Tuesday, April 12, 2011

Dual role of TLR8 during the engulfment of Lyme disease spirochetes by human monocytes

For the first time scientists have shown that Toll-like receptor 8 (TLR8), a microbial RNA sensor located inside phagocytes, detects what is primarily an extracellular pathogen, the Lyme disease spirochete Borrelia burgdorferi.1  As one may expect, the phagocytes secreted a mixture of inflammatory cytokines in response to the spirochete.  But they also expressed at least one of the type I interferons (IFNs), which until recent years were thought to be produced only in response to viral and intracellular bacterial infections.2

ResearchBlogging.orgThe phagocytes used for the study were human monocytes, which are the more easily available bloodstream form of the macrophages found in our tissues.  Macrophages are designed to capture and engulf microbial pathogens invading our bodies.  While sopping up the invaders, the macrophages send out warning signals in the form of cytokines and other inflammatory molecules to alert nearby cells and to get the immune system to send more immune cells to help defend the tissue under attack.

Most of the macrophage's microbial sensors belong to a family of related membrane proteins called Toll-like receptors (TLRs).  TLR1, TLR2, TLR4, and TLR6 span the plasma membrane, whereas TLR3, TLR7, TLR8, and TLR9 are located in membrane structures inside the cell.  Each TLR recognizes a specific component of microbes.  For example, TLR4 latches onto LPS; TLR2 forms a complex with TLR1 or TLR6 to bind the lipidated amino terminus of lipoproteins; TLR7 and TLR8 recognize single-stranded RNA; and TLR9 recognizes DNA.  Engagement of a TLR by a microbial component triggers a signaling cascade within the cell leading to the transcription of genes encoding inflammatory cytokines, which are then secreted.  Stimulation of TLR3, TLR4, TLR7, TLR8, and TLR9 can also activate transcription of genes encoding type I IFNs.  The exact response of the macrophage depends on which TLRs are engaged by the pathogen.3

In the PNAS paper by Cervantes and colleagues, the authors searched for the sensors triggered by B. burgdorferi.1 It had been known for a decade that one of the sensors of B. burgdorferi is TLR2, which recognizes the many lipoproteins that populate the surface of the spirochete.  However, TLR2 can't be the only B. burgdorferi sensor in macrophages because a more recent study showed that mouse macrophages missing its Tlr2 gene continued to produce inflammatory cytokines, albeit at lower levels, while engulfing B. burgdorferi.4 This same study also showed that B. burgdorferi stimulated human monocytes to transcribe genes encoding type I IFNs and a number of genes known to be induced by type I IFNs.

The investigators first examined the effects of blocking phagocytosis of B. burgdorferi by treating the monocytes with cytochalasin D, a chemical that blocks phagocytosis.  They found that cytochalasin D prevented transcription of the gene encoding the type I interferon IFN-β and reduced the amount of the inflammatory cytokine TNFα secreted from the monocytes.  The little bit of TNFα that continued to be produced was probably a consequence of TLR2 being stimulated by B. burgdorferi lipoproteins on the surface of the monocytes.

Since the spirochetes had to be engulfed to observe the monocyte's complete response, an intracellular sensor must participate in sensing B. burgdorferi.  The investigators therefore focused their attention on the intracellular nucleic acid sensors TLR7, TLR8, and TLR9.  Earlier studies by many other labs have shown that engagement of these intracellular TLRs activated production of type I IFNs, so it made sense to examine these TLRs.

To figure out which TLR functioned as the intracellular sensor of B. burgdorferi, the investigators used synthetic fragments of DNA that specifically blocked each TLR without interfering with phagocytosis.  When they applied the inhibitors individually to the monocytes, they found that only the TLR8 inhibitor blocked induction of the IFN-β gene by B. burgdorferi (see right half of panel A below).  A synthetic DNA fragment that does not inhibit any TLR failed to block induction of IFN-β transcription.  Therefore, TLR8 was implicated as being the intracellular sensor that detects B. burgdorferi.  As a control, LPS, which is sensed by TLR4 but not TLR8, continued to induce synthesis of the IFN-β transcript in the presence of the TLR8 inhibitor (left half of panel A).

B. burgdorferi was added at a 10:1 ratio (bacteria:monocytes).  Following the 4-hour incubation period, IFN-β transcript levels were measured by quantitative real-time RT-PCR.  ODN, a control synthetic oligodeoxynucleotide; N.S., not significant; *, P < 0.05; **, P < 0.01 (Mann-Whitney U test)

When the investigators looked at the inflammatory cytokines being produced by the TLR8-inhibited monocytes, they discovered that TLR8 had another role in the monocyte's response to B. burgdorferi.  Blocking TLR8 reduced (but did not eliminate) the secretion of the inflammatory cytokines TNFα, IL-6, IL-1β, and IL-10 (see panel B below).  These results indicated that TLR2 and TLR8 both had to send signals the nucleus to maximize the amount of cytokines produced during engulfment of the spirochete.  The authors also found by indirect immunofluorescence microscopy that TLR2 and TLR8 were together in the phagosomal membrane surrounding the engulfed B. burgdorferi, which they saw were being destroyed.

The amount of cytokines secreted by the human monocytes was measured following the 4-hour incubation period.  "Un," no spirochetes added; "Bb," B. burgdorferi added at a 10:1 ratio (bacteria:monocytes)

So here's what the authors believe is happening during the encounter of human monocytes with B. burgdorferi.  The monocyte first contacts the spirochete at the surface of the plasma membrane.  The lipoproteins on the surface of the spirochete activate the TLR2 sensor on the surface of the monocyte, but the low local concentration of TLR2 in the plasma membrane hampers its full signaling potential.  As the monocyte engulfs the spirochete by phagocytosis, TLR8 and TLR2 are recruited to the phagosomal membrane surrounding the spirochete, which at this point is being destroyed by antimicrobial substances being dumped into the phagosome.  Destruction of the spirochete releases its RNA to stimulate TLR8, while the crowding of TLR2 in the phagosomal membrane enhances the signaling stimulated by lipoproteins.  TLR8 and TLR2 work together to send a signal to the nucleus to activate transcription of numerous genes, including those encoding inflammatory cytokines.  Signaling from TLR8 by a separate pathway also stimulates transcription of type I interferons.

So is TLR8 relevant to Lyme disease?  The authors make the reasonable assertion that TLR8 activation benefits those infected with B. burgdorferi in part by turning on the type I IFN response.

Given that type I IFNs can shape a variety of downstream inflammatory responses through positive and/or negative regulation of hundreds of additional genes involved in secondary host defenses, TLR8 activation is likely to play a critical role in clearance of the spirochete and more importantly, disease control. [emphasis mine]

Unfortunately, the research literature tells us that type I IFNs have a dark side.  Although the beneficial role of type I IFNs in fighting off viral infections is well established, whether they help or hurt us during bacterial infections is not always obvious.  Type I IFNs are clearly essential in combating some bacterial infections such as lethal bloodstream infections caused by group B streptococci, Streptococcus pneumoniae, and E. coli.2  As for Lyme disease, type I IFNs may help kill spirochetes, but they also promote joint inflammation in infected mice.5  To determine whether TLR8 contributes to Lyme arthritis, scientists will need to perform B. burgdorferi infection studies with Tlr8-knockout mice.

Featured paper

1. Cervantes, J.L., Dunham-Ems, S.M., La Vake, C.J., Petzke, M.M., Sahay, B., Sellati, T.J., Radolf, J.D., & Salazar, J.C. (2011). Phagosomal signaling by Borrelia burgdorferi in human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-β Proceedings of the National Academy of Sciences, 108 (9), 3683-3688 DOI: 10.1073/pnas.1013776108

Key references

2. Mancuso G, Midiri A, Biondo C, Beninati C, Zummo S, Galbo R, Tomasello F, Gambuzza M, Macrì G, Ruggeri A, Leanderson T, & Teti G (2007). Type I IFN signaling is crucial for host resistance against different species of pathogenic bacteria. Journal of Immunology, 178 (5), 3126-3133 PMID: 17312160

3. Kawai, T., & Akira, S. (2010). The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors Nature Immunology, 11 (5), 373-384 DOI: 10.1038/ni.1863

4.Salazar, J.C., Duhnam-Ems, S., La Vake, C., Cruz, A.R., Moore, M.W., Caimano, M.J., Velez-Climent, L., Shupe, J., Krueger, W., & Radolf, J.D. (2009). Activation of human monocytes by live Borrelia burgdorferi generates TLR2-dependent and -independent responses which include induction of IFN-β PLoS Pathogens, 5 (5) DOI: 10.1371/journal.ppat.1000444

5. Miller JC, Ma Y, Bian J, Sheehan KC, Zachary JF, Weis JH, Schreiber RD, & Weis JJ (2008). A critical role for type I IFN in arthritis development following Borrelia burgdorferi infection of mice. Journal of Immunology, 181 (12), 8492-8503 PMID: 19050267

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