TLR signaling augments macrophage bactericidal activity through mitochondrial ROS: How ROS impact the Innate Immune Response

            This paper, published in 2011 in Nature by a research team out of Yale University School of Medicine lead by Dr. A Phillip West investigated the potential role that macrophages, which are phagocytotic cells that participate in the innate immune response, play in attacking bacteria. Prior to this paper, the link between reactive oxygen species (ROS) and phagocytes involved in the innate immune response had already been connected (Lambeth, 2004). However, Dr. West and his team attempted to illustrate the pathway in which ROS originated from the mitochondria contribute to the ability of the macrophage to combat bacteria.

            To set the stage, reactive oxygen species play numerous roles within aerobic organisms, which are organisms that create energy by use of oxygen as the final electron receptor (Hulbert et al, 2007). Aerobic metabolism produces reactive oxygen species as a byproduct of producing the proton gradient needed to make ATP, which occurs along the inner membrane of the mitochondria (Hulbert et al, 2007). These ROS can harm an organism, as they are extremely reactive species that can damage a cells macromolecules, such as lipids, DNA, and proteins (Hulbert et al, 2007). However, ROS can also function as signaling molecules and play an important role within the innate immune response (Lambeth, 2004). The innate immune response is a general immune response utilized by an organism at the first sign of damage or infection, as, although a generalized attack, it is quick and sometimes sufficient (Mak et al, 2014). Macrophages play an important role in the innate immune by engulfing, through phagocytosis, and killing microorganisms (Mak et al, 2014). Since the ROS produced within the mitochondria have been thought to only play a negative role, Dr. West and his team thought to investigate the possible role that mROS could play in the macrophages overall ability to destroy invading bacteria.

            To observe this, West and his team started by answering the most broad question of their research project, what links mROS production to the innate immune response? West et al (2011) hypothesized that the link may be related to a group of Toll-like receptors, specifically TLR1, TLR2, and TLR4.  A Toll-like receptor is a microbial sensing proteins that are embedded in the membranes of cells and are utilized by the cell to patrol for potential dangers to the organism (Christmas, 2010). There are, so far, ten different toll-like receptors (TLR), but TLR1, TLR2, and TLR4 were selected by Dr. West and his team because these three are part of a group that can bind to parts of microbial cell walls and membranes that are only found in pathogens (Christmas, 2010). These three TLRs are also located on the cell’s plasma membrane, thus these three could then help understand the connection between the activation of these receptors and mROS generation (Christmas, 2010). To activate the specific TLRs in the macrophages, macrophages were obtained by the ATCC (a company that provides researchers with immunological research tools) from tumor cells in mice and were then exposed to specific molecules that bind to a TLR and turn it on, which are called agonists. These included lipopolysaccharides (LPS), which turn on TLR 4, the synthetic lipopeptide Pam3CSK4, which turns on TLR 1/2, lipotechoic acid, which turns on TLR 2 (West et al, 2011). Other TLR receptors that do not bind to microbial cell walls or membranes and are located on the membrane of the endosome were also looked, such as TLR 3,7,8,9 and each were also turned on by their respective molecules (West et al, 2011; Christmas, 2010). Bone marrow derived macrophages (BMM) were looked at in a similar manner. To do this, the researchers plated each cell type on a well plate and mixed them with each type of agonist separately. Dyes that colored the mROS and hydrogen peroxide were used to be able to calculate concentration from each sample’s fluorescence intensity value (FACS). The researchers found that stimulation of the surface TLR receptors triggered the production of mROS while the endosomal TLRs did not (West et al, 2011)(Figure 1). The authors concluded that the cell surface TLRs increased ROS generation because these are the receptors that interact with the bacteria directly. Endosomal TLRs sense viral mediators and thus, it would not make sense for the end to increase ROS production if the purpose of the ROS is to help signal the macrophage to phagocytose a bacteria. To learn more about how TLRs bind to pathogens and induce an immune signal, click here.

Figure 1: A. RAW macrophages cell surface TLRs interacting with their agonists produced more ROS than endosomal TLRs interacting with their agonists, as illustrated by their fluorescence intensity values. The cell surface TLR samples fluoresced more intensely in the ROS dye than the endosomal TLR samples, indicating more ROS was produced in the former. B.  BMM macrophages cell surface TLRs increased hydrogen peroxide production while endosomal TLR 9 did not. The same technique described above was used to determine these results.

             Next, armed with the knowledge that ROS production is up-regulated by activation of cell surface TLRs, West and his team investigated if mitochondria could be moved near to internal vacuoles containing the phagocytized bacteria. Previous studies have indicated that mitochondria are recruited inside a cell to locate closer to vacuoles containing bacteria, one such study by Sinai, Webster, and Joiner (1997) can be found here if further inquiry is desired.  To potentially observe this phenomena and thus explain how mROS play a role in the innate immune response, West and his team exposed BMM to coated beads covered in pathogen-associated molecular patterns (PAMPs).  A PAMP is a functional component of a microorganism that allows host cells to differentiate that microorganism as “not self” and thus deal with it effectively (Tang et al, 2012). West and his team witnessed that BMM that consumed beads coated in a TLR 1/TLR 2 agonist and TLR 4 agonist, mitochondria were recruited to the bead (Figure 1c).

Figure 1c. The recruitment of mitochondria inside BMM. The bead are colored in red and in rows 1 and 2, mitochondria are colored green. In the last row, mitochondria are colored yellow. In BMM that encapsulated beads coated in Pam3csk4 and LPS, mitochondria are seen to surround the bead, an effect not seen in the uncoated bead trial.

            These two results led West and his team to hypothesize that since the mitochondria move towards areas containing the agonists of TLR 1/2/4, TLR signaling from the 1/2/4 pathways should also occur. To begin to examine this hypothesis, West and his team measured a known intermediate in the cell signaling pathway in the above TLRs, TRAF6 (West et al, 2011). The researchers found that macrophages stimulated agonists for the three TLRs had increased expression of TRAF6 in their mitochondria (West et al, 2011). The researchers connected this upregulation of TRAF6 to the evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), which is a protein that has been connected to building the mitochondrial respiratory chain, the same chain in which mROS are produced (Vogel et al, 2007). TRAF6 was able to interact with ECSIT after ECSIT localized in the mitochondrial outer membrane when exposed with LPS, the agonist in the TLR 4 pathway (West et al, 2011). This places ECSIT in a better location to interact with TRAF6, which enables the macrophage to localize around PAMP-coated beads (West et al, 2011). West and his team then used this information to investigate a potential link between ECSIT and the concentration of mROS being created. The research team did this by taking BMM from mice heterozygous for ESCIT, meaning these macrophages contained around 40% less ECSIT (West et al, 2011). The researchers discovered that without the correct levels of ECSIT and also depleted levels of TRAF6, macrophages produced remarkably less ROS. Thus without both proteins functional, ROS is not generated at the heightened level (West et al, 2011).

            Wanting to test their findings on a larger scale, West and his team investigated macrophages that were deficient in ECSIT and TRAF6 that were subsequently exposed to the bacteria Salmonella typhimurium (West et al, 2011). The team found that macrophages missing ECSIT contained more Salmonella than control cells with normal levels or lower levels of ECIST (Figure 4a-c). This illustrates even a small amount of ECSIT is enough to elicit the response witnessed in control cells, but a lack of ECSIT prevents this action (West et al, 2011). To push this further, West and his team tested their hypothesis on transgenic mice that were created to overexpress catalase, an enzyme that breaks down the most prominent mROS, hydrogen peroxide (West et al, 2011). Macrophages derived from these mice were tested against wild type macrophages in order to see if less hydrogen peroxide, a type of ROS, would affect the macrophages’ ability to combat bacterial pathogens and thus show that mROS generation is important in the innate immune response (West et al, 2011). True to pattern, the macrophages from the transgenic mice contained more Salmonella than the wild type (West et al, 2011) (Figure 4). ECSIT heterogeneous mice were also found to contain higher Salmonella loads than the wild type mice (West et al, 2011)(Figure 4).

Figure 4: A-C. Wild Type Macrophages and the ECSIT +/- Macrophages infected with Salmonella typhimurium. A. Illustrates the nuclei in each cell using a stain called DAPI in the wild type or ECSIT +/-. B. Illustrates the amount of ECSIT and protein present from both the Salmonella typhimurium using western blotting. C. Illustrates the colony count for Salmonella typhimurium in the hours following exposure. D-E. Wild Type or BMM macrophages from catalase deficient or wild type. D. Illustrates the amount of Catalase and protein derived from Salmonella typhimurium present using western blotting. E. Illustrate the nuclei in each cell using a stain called DAPI in the wild type or MCAT macrophages. F-G. Illustrate the spleens and livers from MCAT, Wild Type, and ECSIT +/- and the levels of intracellular bacterial coloney forming units. 

All in all, the results of this study by West et al (2011) illustrated that macrophages exposed to agonists that trigger TLR 1/2/4 lead to mitochondrial movement to the site of the bacterial PAMP and the interaction between TRAF6 and ECSIT upregulate the production of ROS (West et al, 2011). Macrophages deficient in either TRAF6, ECSIT, or contained high levels of catalase were unable to destroy intracellular bacteria, showing that mitochondrial ROS are needed to aid the macrophage in destroying bacteria and protecting the host. ROS are needed for the innate immune response, and West and his team illustrated that the mitochondria not only produce ROS, but are important immune signaling centers for the innate immune response. Future directions and applications for this material could include investigating the interplay between oxidative stress and antioxidant concentration and the production of mROS for the innate immune response, as a balance between both processes would be needed for a cell to prevent macromolecule damage, but to also correctly respond to bacterial PAMPS. 

References

Christmas, P. (2010) Toll-Like Receptors: Sensors that Detect Infection. Nature

Education 3(9):85.

Hulbert, A. J., Pamplona, R., Buffenstein, R., & Buttemer, W. A. (2007). Life and death:

metabolic rate, membrane composition, and life span of animals. Physiological reviews, 87(4), 1175-1213.

Lambeth, J. D. (2004). NOX enzymes and the biology of reactive oxygen. Nature Reviews

Immunology, 4(3), 181.

Mak, T.W., Saunders, M.E., & Jett, B.D. (2014). Primer to the Immune Response. Burlington, MA:               Elsevier Inc. 
Tang, D., Kang, R., Coyne, C. B., Zeh, H. J., & Lotze, M. T. (2012). PAMPs and DAMPs:                             Signal 0s that Spur Autophagy and Immunity. Immunological Reviews, 249(1), 158–175.                     http://doi.org/10.1111/j.1600-065X.2012.01146.x

Vogel, R. O., Janssen, R. J., van den Brand, M. A., Dieteren, C. E., Verkaart, S., Koopman, W. J.,                   ... & Nijtmans, L. G. (2007). Cytosolic signaling protein Ecsit also localizes to mitochondria               where it interacts with chaperone NDUFAF1 and functions in complex I assembly. Genes &               development, 21(5), 615-624.

West, A. P., Brodsky, I. E., Rahner, C., Woo, D. K., Erdjument-Bromage, H., Tempst, P., ... &

Ghosh, S. (2011). TLR signalling augments macrophage bactericidal activity through                          mitochondrial ROS. Nature, 472(7344), 476.