Are NETs involved in fighting Leptospira interrogans infections?

Neutrophils are the most abundant white blood cells in the bloodstream.  As the first immune cells to be recruited to infected tissues, they play a key role in the fighting microbial intruders.  It's long been known that they engulf microbes by phagocytosis, which results in the microbes being imprisoned within phagosomes inside the neutrophil.  Deadly proteases, antimicrobial proteins, and reactive oxygen species are released into the phagosome to kill the microbes.

Another means used by neutrophils to kill microbes was discovered just a decade ago.  When mixed with bacteria, neutrophils cast nets of DNA impregnated with antimicrobial proteins to trap and kill the bacteria.  The web-like DNA goes by the name "neutrophil extracellular trap" (NET).  Several bacteria are known to trigger neutrophils to cast NETs, and NETs have even been observed by microscopy within infected tissues.

Fluorescence staining of a neutrophil exudate in an appendicitis case.  NETs are the fibrous material.  Figure 4H from Brinkmann et al., 2004.  Bar = 50 μm.

A study published last year in PLOS NTD showed that the spirochete Leptospira interrogans is also killed by NETs.  The image below shows the spirochetes trapped in a NET cast by a human neutrophil.

Human neutrophils were cultured with L. interrogans for 3 hours.  Figure 1A from Scharrig et al., 2015.  Bar = 50 μm.

The real question is whether NETs are involved in killing L. interrogans during infection.  To answer this question, the investigators turned to the mouse model of leptospirosis.  They found that the number of spirochetes in the bloodstream more than doubled when the neutrophils in the mice were depleted by injection of a monoclonal antibody targeting a antigen located on the neutrophil surface.  Later in the infection, there was 10-fold more spirochetes in the kidneys of mice whose neutrophils were depleted than in those with normal numbers of neutrophils.  This confirmed that neutrophils were involved in limiting infections by L. interrogans, but did the neutrophils fight the infection by casting NETs?

The investigators used an indirect method to measure the amount of NETs generated during infection.  Neutrophils often expel nuclear DNA in the form of nucleosomes to generate NETs.  (Nucleosomes are assembled by wrapping nuclear DNA around histones.)  For this reason, the investigators measured the levels of free nucleosomes in the bloodstream of infected mice by ELISA. They concluded that NETs were generated by neutrophils in the bloodstream because they detected free nucleosomes in blood drawn from infected mice.  Much less was detected when neutrophils were first depleted with the anti-neutrophil antibody, confirming that the main source of free nucleosomes was neutrophils.

These results don't convince me that NETs are generated by neutrophils during L. interrogans infection.  There could be other reasons for free nucleosomes being present in the bloodstream.  For example, nucleosomes could be released from neutrophils simply dying from their battle against L. interrogans.  More convincing evidence would be direct observation of NETs in infected animals, as done in this study of mice with E. coli blood infections.

References

Scharrig E, Carestia A, Ferrer MF, Cédola M, Pretre G, Drut R, Picardeau M, Schattner M, & Gómez RM (2015). Neutrophil extracellular traps are involved in the innate immune response to infection with Leptospira. PLoS Neglected Tropical Diseases, 9 (7) PMID: 26161745

Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, & Zychlinsky A (2004). Neutrophil extracellular traps kill bacteria. Science (New York, N.Y.), 303 (5663), 1532-5 PMID: 15001782

A tick protein helps Lyme disease spirochetes fight complement

Microbial pathogens attempting to establish an infection face the daunting challenge of overcoming the complement system.  To survive the onslaught of complement proteins, pathogenic microbes express surface structures that resist or manipulate the action of complement.  Not surprisingly, many Lyme disease Borrelia strains express proteins ("CRASPs" and "Erps") that ward off complement.  But they also get help from a protein found in the saliva of the Ixodes tick, according to a study that appeared last August in Cell Host & Microbe.

A Bare-bones Review of the Complement System

The complement system consists of ~30 seemingly innocuous proteins floating in our tissue fluids, with the highest concentrations being found in our bloodstream.  Upon activation, they morph sequentially into several protease complexes (see figure below).  Each protease cleaves specific complement components, generating the subunits for the next protease complex in the cascade.  The cascade ends with assembly of the membrane attack complex, a pore that kills the microbe.  It is important to keep in mind that the membrane attack complex is not the only weapon that the complement system uses to kill invading microbes.  Some of the complement fragments generated by the proteases also help ignite inflammation, in part by attracting phagocytes that engulf the target microbe.

The complement system is triggered when a complex of complement proteins bearing a recognition subunit, either C1q, a ficolin, or mannose-binding lectin (MBL), bind to certain microbial surface molecules.  C1q can also attach to the Fc region of antibodies bound to the microbe.  Binding of the recognition subunit to the microbe activates the protease component of the complex.  The protease (C1r/C1s and MASP in the figure below) cleaves the complement components C4 and C2, leading to the formation of another protease complex on the surface of the microbe, a C3 convertase with the composition C4b-C2a.  Although the classical pathway is triggered by C1q and the lectin pathway by MBL and the ficolins, note that both pathways lead to formation of the C3 convertase.

There is also a third pathway.  The alternative pathway is triggered by covalent binding of exposed hydroxyl groups on membrane surfaces to C3b, which is generated by the slow, spontaneous cleavage of C3.  Of course all cell surfaces, whether pathogen or host, bear hydroxyl groups.  To prevent tissue damage, complement regulators quickly inactivate any C3b molecules that end up binding host cells.  C3b molecules that end up bound to the microbe capture factor B, which is subsequently cleaved by the protease factor D, resulting in the formation of another type of C3 convertase, one that comprises the C3b and Bb subunits.

Regardless of its composition, C3 convertase cleaves C3, generating even more C3b, which end up sticking covalently to the microbial surface.  C3b is a key player in the complement system.  C3b flags the unlucky microbe for destruction by phagocytes, which are attracted to the site of infection by soluble protein fragments generated by the complement proteases.  In addition, C3b can also join with either type of C3 convertase, resulting in yet another protease complex called the C5 convertase.  Cleavage of C5 by C5 convertase releases C5b, which assembles the complement proteins C6 through C9 into the lethal membrane attack complex.

A "simplified" view of complement activation.  Ficolins are not shown.  Source

What They Did

In an earlier study, Schuijt and colleagues identified the Ixodes scapularis tick protein TSLPI as a target for antibodies generated by rabbits that were immunized with tick saliva.  Since TSLPI also possessed anti-complement activity, its properties were examined further by the investigators.

Since TSLPI is actually a glycoprotein, the investigators mass produced TSLPI in an insect cell line so that the recombinant protein, like the native one, would be decorated with sugar molecules.  Following production of TSLPI, the glycoprotein was purified for use in the test-tube experiments described below.

Serum contains all the components necessary to execute the complement cascade, including assembly of the membrane attack complex (MAC).  An easy way to test whether a bacterial strain is susceptible to complement-mediated killing by MAC is to see how well it survives in serum.  When a strain of Borrelia garinii, a European Lyme disease species, was mixed with human serum, the MAC assembled on the surface of the spirochete, and the spirochetes perished.  Addition of TSLPI allowed the spirochetes to survive in serum.

As I mentioned earlier, the complement system also has other methods of killing microbes.  Some of the complement components that end up attached to the microbe are recognized by phagocytes attracted to the infection site by the complement cleavage products C3a and C5a.  This activity, known as opsonophagocytosis, is not reflected in the serum killing assay.   To see whether activation of complement produced products that promoted phagocytosis of B. garinii, the investigators added human neutrophils to the mixtures of B. garinii and serum.  The neutrophils engulfed the spirochetes within minutes.  Fewer spirochetes were engulfed when TSLPI was also present, indicating that TSLPI protected B. garinii from opsonophagocytosis.

A U.S. strain of B. burgdorferi was also tested.  Most B. burgdorferi strains survive in human serum unless antibodies against the spirochete are also present.  Therefore, TSLPI was tested with serum pooled from seropositive Lyme disease patients.  TSLPI promoted survival of B. burgdorferi in immune serum.  TSLPI also limited the number of spirochetes that ended up engulfed when neutrophils were added to the immune serum.

The investigators next tried to figure out which of the three complement pathways was being blocked by TSLPI.  Fortunately simple assays are available to recreate the classical pathway with the CH50 assay and the alternative pathway with the AP50 assay.  Both assays are set up so that activation of the complement pathway leads to the assembly of the membrane attack complex in the plasma membrane of red blood cells, causing them to lyse and release their hemoglobin.  The extent of lysis is determined simply by measuring the absorbance of the freed hemoglobin in a spectrophotometer.

In the CH50 assay, human serum is mixed with sheep red blood cells that are coated with anti-sheep red blood cell antibodies.  The classical pathway is triggered when C1q, the recognition subunit of the C1 protease complex, binds the antibody.  In the figure below, TSLPI or BSA (control) was mixed with different dilutions of human serum prior to addition of the antibody-sensitized red blood cells.  Based on the serum killing assay with B. burgdorferi, one may expect the classical pathway to be hindered by TSLPI.   Surprisingly, the results indicated that TSLPI had no effect whatsoever ("control" vs. rTSLPI in graph below).  The anti-C1q and anti-C3 antibodies, additional controls, blocked the classical pathway, as expected.

Fig. 3C from Schuijt et al., 2011.  Effect of TSLPI on the classical complement pathway determined with a CH50 assay.

The AP50 assay is identical to the CH50 assay except for a couple of modifications.  The red blood cells come from rabbits because sheep red blood cells are not sensitive targets for the human alternative complement pathway.  The other critical difference is that the chelator EGTA is added to prevent activation of the classical pathway, which requires calcium for activity.  The results (below) show that TSLPI failed to block the alternative pathway as well.

Fig. 3D from Schuijt et al., 2011.  Effect of TSLPI on the alternative complement pathway determined with an AP50 assay.

No comparable assay is available to examine the lectin pathway.  Instead the investigators added dilutions of human serum to mannan-coated ELISA plates to look at deposition of the C4b protein, a component of C3 convertase.  Mannan is a polysaccharide made up of the simple sugar mannose and is the molecule recognized by MBL.  C4b will be generated only if the lectin pathway is triggered by binding of MBL to mannan.  C4b binds to the mannan coat.  After the incubation, unbound proteins are washed away, and adherence of C4b is detected by doing a standard ELISA with anti-C4 antibody.  The results indicate that TSLPI blocked the deposition of C4b, and thus the lectin pathway, in a dose-dependent manner (see graph below).

Fig. 3E from Schuijt et al., 2011.  Effect of TSLPI on the mannose arm of the lectin-binding pathway determined by ELISA measurement of C4b deposition onto mannan-coated wells.

Additional experiments showed that TSLPI blocked the very first step in the lectin pathway, binding of MBL to mannan.  TSLPI also interfered with the binding of L-ficolin to its target.

To confirm that the lectin pathway killed Borrelia, the authors looked at the killing activity of serum pooled from individuals deficient in MBL.  Such individuals were not hard to find.  According to the authors, about 25% of the population is deficient in MBL!

A lower percentage of B. garinii ended up being killed in MBL-deficient serum than in normal serum.  TSLPI further reduced the killing activity of MBL-deficient sera, suggesting that recognition of B. garinii by ficolin also triggered the lectin pathway.  The balance of the lethal activity of human serum was accounted for by the classical pathway; C1 esterase inhibitor, which blocks both the classical and lectin pathways, completely eliminated the killing activity of human serum.

The investigators next determined whether TSLPI aided B. burgdorferi infection of laboratory mice.  They knocked down TSLPI expression in Ixodes scapularis by RNA interference and used the altered ticks to transmit B. burgdorferi into mice.  As a control, they also had another group of mice that were inoculated with B. burgdorferi with unmodified ticks, which were producing normal levels of TSLPI.  When the altered ticks were used to inoculate B. burgdorferi, the bacterial loads in the heart, joint, and skin (distant from the site of tick feeding) turned out to be much less than when the unaltered ticks were used.  These results demonstrate that TSLPI protected B. burgdorferi from being killed inside the host.

Even though the complement system encounters pathogens early in infection, all of the in vitro B. burgdorferi experiments were done with serum containing anti-B. burdgorferi antibodies, which are not produced until later during infection.  For this reason, the in vitro experiments fail to shed any light on how complement killed B. burgdorferi in the mouse experiment when TSLPI was not around to provide protection.  The authors surmised that B. burgdorferi is susceptible to killing by complement-mediated opsonophagocytosis, which should not require antibody since complement components such as C3b can serve as opsonins.  Unfortunately, the neutrophil phagocytosis experiment with  B. burgdorferi was conducted only with immune serum.

The commentary written by Marconi and McDowell is also worth a look.  Although they concede that the evidence for the lectin pathway playing a crucial role in killing Borrelia is compelling, they point out that other studies have implicated the classical and alternative pathways in controlling Lyme Borrelia infections.  They also wonder how exactly TSLPI, which most likely acts at the site of the tick bite, influenced the bacterial load in tissues distant from the tick bite.  If you're wondering why the mouse experiment wasn't done with B. garinii, Marconi and McDowell explain that tick-animal models for B. garinii infections are not as developed as they are for B. burgdorferi infections.

The bottom line is that the tick salivary glycoprotein TSLPI helps B. burgdorferi and possibly B. garinii establish an infection. The in vitro experiments suggest that TSLPI facilitates infection by protecting Borrelia from being killed by the lectin complement pathway, although it's not clear how the lectin pathway would kill B. burgdorferi if TSLPI was not around to provide protection.


Featured study

Schuijt, T.J., Coumou, J., Narasimhan, S., Dai, J., DePonte, K., Wouters, D., Brouwer, M., Oei, A., Roelofs, J.J.T.H., van Dam, A.P., van der Poll, T., van't Veer, C., Hovius, J.W., & Fikrig, E. (2011). A tick mannose-binding lectin inhibitor interferes with the vertebrate complement cascade to enhance transmission of the Lyme disease agent Cell Host & Microbe, 10 (2), 136-146 DOI: 10.1016/j.chom.2011.06.010

Marconi, R.T., & McDowell, J.V. (2011). Tick salivary proteins offer the Lyme disease spirochetes an easy ride and another way to hide Cell Host & Microbe, 10 (2), 95-96 DOI: 10.1016/j.chom.2011.08.003 (Commentary)


Other references

Schuijt, T.J., Narasimhan, S., Daffre, S., DePonte, K., Hovius, J.W.R., Veer, C., van der Poll, T., Bakhtiari, K., Meijers, J.C.M., Boder, E.T., van Dam, A.P., & Fikrig, E. (2011). Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display PLoS ONE, 6 (1) DOI: 10.1371/journal.pone.0015926


Related posts

• The Lyme disease spirochete feasts on tick antifreeze
• Designing a Lyme disease vaccine to attack the tick vector
• A fresh approach towards a Lyme disease vaccine:  targeting the tick

Does Borrelia burgdorferi cause an inadequate antibody response by altering B cell activation in the lymph node?

One of the characteristic features of Lyme disease is lymphadenopathy or swollen lymph nodes.  It's not too surprising when a lymph node draining a site of infection swells.  However when investigators looked at the lymph node draining the inoculation site of Borrelia burgdorferi in mice, they found that the spirochete had somehow altered the course of activation of B cells producing the antibodies that targeted the spirochete.

Before discussing the findings in the paper, a review of how the antibody response evolves in the lymph node is in order.  An antibody response to microbial proteins is sparked when antigen from microbes breaching the skin layer flow into the draining lymph node or are carried to the lymph node by dendritic cells.  Lymph nodes are where naive B cells, upon recognition of antigen, differentiate into plasma cells, which secrete large amounts of antibody that target the invading microbe.  The antibodies which bind most tightly to protein antigens are made with T cell help in germinal centers, which emerge from the rare B cells in the lymph node that produce antibody capable of recognizing the antigen.  (I say "rare" here because each B cell in the lymph node produces antibodies with different antigenic specificities to ensure that any microbe that the host may possibly encounter will be recognized by antibodies displayed by at least a few B cells.)  Upon binding the antigen and reception of critical signals from T cells, the B cells migrate to areas in the lymph node containing fixed networks of follicular dendritic cells (FDCs), a type of immune cell with long branched processes that extend out from the body of the cell.  (FDCs differ from the dendritic cells that bring antigen to the lymph node.)  The B cells then start to proliferate wildly, doubling every 6 to 8 hours (faster than B. burgdorferi!).  As the B cell numbers surge, they form germinal centers, which can be identified easily by standard histological stains (see image below).  The lymph node may even swell, depending on how much the B cells proliferate.

Lymph node: (1) capsule; (2) subcapsular sinus; (3) germinal centers; (4) lymphoide nodule; (5) trabeculae.  Source

As the B cells multiply in the germinal center, a process called somatic hypermutation, which is promoted by signals received from T cells, causes a large number of mistakes to be made within the segment of DNA encoding the antigen binding portion of the antibody.  Consequently, the antibodies displayed by some germinal center B cells are no longer able to bind to the microbial antigen whereas those made by other B cells will bind better.  The FDC processes, whose surfaces are loaded with antigen, continuously probe the antibodies expressed by the newly arising B cells.  Since the new B cells are programmed to die unless they express antibody able to bind the antigen displayed by the FDCs, only B cells displaying antibody that bind most tightly to the antigen will survive.  This process by which B cells expressing the antibodies with the highest affinity for antigen are selected is called affinity maturation.  Somatic hypermutation and affinity maturation can only occur in germinal centers.  The B cells also undergo class switching, in which the class of antibody expressed by the B cells switches from IgM and IgD, which are expressed by naive B cells, to IgA, IgE, or one of the IgG subclasses.  The exact switch that occurs is governed by the cytokines that the B cells are exposed to.  Which cytokines are present depends on the nature of the infection.  Eventually the B cells expressing high-affinity antibodies of the appropriate class differentiate into antibody-secreting plasma cells, which are released from the lymph node to circulate throughout the body and fight the infection.

So what happens in the lymph nodes during a B. burgdorferi infection?  When the investigators inoculated B. burgdorferi into or underneath the skin of mice, the lymph node draining the site swelled considerably, enlarging by more than a factor of 10 by the tenth day of infection.

From Fig. 2 of Tunev et al., 2011.  Arrow points to lymph node draining the inoculation site.  Source

What the investigators saw when they looked at lymph node sections under the microscope was very different from the textbook description of T-cell dependent B cell activation that I gave above.  First of all, live B. burgdorferi was found in the lymph node draining the site of infection in mice.  This is unusual since phagocytes would normally greet and destroy any microbe that managed to find its way into the lymph node.

From Fig. 3 of Tunev et al., 2011.  Day 8 of infection.  The arrows point to intact extracellular B. burgdorferi in the subcapsular sinus of the lymph node, which was culture positive beginning on day 1 of infection.   Source

Second, massive proliferation of B cells accounted for lymph node swelling, but the expansion of B cell numbers wasn't occurring in well-defined germinal centers. The authors also noted that T cells were not increasing in number.  These observations suggested that T cells, which are required for germinal centers to form, were not fully participating in B-cell activation.  The lack of germinal centers suggested that somatic hypermutation and affinity maturation were not occurring.

From their observations, the authors speculated that B. burgdorferi somehow subverted B cell activation in the lymph node so that the end result was a large number of plasma cells secreting antibodies of poor quality.  By poor "quality," I assume that the authors meant that the affinity of the antibody for B. burgdorferi proteins was low and that the "wrong" subclasses of IgG antibodies were expressed.  The most abundant IgG subclasses being produced in the draining lymph node at its most swollen state were IgG2b and IgG3.  Whether other IgG subclasses would be more effective at clearing B. burgdorferi from the host and whether the affinities of the antibodies for B. burgdorferi proteins were poor still need to be determined experimentally.  Perhaps a classic T-cell dependent B cell response involving the formation of germinal centers accompanied by somatic hypermutation, affinity maturation, and appropriate class switching would have led to production of "high" quality antibodies.  If the authors are correct, they have revealed yet another means by which B. burgdorferi could persist in the host.


Featured paper

Tunev SS, Hastey CJ, Hodzic E, Feng S, Barthold SW, and Baumgarth N (May 2011).  Lymphadenopathy during Lyme borreliosis is caused by spirochete migration-induced specific B cell activation.  PLoS Pathogens 7(5):e1002066.  DOI: 10.1371/journal.ppat.1002066

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

The 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


Related posts

• Antigen presentation in the bloodstream:  How invariant NKT cells are activated by Lyme disease spirochetes

Antigen presentation in the bloodstream: How invariant NKT cells are activated by Lyme disease spirochetes

The spirochete Borrelia burgdorferi is the tick-borne agent of Lyme disease, which affects the joints, nervous system, and heart.  After being deposited into the skin by an infected tick, the spirochete must enter the bloodstream so that it can circulate in the blood to gain access to its target organs.

The host doesn't sit idly as B. burgdorferi establishes an infection.  Invariant natural killer (iNKT) cells are one of the tools deployed by the immune system in its battle against the Lyme spirochetes.  Scientists know this because B. burgdorferi-infected mice lacking iNKT cells ended up with more spirochetes in their tissues and greater joint swelling than mice with a complete immune system.²

iNKT cells are an odd type of T cell.  Like other T cells, iNKT cells have a T cell receptor (TCR), yet they also express protein markers used to identify natural killer (NK) cells.  What makes the iNKT cell invariant is its TCR α chain, which comes only in the version dubbed Vα14 in mice and Vα24 in humans.  Even the β chain of the TCR of iNKT cells is restricted to three types in mice and just one in humans.  The lack of variation is unusual because the α and β TCR chains of conventional αβ T cells come in many forms in each individual, resulting in millions of varieties of TCRs.  This enables conventional αβ T cells to recognize a wide range of microbial peptide antigens when displayed by an MHC molecule on the surface of an antigen-presenting cell (see figure below).  In contrast, the TCRs of iNKT cells recognize a limited set of glycolipids displayed by the antigen-presenting cell's CD1d molecule, which structurally resembles MHC.  So far these glycolipids have been found only in Sphingomonas and B. burgdorferi.

Antigen recognition by T cells.  The "X" represents variable T cell receptor chains.
Figure 1 from ref. 3.

The structures of the B. burgdorferi glycolipids recognized by iNKT cells are shown below.  BbGL-IIc is recognized by mouse iNKT cells, and BbGL-IIf reacts with human iNKT cells.⁴

Structures of B. burgdorferi glycolipid antigens recognized by iNKT cells.  Figure 3d from ref. 3.

iNKT cells are activated when their TCR binds to BbGL-II complexed with CD1d.⁴  The activated iNKT cells secrete cytokines that elicit the appropriate immune response against the spirochetes.  How these cytokines promote killing of B. burgdorferi remains unknown.

To view the process of iNKT cell activation, scientists have recently obtained video footage of the early stages of the immune response to Borrelia burgdorferi circulating in the bloodstream of mice.¹  The study by Lee et al., which appeared in the April issue of Nature Immunology, complements two earlier studies that revealed how the Lyme disease spirochete escapes from the bloodstream of mice to invade the surrounding tissues.^5,6

The investigators employed fluorescence video microscopy to watch the immune cells in action following injection of an engineered B. burgdorferi strain expressing green fluorescent protein (GFP) into the bloodstream.  Although the spleen is better known for filtering bloodstream pathogens, the liver was selected for observation because iNKT cells make up 30% of the T cells in the liver.  In contrast, iNKT cells represent only 2.5% of T cells in the spleen.  Moreover, mice missing their spleen were able to limit B. burgdorferi infection as well as mice having a spleen, suggesting that the spleen is not critical in fighting bloodstream B. burgdorferi.

iNKT cells reside in the liver's sinusoids, which are the specialized capillaries that carry blood through the liver.  Similar to what other investigators have observed, the authors saw iNKT cells creeping along the inner surface of the liver sinusoids in healthy mice (see video below).

iNKT cells crawling within the liver sinusoids of a mouse genetically altered to express green fluorescent protein (GFP) in iNKT cells.  The iNKT cells glow bright green.  The elapsed time is shown at the top right.  Video 2 from ref. 1.

The investigators wanted to figure out which of the antigen-presenting cells found in the liver presented borrelial glycolipid to iNKT cells.  The answer?  After the spirochetes were injected into the bloodstream, they were quickly captured by Kupffer cells, the specialized blood-filtering macrophages that also reside in the liver sinusoids (see figure below).  Unlike iNKT cells, Kupffer cell remained stationary.

Capture of fluorescent B. burgdorferi (thin green bodies) by Kupffer cells (arrowhead).  Kupffer cells are stained red.  B. burgdorferi that avoided capture can be seen bound to the endothelium, trying to escape from the bloodstream into the liver tissue (arrow).  Figure 2e from ref. 1.

During the next several hours, the captured spirochetes were engulfed and broken up by the Kupffer cells so that BbGL-II could be loaded onto CD1d and displayed on the cell surface.  At 8 hours post injection, iNKT cells started to cluster and form stable contacts with Kupffer cells.  The iNKT cells were attracted to Kupffer cells churning out the chemokine CXCL9, a potent iNKT cell attractant.  The evidence for this was that injection of antibodies against the CXCL9 receptor, located on the iNKT cell surface, blocked clustering of iNKT cells.  Interaction of the Kupffer and iNKT cells was accompanied by increased blood and liver levels of the cytokine IFN-γ (interferon-gamma), a sign that the iNKT cells were being activated.

Left panel:  Liver 24 hours after injection of a GFP+ strain of B. burgdorferi into the bloodstream of a mouse.  Arrows indicate spirochetes (thin green bodies) that were not captured.  Kupffer cells are stained a red. The iNKT cells are the large bright green bodies.  The bright iNKT clusters overwhelm the faint red Kupffer cells, which are difficult to see.  Right panel:  To obtain a more convincing image showing contact between Kupffer cells and iNKT cells, a 3D reconstruction of the optical sections through the liver was performed.  Rotation of the image reveals interactions between Kupffer and iNKT cells.

Not all spirochetes were captured.  The investigators saw B. burgdorferi escaping from the sinusoids into the surrounding liver tissue even as other spirochetes were trapped by nearby Kupffer cells (see figures above).  Spirochetes circulating throughout the host probably escaped into other organs in the same manner.  Indeed, large amounts of  B. burgdorferi DNA were detected by PCR in several organs, including the liver, three days after the spirochetes were injected.  Although one doesn't usually think about the effects of Lyme disease on the liver, the authors pointed out that a mild hepatitis is common in Lyme disease patients.  In one prospective study, 40% of Lyme disease patients had at least one liver test abnormality.⁷

By now you may be wondering why the liver would devote such a high percentage of its T cells towards recognizing glycolipids that aren't found on most bacteria.  One answer is that microbes lacking the proper glycolipids may activate iNKT cells indirectly.³  For example, Salmonella typhimurium uses its LPS to coax antigen-presenting cells into making an endogenous glycolipid that gets presented to the iNKT cell by CD1d.⁸  It is also possible that glycolipids that are recognized by iNKT cells are present in other bacteria but are yet to be discovered.


Featured paper

1. Lee, W.Y., Moriarty, T.J., Wong, C.H.Y., Zhou, H., Strieter, R.M., van Rooijen, N., Chaconas, G., & Kubes, P. (2010). An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells Nature Immunology, 11 (4), 295-302 DOI: 10.1038/ni.1855

Other references

2.  Tupin, E., Benhnia, M.R., Kinjo, Y., Patsey, R., Lena, C.J., Haller, M.C., Caimano, M.J., Imamura, M., Wong, C., Crotty, S., Radolf, J.D., Sellati, T.J., and Kronenberg, M. (2008).  NKT cells prevent chronic joint inflammation after infection with Borrelia burgdorferi.  Proc. Natl. Acad. Sci. USA 105(50):19863-19868.  DOI: 10.1073/pnas.0810519105

3.  Tupin, E., Kinjo, Y., and Kronenberg, M. (2007).  The unique role of natural killer T cells in the response to microorganisms.  Nature Reviews Microbiology 5(6):405-417.  DOI: 10.1038/nrmicro1657

4.  Kinjo, J., Tupin, E., Wu, D., Fujio, M., Garcia-Navarro, R., Benhnia, M. R., Zajonc, D.M., Ben-Menachem, G., Ainge, G.D., Painter, G.F., Khurana, A., Hoebe, K., Behar, S.M., Beutler, B., Wilson, I.A., Tsuji, M., Sellati, T.J., Wong, C., and Kronenberg, M. (2006).  Nature Immunology 7(9):978-986.  DOI: 10.1038/ni1380

5.  Moriarty, T.J., Norman, M.U., Colarusso, P., Bankhead, T., Kubes, P., and Chaconas, G. (June 20, 2008).  Real-time high resolution 3D imaging of the Lyme disease spirochete adhering to and escaping from the vasculature of a living host.  PLoS Pathogens 4(6):e1000090.  DOI: 10.1371/journal.ppat.1000090

6. Norman, M.U., Moriarty, T.J., Dresser, A.R., Millen, B., Kubes, P., and Chaconas, G. (October 3, 2008). Molecular mechanisms involved in vascular interactions of the Lyme disease pathogen in a living host.  PLoS Pathogens 4(10):e1000169.  DOI: 10.1371/journal.ppat.1000169

7.  Horowitz, H.W.,  Dworkin, B., Forseter, G., Nadelman, R.B., Connolly, C., Luciano, B.B., Nowakowski, J., O'Brien, T.A., Calmann, M., Wormser, G.P. (June 1996).  Liver function in early Lyme disease.  Hepatology 23(6):1412-1417.  DOI: 10.1002/hep.510230617

8.  Mattner J., DeBord, K.L., Ismail, N., Goff, R.D., Cantu III, C., Zhou, D., Saint Mezard, P., Wang, V., Gao, Y., Yin, N, Hoebe, K., Schneewind, O., Walker, D., Beutler, B., Teyton, L, Savage, P.B., and Bendelac, A. (March 24, 2005).  Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections.  Nature 434(7032):525-529.  DOI: 10.1038/nature03408

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