages act as a portal for S. aureus to disseminate to visceral organs such as the kidney (video 5,6).
References: Surewaard BGJ et al., J Exp Med, 2017; Jorch SK et al., JCI, 2019
liver1.mp4Video 3. Capture of S. aureus by Kupffer cells.
liver2.mp4Video 4. Intracellular accumulation of MRSA in KCs.
JCI127286.sdv3.mp4Video 5. Isolated peritoneal macrophages 46 h post infection get overgrown by S. aureus
JCI127286.sdv7.mp4Video 6. 3D reconstruction of S. aureus on the kidney capsule 48 h post-infection.
Neutrophils intercept disseminating S. aureus in the lymph node
Recent work by our laboratory has focused on mechanisms in the lymph node that prevent systemic dissemination of Staphylococcus aureus (Bogoslowski et al., PNAS, 2018):
Staphylococcus aureus (S. aureus) is a bacterium causing many community-acquired skin infections. However, as dissemination from skin infections is rare, we were interested in understanding what mechanisms the body has in place to prevent escape of bacteria from skin infections via lymphatics to the rest of the body. We found that after infection, neutrophils were almost immediately recruited to the lymph nodes via specialized blood vessels to cut bacteria off at the pass. Neutrophils worked together with resident macrophages to prevent S. aureus spread, as removing both these immune cells from the lymph nodes resulted in bacterial dissemination to peripheral organs. This study demonstrates the importance of the lymph node neutrophils in early infection to prevent systemic dissemination of bacteria.
Figure. Neutrophils primarily use blood vessels to enter lymph nodes. PopLN blood vessels at 3 hours post infection with S. aureus MW2 in an LysM EGFP reporter mouse. Green, neutrophils; red, i.v. TRITC-dextran. Image displayed as focus stacking of six 2-μm image slices (scale bar: 52 μm). (Bogoslowski et al., PNAS, 2018)
Neutrophil Extracellular Traps
Neutrophils are the first line of innate immune defense against infection. In addition to the more traditional mechanism of phagocytosis to kill bacteria, activation of neutrophils causes the release of web-like structures of DNA (neutrophil extracellular traps, NETs). Our lab has been studying the formation of NETs both in vitro and in vivo. Our studies demonstrate that NETs assist in trapping intravascular bacteria as well as to limit tissue pathogen dissemination.
NETs in Severe infection and Sepsis
Sepsis kills approximately 300,000–500,000 North Americans a year, and, regardless of the source of the infection, organs such as the lung and the liver are almost always involved. It is now well appreciated that neutrophils become activated and lodge primarily in the capillaries of the lungs and the sinusoids of the liver. Our lab discovered that NETs can occur during severe infection and can be found in humans with septic shock. Furthermore intravascular NETs were able to capture bacteria within the blood and plasma from septic humans could initiate further NETs.
Infection - NETS Figure1: Novel model of bacterial trapping in the microvasculature. (a) Prior to detection of bacteria inactivated neutrophils and platelets are circulating through the microvasculature. The presence of E. coli leads to TLR4 activation. (b) Neutrophils detect LPS and are recruited to the endothelium lining the microvasculature. TLR4-activated platelets are then recruited to the adherent neutrophils, where they bind to the immobilized neutrophils. (c) This leads to robust neutrophil activation and NET formation. A greater number of E. coli are now trapped within the microvasculature by the NETs, where they can be killed and cleared. (Journal of Thrombosis and Haemostasis 2008).
NETs formation requires platelet TLR4
We discovered that platelets, via TLR4, function as a barometer for systemic infection, binding avidly to sequestered neutrophils. This leads to the rapid (within minutes) formation of NETs that maintain their integrity under flow conditions and ensnare bacteria in the circulation. Our data also suggest that this event only happens under extreme conditions, such as severe sepsis, and occurs at the expense of injury to endothelium and tissues.
Infection - NETS Figure 2: LPS-induced platelet adhesion to neutrophils resulted in NET formation. Representative images of neutrophils visualized by white light through an orange filter using darkfield illumination and fluorescence microscopy using Sytox Green to stain extracellular DNA green. (Nature Medicine 2007).
Mechanisms of in vitro NETosis
Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported that NETs take up to 3-4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5-60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton-Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton-Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.
Infection - NETS Figure 3: Scanning electron microscopy of NETs after incubation with S. aureus. Neutrophils and S. aureus were incubated for 1 h on autologous plasma-coated slides, fixed with glutaraldehyde, stained with a gold film and studied with scanning electron microscopy. A, Neutrophils alone. B, Neutrophils stimulated with PMA. C–F, Neutrophils incubated with S. aureus: (D) S. aureus was covered by NETs; (E) NETs formed lattice-like structures and entangled S. aureus; (F) NETs were seen emanating from a small area on the neutrophil surface and fused to other NETs. The images are representative of three experiments with neutrophils from different donors. (Journal of Immunology 2010).
Infection - NETS Figure 4: Transmission electron microscopy of NET formation showing nuclear envelope alterations. Neutrophils and S. aureus were prepared as before, and after fixation with glutaraldehyde they were processed and studied with transmission electron microscopy. Images were obtained from samples with neutrophils alone (A) and neutrophils incubated with S. aureus (B–E). B and C show neutrophils displaying separation of inner nuclear membrane (INM) from outer nuclear membrane (ONM). The nuclear pore complex (NPC) is shown as a marker of INM and ONM. There are small DNA strands with characteristic, previously published “beads on a string” appearance within the lumen between the INM and ONM measuring an average of 11 nm in diameter (strand in C, arrowhead, is magnified in D, arrowheads display the repeated array or “beads on a string”). Cells with nuclear dilations at early time points are surrounded by NETs (E). Original magnifications ×10,000 (A), ×20,000 (B), ×30,000 (C), ×80,000 (D), ×20,000 (E), and ×10,000 (F) using 60 kV voltage. Sections were stained with uranyl acetate and lead citrate. (Journal of Immunology 2010).
References:
Bogoslowski A, Butcher EC, Kubes P. Neutrophils recruited through high endothelial venules of the lymph nodes via PNAd intercept disseminating Staphylococcus aureus. Proc Natl Acad Sci U S A. 2018 Mar 6;115(10):2449-2454. doi: 10.1073/pnas.1715756115. Epub 2018 Jan 29.
Pilsczek FH, Salina D, Poon KK, Fahey C, Yipp BG, Sibley CD, Robbins SM, Green FH, Surette MG, Sugai M, Bowden MG, Hussain M, Zhang K, Kubes P. A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol. 2010 Dec 15;185(12):7413-25.
Clark SR, Ma AC, Tavener SA, McDonald B, Goodarzi Z, Kelly MM, Patel KD, Chakrabarti S, McAvoy E, Sinclair GD, Keys EM, Allen-Vercoe E, Devinney R, Doig CJ, Green FH, Kubes P. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med. 2007 Apr;13(4):463-9.
Ma, AC, Kubes, P. Platelets and neutrophils interact to create bacterial traps in sepsis. Journal of Thrombosis and Haemostasis, 2008 Mar; 6(3):415-20.
Lee WY, Moriarty TJ, Wong CH, Zhou H, Strieter RM, van Rooijen N, Chaconas G, Kubes P. An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells. Nature Immunology Volume:11, Pages:295–302 (2010).