Publication: Human-specific mutations and positively-selected sites in MARCO confer functional changes.

First author on the publication, PhD student Kyle Novakowski of Dr. Dawn Bowdish’s lab.
A common element that links ancient fish that dwell in the darkest depths of the oceans to land mammals, Neanderthals, and humans is the necessity to defend against pathogens. Hundreds of millions of years of evolution have shaped how our innate immune cells, such as macrophages, detect and destroy microorganisms.

In a new study led by Dr. Dawn Bowdish (in collaboration with Dr. Brian Golding) and her PhD student Kyle Novakowski, the team identified novel sites within a macrophage receptor, MARCO, that are under positive selection and are human-specific. The team demonstrated the importance of these sites by site-directed mutation and showed a reduction in cellular binding and uptake of pathogens. These findings demonstrate how small genetic changes in humans can influence how we defend ourselves against pathogens.

Read the full publication in Oxford University Press.

Human-specific mutations and positively-selected sites in MARCO confer functional changes. Novakowski KE, Yap NVL, Yin C, Sakamoto K, Heit B, Golding GB, Bowdish DME. Mol Biol Evol. 2017 Nov 20. doi: 10.1093/molbev/msx298.
PMID: 2916561

Publication: A naturally occurring transcript variant of MARCO reveals the SRCR domain is critical for function

Macrophages play a critical role in innate immunity by detecting, engulfing and destroying pathogenic bacteria and alerting neighbouring immune cells to join the fight against infection. They have many different receptors on their cell surface that allow them to carry out these important processes. A particular group of receptors called Scavenger Receptors are vital to this response. A recent study published in Immunology and Cell Biology by PhD student Kyle Novakowski from the laboratory of Dr. Dawn Bowdish has uncovered a mechanism by which a specific scavenger receptor contributes to macrophage-specific antibacterial immunity.

Scavenger Receptors are evolutionarily ancient and have evolved to recognize a wide array of pathogens by detecting ligands that are common across many pathogenic organisms. A particularly important Scavenger Receptor is Macrophage Receptor with Collagenous Structure, or MARCO. MARCO has been shown to significantly contribute to the clearance of Streptococcus pneumoniae colonization of the nose and in models of pneumococcal pneumonia. The NSERC-funded study took a unique approach to functionally characterizing how MARCO contributes to innate immunity by studying a naturally-occurring variant of the receptor. The study highlighted the importance of a particular domain of the receptor that is required for macrophages to bind and internalize ligands. The study also showed that the domain is necessary to enhance the pro-inflammatory response to pathogenic Streptococcus pneumoniae and can enhance cellular adhesion; both vital to proper macrophage functions.

To read the article, please click here.

Manuscript: The evolution of the scavenger receptor cysteine-rich domain of the class A scavenger receptors

Do you work out? Cause you’re built like a rock! A rock like Dwayne “The Rock” Johnson! You have an impenetrable body thanks to your complex immune system. So how did you get such a sophisticated immune system?

In the Bowdish lab, we do more than just macrophage biology; we also study the evolution of the immune system! The scavenger receptors are a group of receptors that play an important role in your immune system by binding harmful bacteria. Our most recent publication by Yap et al., looks at how these receptors evolved and how evolution has changed their function. These receptors are found in various forms of life such as sharks, frogs, and mammals, but the function and appearance of these receptors has changed over time. Check out the open access….

Yap N, Whelan FJ, Bowdish DM and Golding B (2015). The Evolution of the Scavenger Receptor Cysteine-Rich Domain of the Class A Scavenger Receptors. Front. Immunol. 6:342. doi: 10.3389/fimmu.2015.00342

Yap N, Whelan FJ, Bowdish DM and Golding B (2015). The Evolution of the Scavenger Receptor Cysteine-Rich Domain of the Class A Scavenger Receptors. Front. Immunol. 6:342. doi: 10.3389/fimmu.2015.00342

Abstract

The class A Scavenger Receptor (cA-SR) family is a group of five evolutionarily related innate immune receptors. The cA-SRs are known for their promiscuous ligand binding; as they have been shown to bind bacteria such as Streptococcus pneumoniae, and Escherichia coli, as well as different modified forms of low-density lipoprotein. Three of the five family members possess a Scavenger Receptor Cysteine Rich (SRCR) domain while the remaining two receptors lack the domain. Previous work has suggested that the Macrophage Associated Receptor with COllagenous structure (MARCO) shares a recent common ancestor with the non-SRCR-containing receptors; however the origin of the SRCR domain within the cA-SRs remains unknown. We hypothesize that the SRCR domains of the cA-SRs have a common origin that predates teleost fish. Using the newly available sequence data from sea lamprey and ghost shark genome projects, we have shown that MARCO shares a common ancestor with the SRCR-containing proteins. In addition, we explored the evolutionary relationships within the SRCR domain by reconstructing the ancestral SRCR domains of the cA-SRs. We identified a motif that is highly conserved between the cA-SR SRCR domains and the ancestral SRCR domain that consist of WGTVCDD. We also show that the GRAEVYY motif, a functionally important motif within MARCO, is poorly conserved in the other cA-SRs and in the reconstructed ancestral domain. Further, we identified three sites within MARCO’s SRCR domain which are under positive selection. Two of these sites lie adjacent to the conserved WGTVCDD motif, and may indicate a potential biological function for these sites. Together these findings indicate a common origin of the SRCR domain within the cA-SRs; however different selective pressures between the proteins may have caused MARCOs SRCR domain to evolve to contain different functional motifs when compared to the other SRCR-containing cA-SRs.

Love RJ, Patenaude M, Dorrington M, Bowdish DM, Hoare T, Jones KS. An investigation of scavenger receptor A mediated leukocyte binding to polyanionic and uncharged polymer hydrogels. J Biomed Mater Res A. 2015 May;103(5):1605-12. doi: 10.1002/jbm.a.35297.

Love RJ, Patenaude M, Dorrington M, Bowdish DM, Hoare T, Jones KS. An investigation of scavenger receptor A mediated leukocyte binding to polyanionic and uncharged polymer hydrogels. J Biomed Mater Res A. 2015 May;103(5):1605-12. doi: 10.1002/jbm.a.35297.

Abstract
Cell adhesion to biomaterials can be mediated in part by mechanisms aside from the traditionally recognized opsinization and integrin binding mechanisms. In this study, we investigated the role of scavenger receptor A (SR-A) in leukocyte binding to a series of well-controlled polyanionic and uncharged hydrogels based on a poly(N-isopropylacrylamide) backbone. The hydrogels were injected in the peritoneal cavity of SR-A knockout (KO) and wild-type mice using a minimally invasive procedure and allowed to set in situ. After 24 h, the hydrogels were recovered and analyzed, the peritoneal cavity was lavaged, and cytokine concentrations were assessed by ELISA. The polyanionic hydrogels retrieved from the KO animals were found to be completely devoid of adherent leukocytes, which were present in other materials regardless of the mouse strain in which they were injected. Results from a subsequent in vitro cellular adhesion study with a RAW264.7 cell line failed to yield a similarly definitive role for SR-A in the cellular binding of a polyanionic hydrogel. Taken together, the results of this study show that SR-A mediates leukocyte adhesion to a polyanionic hydrogel in the peritoneal cavity, but other adhesion mechanisms contribute to cellular binding in vitro. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 1605–1612, 2015.

PradhuDas et al. Standardizing Scavenger Receptor Nomenclature. J Immunol. 2014 Mar 1;192(5):1997-2006.

PrabhuDas M, Bowdish D, Drickamer K, Febbraio M, Herz J, Kobzik L, Krieger M, Loike J, Means TK, Moestrup SK, Post S, Sawamura T, Silverstein S, Wang XY, El Khoury J. Standardizing scavenger receptor nomenclature. J Immunol. 2014 Mar 1;192(5):1997-2006. doi: 10.4049/jimmunol.1490003.

PradhuDas et al screen shot

MARCO is required for TLR2- and NOD2-mediated resonses to Streptococcus pneumoniae and clearance of pneumococcal colonization in the murine nasopharnyx. 2013. Dorrington et al. J. Immunol.

Click image for .pdf of paper.

Dorrington JI

Despite having multiple vaccines against Streptococcus pneumoniae available today, over a million people die each year due to pneumococcal infections. Mike Dorrington, a Ph.D. candidate in the Bowdish lab, is attempting to understand how to produce better vaccines by gaining a better grasp on how the immune system fights these bacteria. Mike has recently published a manuscript entitled “MARCO is required for TLR2- and NOD2-mediated responses to Streptococcus pneumoniae and clearance of pneumococcal colonization in the murine nasopharynx” in the Journal of Immunology. Mike’s work focuses on the importance of macrophage scavenger receptors in immune protection against S. pneumoniae, the most common cause of bacterial pneumonia. This manuscript provides us with evidence that Macrophage Receptor with Collagenous structure (MARCO), a class A scavenger receptor, plays an integral role in establishing and maintaining the appropriate innate immune response to the bacteria in its preferred niche, the nasal passage.

     S. pneumoniae is a very common pathogen that causes fatal disease in children under the age of 5 (where it often causes meningitis) and adults over the age of 65 (where it most often presents in pneumonia). Before infectious disease occurs, bacteria colonize the nasal passages of individuals where they replicate. If the bacteria are able to persist for long enough, they will then move to the lungs, blood, or meninges and cause potentially life-threatening disease. It has previously been shown that the clearance of the bacteria from the nasal passages was dependent on an influx of macrophages to the site. These cells are able to internalize and kill the bacteria efficiently. MARCO is expressed by these active macrophages and has been shown to play a role in the recognition of the bacteria.

Mike’s work shows that mice who lack MARCO expression are unable to clear bacterial colonization in a timely fashion. This is due to a decrease in a number of innate immune functions. First, MARCO-deficient mice have significantly less recruitment of innate immune cells such as neutrophils and macrophages to the site of colonization. Without these cells, the bacteria are free to thrive and replicate in the nasal passage, increasing the chance that they will travel to further tissues and cause disease. MARCO-deficient mice also present with less inflammation than they’re wild-type counterparts, as seen by a paucity of pro-inflammatory cytokines and chemokines including, surprisingly, type I interferons (cytokines associated with antiviral immunity). These data are supported by experiments performed in vitro using macrophage populations from MARCO-deficient and wild-type mice. When these cells are stimulated with S. pneumoniae, the MARCO-deficient macrophages produce less cytokines and chemokines. These cells are also less able to internalize the bacteria, a key step in the destruction of the pathogens.

A potentially ground-breaking finding that comes from Mike’s work is that MARCO is able to modulate the activity of other important innate immune receptors. Mike has shown that NF-kB activation in S. pneumoniae-stimulated cells expressing MARCO along with TLR2 and its co-receptor CD14 is much higher than cells not expressing MARCO. This is also true of cells expressing MARCO as well as NOD2 when compared to those expressing just NOD2. As NF-kB is a central regulator of immune function, this represents a very important step in our understanding of antibacterial innate immune responses in the nose.

Mike’s work on MARCO will continue as he attempts to uncover the mechanism by which MARCO increases NF-kB activation by these other receptors. It is his hope to be able to apply these advances in the basic science to vaccine development in order to generate an effective strain-independent vaccine against S. pneumoniae infection.

“The Evolution of the Class A Scavenger Receptors” 2012. Whelan et al. BMC Evol Biol

Whelan et al. BMC Evolutionary Biology 2012, 12:227 http://www.biomedcentral.com/1471-2148/12/227

The scavenger receptors are an evolutionarily ancient family of proteins required for host defence and homeostasis but teasing apart their function and even their structure has been challenging. The goal of this manuscript was to use evolution as a guide to discover how the class A scavenger receptor family was formed and to identify regions of conservation and hence probable functional importance for future study. Phagocytic receptors such as the class A scavenger receptors are integral members of the innate immune response, which is conserved in all classes of life and after reproduction and nutrient acquisition is probably the major most fundamental requirement for survival.

There are essentially only four basic mechanisms of the innate immune system – agglutination (e.g. lectins), lysis/neutralization (e.g complement, antimicrobial peptides), phagocytosis (e.g. scavenger receptors), and pro-inflammatory signalling (e.g. the toll like receptors). The fact that these processes are ancient and have been so strongly preserved is a testament to their importance. Of these, phagocytosis is likely the most ancient process and was probably adapted from its original purpose of nutrient ingestion . One might hypothesize that phagocytosis was truly the genesis of the immune system since our single celled ancestors had to distinguish  between “self” and “non-self” in order to distinguish between food and their own daughter cells.  From there phagocytosis became essential to fundamental processes such as embryonic development, pathogen recognition, and homeostatic clearance of senescent cells. Without phagocytosis, the transition to more complicated life forms could not have occurred.

Although there have been excellent evolutionary analyses of the lectins, toll like receptors and complement pathways, very little is known about the evolution of the phagocytic receptors. The class A scavenger receptors are an excellent example of these multifunctional receptors as they are involved in both host defence and homestasis. Since the phagocytic receptors in general and the scavenger receptors in particular are a diverse group of proteins,it has been challenging to understand how members within a group are related. Indeed, the first goal of this manuscript was to definitively demonstrate that the members of the class A scavenger receptors, which had been grouped together based on a ragtag combination of ligand binding and some degree of amino acid similarity, were actually a family at all.  Since we were able to trace a probable path of gene duplication and consequent functionalization, we are confident that the 5 members (SRAI/II, MARCO, SCARA3/4/5) are actually related.  Interestingly the class A scavenger receptors may have acquired their long stalk like form with a single scavenger receptor cysteine rich domain (SRCR) around the time of the evolution of fish since, although SRCR domain can be found in invertebrates and single celled organisms, we could not find anything that resembled a modern class A scavenger receptor in any genomes of evolutionarily more ancient organisms such as jellyfish, lampreys and insects.

Because elucidating the function of the specific domains of the scavenger receptors has been so challenging (even the function of the SRCR domain is unclear), ultimately we want to use evolution as a guide to which domains are functionally important (i.e. conserved). In this regard we found that there is a common conserved region in the collagenous domain, which in the type member SRAI, is believed to be the ligand binding domain. In addition conserved domains were identified in the cytoplasmic tail and the coiled-coiled domain. Future experiments will be performed to determine if these domains are necessary for structure, expression, cellular localization or phagocytic function.