J Neuroimmunol. 2016 Jun 15;295-296:12-7. doi: 10.1016/j.jneuroim.2016.03.019.

TLR4 induces CCR7-dependent monocytes transmigration through the blood-brain barrier.

Paradis A1, Bernier S1, Dumais N2.
  • 1Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada.
  • 2Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada. Electronic address: Nancy.Dumais@USherbrooke.ca.



In this study, we examined whether bacterial pathogen-associated molecular patterns recognized by toll-like receptors (TLRs) can modify the CCR7-dependent migration of human monocytes. MonoMac-1 (MM-1) cells and freshly isolated human monocytes were cultivated in the presence of agonists for TLR4 (which senses lipopolysaccharides from gram-negative bacteria), TLR1/2 (which senses peptidoglycan from gram-positive bacteria), and TLR9 (which recognizes bacterial DNA rich in unmethylated CpG DNA). CCR7 mRNA transcription was measured using quantitative reverse transcription polymerase chain reaction and protein expression was examined using flow cytometry. CCR7 function was monitored using migration and transmigration assays in response to CCL19/CCL21, which are natural ligands for CCR7. Our results show that TLR4 strongly increases monocyte migratory capacity in response to CCL19 in chemotaxis and transmigration assays in a model that mimics the human blood-brain barrier, whereas TLR1/2 and 9 have no effect. Examination of monocyte migration in response to TLRs that are activated by bacterial components would contribute to understanding the excessive monocyte migration that characterizes the pathogenesis of bacterial infections and/or neuroinflammatory diseases. Copyright © 2016 Elsevier B.V.

KEYWORDS: Blood-brain barrier; CCR7-dependent migration; Monocytes; TLR

PMID: 27235343



Toll-like receptors in the innate immune response

Toll-like receptors (TLRs) play a significant role in pathogen recognition and the initiation of innate immune and inflammatory responses [1-4]. Importantly, they are expressed not only by immune cells, but by epithelial cells, which come in direct contact with pathogens. A total of ten functional TLRs have been identified in humans, which are characterized by differences in their ligand specificities, expression patterns, signaling pathways, and target genes [5]. All TLRs respond to pathogen-associated molecular patterns (PAMPs); however, PAMPs can be derived from viruses, pathogenic bacteria, or fungi, as well as from parasitic protozoa [5, 6]. TLRs on the cell surface recognize ligands from extracellular microbes; specifically, peptidoglycan is recognized by TLR1/TLR2, lipoprotein is recognized by TLR2/6, lipopolysaccharide (LPS) is recognized by TLR4, and flagellin is recognized by TLR5. In contrast, TLR3, TLR7, TLR8, and TLR9 are located in intracellular vesicles where they recognize microbial nucleic acids. Stimulation of all TLRs activates the mitogen-activated protein kinase (MAPK) and Nuclear Factor-κB (NF-κB) signaling pathways, both of which are critical for an effective immune response. TLR stimulation has been reviewed recently [7-10].


Ccr7-dependent migration of monocytes

Inflammatory monocytes are rapidly recruited to sites of inflammation, where they serve critical roles. However, their excessive and/or prolonged recruitment can hinder the resolution of inflammation and is a hallmark of numerous diseases. Chemokines, CCL19 and CCL21, which are necessary for cellular migration, are expressed both by lymphatic endothelia and within the lymph nodes, where they are produced by stromal cells, endothelial cells, and dendritic cells (DCs) [1114]. CCL19 and CCL21 are the natural ligands of CCR7, a G protein-coupled receptor, which is expressed by DCs [15], T and B cells [16], and monocytes [17]. Normally, CCR7 is responsible for the proper migration of immune cells to secondary organs and, subsequently, their positioning within defined functional compartments. Our group has previously demonstrated that CCR7 expression and functionality in monocytes are modulated by prostaglandin E2 PGE2 [17, 18], LXR [19] and HIV-1 [20].


The principal goal of the study

To gain novel insights into the molecular and cellular events leading to CCR7 expression in monocytes, we explored the role of TLR activation in the CCR7-dependent migration of monocytes across the blood-brain barrier (BBB).


What we have shown

Mono-Mac-1 (MM-1), a human cell line with properties of blood monocytes, was used as a model system to study monocytic functions in vitro. A series of TLR agonists that mimic bacterial products were used to stimulate MM-1, and CCR7 transcription was determined by real-time qPCR. Our results showed that TLR1/2 and TLR4 agonists significantly upregulated CCR7 mRNA transcription, whereas activation of TLR9 had no effect. However, CCR7 cell surface expression was upregulated by both PAM3CSK4 (TLR1/2) and CpG oligodeoxynucleotides (TLR9), but not by LPS (TLR4), after 48h of stimulation. Finally, we performed chemotaxis assays on MM-1 cells stimulated with TLR agonists, to test the functionality of CCR7, and found that only TLR4-stimulated cells migrated in response to CCL19 and CCL21. Although we were unable to demonstrate a correlation between mRNA transcription, protein expression, and functionality of CCR7, our results are in accordance with other studies. Specifically, the enhanced migration of DCs and monocyte-derived DCs (MoDCs) in response to PGE2 has been shown to be mediated by an alternative mechanism that is independent of CCR7 expression [21, 22]. Additionally, in freshly isolated human monocytes, only TLR4 activation increases CCR7 expression, relative to untreated cells. Interestingly, and unlike the effect observed in the MM-1 cell line, migration assays revealed that following LPS stimulation human monocytes migrate in response to CCL19, but not CCL21.


In our previous work, we optimized a two-dimensional model of the human BBB using primary human brain microvascular endothelial cells (HBMEC) and human neuronal astrocytes (NHA) [23]. With this model, we demonstrated that TLR4 activation increased monocyte transmigration across the BBB, toward CCL19. Similarly, here we report that the TLR4 agonist significantly modulated the transmigratory potential of MM-1 cells and human monocytes in response to CCL19. A review of our results are presented in Figure 1.


The importance of our study

Monocytes are important orchestraters of the immune system. Our group has demonstrated that monocytes express CCR7, an important determinant of cell migration, including transmigration across the BBB. These findings are particularly important in the context of mood disorders (MDs), since accumulation of monocytes in the brain has been observed in these patients [24-30]. MDs frequently co-occur with medical illnesses that involve inflammatory pathophysiologic mechanisms, such as autoimmune diseases [31]. The link between inflammation and MDs has been demonstrated in both human and animal studies [32-36]. Importantly, patients with an autoimmune disease are 45% more likely to have an MD, while any history of infection increases the risk of MDs by 62% [31]. To date, we are the first group to show that TLR4 activation increases CCR7-dependent migration of monocytes [37]. Thus, our investigation will provide information crucial for the development of strategies to selectively target the migration of monocytes in the context of infection and inflammation.



Figure 1. CCR7-dependent transmigration through BBB of monocytes induced by TLR4 activation.




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