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research pov_lyme arthritis_Peptidoglycan

Research POV: Lyme Arthritis and Peptidoglycan

Important study identifies the Borrelia burgdorferi peptidoglycan as an immunogen likely to cause Lyme arthritis in some patients

 

by Mayla Hsu, Ph.D., Director of Research and Science, GLA

A longstanding mystery in Lyme disease is why some antibiotic-treated patients continue to suffer long-term symptoms, while others recover. It is possible that a hyperactive immune system in some patients causes inflammation-related tissue damage. Another possibility is that the bacteria, Borrelia burgdorferi, may still be alive in compartments of the body that are not readily accessible to antibiotics, and the bacteria’s continued replication causes ongoing symptoms.

A recent article found that a component of the bacterial cell wall, peptidoglycan (PG), might be driving the persistent inflammation that causes Lyme arthritis even after antibiotic treatment. The authors, who include a GLA-funded researcher, found B. burgdorferi PG antibodies in the synovial fluid that surrounds the knees of Lyme arthritis patients, which is evidence of an ongoing immune response against bacteria or fragments of its cell wall. In contrast, they did not find significant antibodies against PGs of any other bacteria. A strength of this study is that they compared synovial fluid from Lyme arthritis patients with that of other types of non Lyme-related arthritis. Only Lyme arthritis patients had synovial fluid B. burgdorferi PG antibodies.

Blood was also collected from the same Lyme patients whose synovial fluid was studied. Here, they found that sera (serums) of Lyme arthritis patients also had PG antibodies, although the levels were much lower than in the synovial fluid. Moreover, Lyme arthritis patient sera had much higher levels of PG antibodies than any detected in control patient sera. These findings indicate a continued immune response against PG, particularly in the synovial joints, specifically against B. burgdorferi.

Another asset of this study was its analysis of patient samples both before and after antibiotic treatment. The authors designed a way to detect PG itself, not only antibodies, in synovial fluid and sera. Using this test, they could not find PG in control synovial fluid, nor in Lyme arthritis patient sera. However, it was present in 92% of Lyme arthritis synovial fluids, both before and after treatment with antibiotics; and this was true of patients who had been treated with oral as well as intravenous antibiotics. In addition, the amount of PG strongly correlated with the level of PG antibodies in the same synovial fluid samples. This finding suggests that the bacterial cell wall itself is present in the synovia of patients, and that its presence elicits antibodies against PG.

Peptidoglycan_figure for blog
Comparison of B. burgdorferi PG antibodies for serum and synovial fluid, from Lyme arthritis patients (red) and from controls (black) (healthy and non-Lyme arthritis)

Do these results mean that B. burgdorferi is actively replicating in the patients in whom PG or PG antibodies were found? Testing for bacterial DNA was done on synovial fluid and blood, both before and after antibiotic treatment. Although both compartments were often positive for bacterial DNA before therapy, almost all samples became negative after therapy. This finding suggests that PG and PG antibodies persisted even after eliminating actively replicating B. burgdorferi at these sites, but it does not rule out bacterial replication elsewhere, and leaves open the question of how bacterial cell wall components could remain long after the bacterial DNA is no longer detectable.

This study also identified proinflammatory cytokines in synovial fluid, which play an important role in mediating immune responses. In vitro, the addition of purified B. burgdorferi PG to cultured peripheral blood mononuclear cells (PBMCs) caused increased levels of inflammatory cytokines like TNFα, IL1α, and IFNγ. These and others were also significantly elevated in the synovial fluid of Lyme arthritis patients, suggesting a possible mechanistic link between PG and inflammation. Finally, injection of purified PG in mice resulted in ankle swelling and inflammatory changes that are consistent with the established mouse model for Lyme arthritis.

In sum, these results make a strong case that B. burgdorferi PG may be responsible for driving inflammation and continued symptoms in post-antibiotic Lyme arthritis. Whether it is also implicated in promoting other long-term symptoms remains to be seen. These findings open up a new avenue of inquiry that may yield fruitful insights that will help us to better understand and care for people suffering from persistent symptoms.


Learn more about GLA’s research initiatives and accomplishments:

Research Report
Published Research Findings
Current Grantees 
First Observational Study for Lyme Disease Treatment
Post-treatment Lyme: Two Million by 2020
Blog: Why Good Science is Crucial

Ixodes scapularis ticks

GLA POV: Microbiome analysis of Ixodes scapularis ticks from New York and Connecticut

by Timothy Sellati, Ph.D., Chief Scientific Officer, GLA

GLOBAL LYME ALLIANCE’S CHIEF SCIENTIFIC OFFICER OFFERS PERSPECTIVE ON NEW RESEARCH OUT OF COLUMBIA UNIVERSITY WITH ANALYSIS OF BLACKLEGGED TICKS

 

Ixodes scapularis, the blacklegged tick, is among the most clinically important tick species in the U.S. Not only does this tick transmit the most common vector-borne infectious agents in the U.S., Borrelia burgdorferi sensu stricto (s.s.) and B. mayonii, which cause Lyme disease, but it also transmits the greatest diversity of pathogens of any tick within the US. In addition to Lyme disease, other tick-borne illnesses include babesiosis (Babesia microti), anaplasmosis (Anaplasma phagocytophilum), Borrelia miyamotoi disease (B. miyamotoi), ehrlichiosis (Ehrlichia muris eauclairensis) and tick-borne encephalitis (Powassan virus).

It is this diversity of pathogens and the public health concern they provoke that spurred Rafal Tokarz (a Global Lyme Alliance-funded investigator) and colleagues to use high-throughput sequencing of the tick microbiome to catalogue the full range of microbes (i.e., bacteria, archaea, protists, fungi and viruses) found in ticks collected throughout New York and Connecticut.

Dr. Tokarz’s lab examined 197 individual I. scapularis adults and pools from 132 adults and 197 nymphs. Focusing on Borrelia, they found two species in the ticks tested, B. burgdorferi s.s. and B. miyamotoi, which were found in 56.3% and 5.07% of individual ticks, respectively. Importantly, these results were consistent with previously published surveillance studies of I. scapularis by the Tokarz group and others. Across the five sites where ticks were collected, the prevalence of B. burgdorferi s.s. ranged from 40% to 62.5%. Two other human pathogens transmitted by black-legged ticks, B. mayonii and Ehrlichia muris eauclairensis, have been detected in up to 3% of I. scapularis in the Upper Midwest, but were not detected in the ticks collected for this study, suggesting these pathogens have yet to establish themselves, at least at detectable levels, in the northeast.

Another human infectious agent, a protozoan parasite, B. microti was found in 7.6% of ticks, but two other species of Babesia, B. duncani and B. divergens, which also have been implicated in babesiosis in the U.S., were not found.  The absence of B. duncani and B. divergens sequences in their data suggests that I. scapularis is not a vector for these species, at least not in the northeast.

Reports of Bartonella DNA in tick species (i.e., I. scapularis, I. affinis, and I. pacificus), coupled with detection of this bacterium in patients with Lyme disease, have led to the perception that Bartonella is a tick-transmitted pathogen. Recently, there has been an upsurge in Bartonella testing of patients with a suspected tick-borne illness. However, Bartonella sequences were not found in the hundreds of ticks tested as part of this study. The authors speculated that differences between their findings and published reports might have several explanations, which they sought to investigate. One possibility was that the presence of microbial DNA within a tick does not conclusively establish the tick’s competence as a vector, or the viability of the microbe within the tick. The DNA may instead be a remnant of a previous blood meal, and there’s enough of it to be detectable by sensitive genetic techniques such as PCR. Another possibility is that in previous studies, the Bartonella detection techniques were not specific enough. In a 2004 study by Adelson et al., 33% of I. scapularis nymphs in New Jersey had Bartonella DNA. However, the ‘molecular tools’ used for PCR screening could not discriminate between Bartonella DNA and that of a wide range of soil bacteria. So, trace amounts of soil bacterial contaminants in ticks could explain the wrong conclusion that I. scapularis harbors Bartonella. To test this hypothesis, Tokarz’s group designed molecular tools that could distinguish between Bartonella and soil bacterial DNA. Again, they confirmed a lack of Bartonella in the 45 ticks from their study but using the Adelson group’s reagents, an apparent 13% of ticks from NY and CT were now positive for Bartonella.  These findings point to the possibility of false positive results if reagents used for PCR-based tests are not optimized for specificity.

These results do not, however, exclude the possibility that a subset of Lyme disease patients may indeed have a Bartonella infection. In fact, well-accepted means of exposure to Bartonella include contact with infected domestic cats or flea bites. Thus, a conclusion one can draw from this current study is that Lyme patients diagnosed with Bartonella are more likely infected by cats or fleas and not by I. scapularis ticks.  Although not mentioned in this study, the results presented offer a cautionary tale regarding PCR-based diagnosis of Bartonella or any other pathogen. One must be certain that the molecular tools used to detect Bartonella DNA are highly specific to that organism. Care must be taken to avoid detecting a person’s likely exposure to a wide variety of soil bacteria, something that can easily occur in daily life.  The use of non-specific molecular tools in this diagnostic scenario might generate false positives – a possibility that will hopefully be explored by other researchers and diagnostic testing companies.

The survey also revealed a high co-infection rate with 19% of all ticks being co-infected with known pathogens (A. phagocytophilum, B. burgdorferi s.s., B. miyamotoi, B. microti, and Powassan virus). In addition to bacteria, the tick microbiome was found to include a wide variety of common viruses [e.g., phleboviruses 1 and 2 (73%), South Bay virus (22%), Suffolk (10%), and the pathogenic Powassan virus (3.6%) ticks] as well as three rare viruses and even a filarial worm.

In conclusion, this new study provides insight into the impressive microbial diversity within I. scapularis in NY and CT. In conjunction with recently published microbiome studies from other geographical sites, scientists now possess a better understanding of the spectrum of agents harbored by I. scapularis, which can serve to focus research and clinical treatment.  Recognition of the multitude of pathogens found within black-legged ticks also should drive efforts to develop blood and urine based multi-pathogen diagnostic tests instead of just asking whether a patient has Lyme disease or not.  GLA is pioneering the advancement of novel “NextGen” multi-pathogen diagnostic tools through funding of companies and researchers at the top academic institutions in the country.


Read study here.