TLR4 induces CCR7-dependent monocytes transmigration through the blood-brain barrier.
- 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 . 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) [11–14]. CCL19 and CCL21 are the natural ligands of CCR7, a G protein-coupled receptor, which is expressed by DCs , T and B cells , and monocytes . 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  and HIV-1 .
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) . 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 . 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% . To date, we are the first group to show that TLR4 activation increases CCR7-dependent migration of monocytes . 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.
1. Luther SA, et al. Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the plt/plt mouse. Proc Natl Acad Sci. (2000) 97:12694-12699.
2. O’Neill LD, et al. The history of Toll-like receptors – redefining innate immunity. Nat Rev Immunol. (2013) 13:453–460.
3. Ahmad‐Nejad P, et al. Bacterial CpG‐DNA and lipopolysaccharides activate Toll‐like receptors at distinct cellular compartments. Eur J Immunol. (2002) 32:1958-1968.
4. Takeda K, et al. Toll-like receptors. Annu Rev Immunol. (2003) 21:335-376.
5. Akira S, Takeda, K. Toll-like receptor signalling. Nat Rev Immunol. (2004) 4:499-511.
6. Colmont CS, et al. Human peritoneal mesothelial cells respond to bacterial ligands through a specific subset of Toll-like receptors. Nephrol Dial Transplant. (2011) 26:4079-4090.
7. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on toll-like receptors. Nat Immunol (2010) 11:373–384.
8. Coll RC, O’Neill LA. New insights into the regulation of signalling by toll-like receptors and nod-like receptors. J Innate Immun (2010) 2:406–421.
9. Arthur JSC, Ley SC. Mitogen-activated protein kinases in innate immunity. Nat Rev Immunol (2013) 13:679–692.
10. Cohen P. The TLR and IL-1 signalling network at a glance. J Cell Sci (2014) 127:2383–2390.
11. Gunn MD, et al. A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc Natl Acad Sci USA. (1998) 95:258-263.
12. Luther SA, , et al. Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the plt/plt mouse. Proc Natl Acad Sci USA. (2000) 97:12694-12699.
13. Ngo VN, et al. Epstein-Barr virus-induced molecule 1 ligand chemokine is expressed by dendritic cells in lymphoid tissues and strongly attracts naive T cells and activated B cells. J Exp Med. (1998) 188:181-191.
14. Stein JV, et al. The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules. J Exp Med. (2000) 191: 61-76.
15. Saeki H, et al. Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes. J Immunol. (1999) 162:2472-2475.
16. Warnock RA, et al. The role of chemokines in the microenvironmental control of T versus B cell arrest in Peyer’s patch high endothelial venules. J Exp Med. (2000) 191:77-88.
17. Coté SC, et al. CCR7-specific migration to CCL19 and CCL21 is induced by PGE(2) stimulation in human monocytes: Involvement of EP(2)/EP(4) receptors activation. Mol Immunol. (2009) 46:2682-2693.
18. Allaire MA, Dumais N. Involvement of the MAPK and RhoA/ROCK pathways in PGE2-mediated CCR7-dependent monocyte migration. Immunol Lett. (2012) 146:70-73.
19. Tanné B, et al. CCR7 Receptor Expression in Mono-MAC-1 Cells: Modulation by Liver X Receptor α Activation and Prostaglandin E2. Int J Inflam. (2015) doi: 10.1155/2015/201571.
20. Coté SC, et al. Exposure to HIV-1 Altered CCR7-Mediated Migration of Monocytes: Regulation by PGE2. Int J Virol AIDS. (2015) 2:011.
21. Legler DL, et al. Prostaglandin E2 is generally required for human dendritic cell migration and exerts its effect via EP2 and EP4 receptors. J Immunol. (2006) 176:966-973.
22. Muthuswamy R, et al. PGE2 transiently enhances DC expression of CCR7 but inhibits the ability of DCs to produce CCL19 and attract naive T cells. Blood (2010) 116 :1454-1459.
23. Paradis A, et al. Optimization of an in vitro human blood–brain barrier model: Application to blood monocyte transmigration assays. MethodsX (2016) 3:25-34.
24. Wohleb ES, et al. β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat. J Neurosci. (2011) 3117:6277-6288.
25. Wohleb ES, et al. Stress-induced recruitment of bone marrow-derived monocytes to the brain promotes anxiety-like behavior. J Neurosci. (2013) 33:13820-13833.
26. Sawada A, et al. Suppression of bone marrow-derived microglia in the amygdala improves anxiety-like behavior induced by chronic partial sciatic nerve ligation in mice. Pain. (2014) 155:1762-17672.
27. Terrando N, et al. Resolving postoperative neuroinflammation and cognitive decline. Ann Neurol. (2011) 70:986-995.
28. Beumer W, et al. The immune theory of psychiatric diseases: a key role for activated microglia and circulating monocytes. J Leukoc Biol. (2012) 92:959-975.
29. D’Mello C, et al. P-selectin-mediated monocyte-cerebral endothelium adhesive interactions link peripheral organ inflammation to sickness behaviors. J Neurosci. 2013 33:14878-14888.
30. Degos V, et al. Depletion of bone marrow-derived macrophages perturbs the innate immune response to surgery and reduces postoperative memory dysfunction. Anesthesiology. (2013) 118:527-536.
31. Benros ME, et al. Autoimmune Diseases and Severe Infections as Risk Factors for Mood Disorders: A Nationwide Study. JAMA Psychiatry. (2013) 70:812-820.
32. Dantzer R, et al. From inflammation to sickness and depression: when the immune system subjugates the brain. Nat Rev Neurosci. (2008) 9:46-56.
33. Eisenberger NI, et al. Inflammation-induced anhedonia: endotoxin reduces ventral striatum responses to reward. Biol Psychiatry. (2010) 68:748-754.
34. Harrison NA, et al. Inflammation causes mood changes through alterations in subgenual cingulate activity and mesolimbic connectivity. Biol Psychiatry. (2009) 66:407-414.
35. Raison CL, et al. Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends Immunol. (2006) 27:24-31.
36. Reichenberg A, et al. Cytokine-associated emotional and cognitive disturbances in humans. Arch Gen Psychiatry. (2001) 58:445-452.
37. Paradis A, et al. TLR4 induces CCR7-dependent monocytes transmigration through the blood-brain barrier. J Neuroimmunol. (2016) 15:12-17.