Chronic Lyme disease is a controversial and poorly understood condition that is thought to occur in some patients who have been infected with the bacterium Borrelia Burgdorferi; this Spirochaete is transmitted by ticks to humans and hides out deep within tissues, making its detection in infected patients very challenging. Only very low numbers of microbes are required to cause illness, and this microbe displays elaborate mechanisms to evade detection by the immune system, including frequent mutational changes in its genetic signature. In some cases, patients with chronic Lyme disease may experience persistent symptoms even after completing standard antibiotic treatment for the infection; these individuals are described as having Post Treatment Lyme Disease, PTLDS (Asch ES et. al. 1994; Feder HM Jr et. al. 2007).
PTLDS symptoms can be highly personal and heterogenous, however, The Infectious Diseases Society of America (IDSA) guidelines describe PTLDS symptoms as fatigue, widespread musculoskeletal pain and cognitive difficulties, resulting in reduced levels of occupational, educational, personal, or social activity (Wormser, G.P. et. al. 2006). There are currently very few clinical tools to diagnose or monitor patients with PTLDS; however, Incite Health™ is leveraging our team’s expertise in immunology and flow cytometry to develop innovative assays to profile patients during intervention therapies to provide actionable, long-term tracking of the immune systems of these individuals, for a better patient outcome.
Diagnosing Lyme Disease
Fig. 1. Outline of Infectious Disease Society of America (IDSA) guidelines of proposed decision tree for clinical decision points commonly applied for patients with suspected Lyme infection.
Since the Borrelia microbes are very difficult to detect directly, most diagnostic tests look for the seroconversion of infected individuals and the presence of anti-Borrelia IgG and IgM antibodies. However, physicians are encouraged to rely both on the clinical symptoms of their patients, coupled with blood tests to formulate a diagnosis and engage in therapeutic intervention.
Strategies in Patient Monitoring
Flow cytometry is a powerful technique that can be used to analyze cells based on their physical and chemical characteristics. While it is not a direct diagnostic tool for Lyme disease, it can be used to monitor changes in the immune response of patients with PTLDS through the profiling of key biomarkers associated with the condition, either in blood or in CSF samples (Xing, J. et. al. 2015).
To monitor patients with PTLDS using flow cytometry, blood samples are collected at regular intervals and analyzed for changes in the numbers and types of immune cells present. This can provide insight into the patient's immune response biases and help to identify any abnormalities or imbalances that may be contributing to their symptoms.
An example of this is the use of flow cytometry to measure the numbers of B cells, T cells, and natural killer cells in the blood, coupled with the expression of specific cell surface markers that are associated with different immune cell subsets (Wood, K. L. et. al 2009). Changes in these parameters over time may indicate shifts in the patient's immune response that could be related to their symptoms, or in response to therapeutic intervention strategies (Stricker, R. B. 2002).
Deeper profiling of patients can be achieved by analyzing cytokine production by certain immune cells, providing information about the patient's inflammatory response, and specifically the immune system mechanisms that are dysregulated. Abnormalities in cytokine production have been reported in some patients with PTLDS, and monitoring these changes using flow cytometry can be central in guiding treatment decisions.
Personalized Approaches To This Unmet Medical Need
PTLDS is a complex and poorly understood condition that is characterized by a wide range of symptoms, including fatigue, joint pain, and neurological problems. There is currently no single biomarker that can definitively diagnose PTLDS, but there are several biomarkers that may be useful in the diagnosis and management of the condition that helps to identify the specific dysfunctional features of the immune response to the infection.
1. Antibodies to Borrelia burgdorferi:
Antibodies to the bacterium Borrelia burgdorferi, which is the causative agent of Lyme disease, are often used as a diagnostic marker for the infection. However, in chronic Lyme disease, the presence of antibodies may be more difficult to interpret, as some patients may continue to produce antibodies even after the infection has been cleared.
Cytokines are signaling molecules that are produced by immune cells in response to infection or inflammation. Patients with PTLDS frequently display alterations in cytokine levels, with studies reporting elevated levels of inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-γ). These cytokines are produced by activated immune cells in response to infection or inflammation and can contribute to tissue damage that results in the manifestation of disease symptoms.
Other studies have reported alterations in anti-inflammatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β) (Widhe, M. et. al. 2002). These cytokines are important for regulating the immune response and preventing excessive inflammation, and alterations in their levels may contribute to the development of chronic inflammation and immune dysfunction in PTLDS.
The cytokine profiles of patients with PTLDS appear to be complex and heterogeneous, with some individuals exhibiting a pro-inflammatory cytokine profile, while others exhibit alterations in anti-inflammatory cytokines or chemokines (Cerar, T. et. al. 2013). It is important to note that the interpretation of cytokine data can be complex, and may vary depending upon the levels measured in serum versus CSF (Grusell, M. et. al. 2002) but flow cytometry provides one of the most comprehensive and quantitative platforms for immune system profiling and will ultimately enable clinicians to understand the significance of these markers more fully in the context of the condition for each patient.
Chemokines are signaling molecules that help to attract immune cells to sites of infection or inflammation. Alterations in chemokine levels have also been reported in patients with PTLDS, with studies showing increased levels of chemokines CXCL9 and CXCL10 as hallmarks of specific chemokine signatures associated with the condition (Soloski MJ, et. al. 2014). These two chemokines are important for recruiting immune cells such as T cells and natural killer (NK) cells to sites of infection or inflammation and have been implicated in the pathogenesis of other chronic inflammatory conditions, such as rheumatoid arthritis and multiple sclerosis. The chemokine CXCL13 has been identified as a potential diagnostic marker for neuroborreliosis- the neurological manifestation of chronic Lyme disease (Rupprecht T. A. et. al. 2005; Senel M, et. al. 2010).
Patients with PTLDS may also display alterations in the expression of several chemokine receptors, including CCR5 and CXCR3, which are important for mediating the migration of immune cells to affected tissues.
4. Natural killer cell function
Natural killer (NK) cells are a type of immune cell that plays a role in controlling viral infections and cancer. Many studies have identified alterations in NK cell function in patients with PTLDS. Natural killer (NK) cells are a type of immune cell that plays an important role in controlling viral infections and cancer. In chronic PTLDS, alterations in NK cell function may contribute to immune dysfunction and persistent symptoms associated with the condition. Decreased NK cell activity in PTLDS patients resulted in reduced cytotoxicity, which is the ability of the cells to kill infected or abnormal cells (Stricker, R. B. & Winger, E. E. 2001). This decrease in NK cell activity was also correlated with the severity of disease symptoms, suggesting that alterations in NK cell function may contribute to the pathogenesis of PTLDS.
NK cell function in PTLDS patients has also been shown to be impaired in response to stimulation with Borrelia burgdorferi antigens. This impaired NK cell response may contribute to the persistence of the infection and the development of chronic inflammation and immune dysfunction associated with the disease (Lima, M. et. al. 2002).
It is increasingly clear that alterations in NK cell function play a role in the pathogenesis of PTLDS, and the most recent research has focused on specific activation markers on these immune cells, with heterogeneity in responses – with some patients displaying a decreased expression of the activating receptor NKG2D on NK cells. NKG2D is important for the recognition and killing of infected or abnormal cells, and its decreased expression on NK cells may contribute to the impaired NK cell function observed in patients with PTLDS. Similarly, some patients appear to exhibit increased expression of the inhibitory receptor NKG2A, which can inhibit NK cell function and therefore contribute to the decreased NK cell activity observed in PTLDS (Katchar K. et. al. 2013). Alterations in the expression of other markers on NK cells, such as CD56 and CD16, have been reported in PTLDS patients. CD56 is a marker of NK cell activation, and decreased expression of this marker on NK cells has been reported in some PTLDS patients. CD16 is a marker of NK cell cytotoxicity, and alterations in its expression on NK cells have also been reported in PTLDS patients. Such changes in the expression of these markers would contribute to NK dysfunction in PTLDS patients and may contribute to some of the symptoms that these individuals display.
Autoantibodies are antibodies that target the body's own tissues and can contribute to autoimmune diseases. Some studies have reported the presence of autoantibodies in patients with PTLDS, which may suggest an autoimmune component to the condition (Fallon, B. A. et. al. 2020). It appears that repeat Borrelia infections may increase the risk of developing autoantibodies including those to neuronal proteins such as anti-lysoganglioside GM1 (Garcia-Monoco et. al 1993), anti-tubulin and anti-D1R, and this is often correlated with increased functional antibody mediated CaMKII activation, supporting an inflammatory process in the brain (Coughlin et. al. 2018). Incite Health offers a customizable flow cytometry-based serology platform, that can be adapted to titer autoimmune antibodies in PTLDS patients.
Example of a potential flow cytometry panel that is offered to monitor the immune response in PTLDS patients:
Core panel markers:
- CD3: a marker for T cells
- CD4: a marker for helper T cells
- CD8: a marker for cytotoxic T cells
- CD19: a marker for B cells
- CD56: a marker for natural killer (NK) cells
- CD14: a marker for monocytes and macrophages
- HLA-DR: a marker for activated immune cells
This panel can be expanded to include markers for specific subsets of T cells and B cells that have been implicated in PTLDS
- CD25: a marker for regulatory T cells (Tregs)
- CD69: a marker for activated T cells
- IgD: a marker for naive B cells
- CD27: a marker for memory B cells
- CD138: a marker for plasma cells
Along with cytokine profiles of these immune cells.
IFN-gamma: a cytokine produced by T cells and NK cells
IL-10: a cytokine produced by regulatory T cells and other cells that can dampen the immune response in PTLDS patients.
Overall, while flow cytometry is not a direct diagnostic tool for Lyme disease, it can be a useful tool for monitoring patients with chronic Lyme disease and providing insight into their immune response to the infection and how this is contributing to their symptoms.
By analyzing the expression of these immune system markers on different immune cell subsets, it is possible to gain insight into the immune responses driving the symptoms of PTLDS within that patient and identify more focused targets for therapeutic intervention (Rebman AW et. al. 2020; Nemeth J et. al. 2016). Studies have shown that patients who react to the Borrelia infection with a high TH1-associated response, display more effective immune-mediated spirochetal killing, whereas individuals who respond to the infection with high TH17-associated immune responses, often develop autoantibodies, that correlate with post-Lyme symptoms. Insights such as these are providing a new paradigm for the study of postinfectious symptoms in PTLDS patients (Klemen, S. et. al. 2014)
There needs to be a shift in thinking that one size fits all when it comes to treating Lyme Disease and PTLDS. By profiling each patient’s signature immune response to the infection, it will be possible to identify early on those individuals who are most at risk of poor outcome and the development of neuroborreliosis, as well as hone in on the areas of dysregulated immune response to the infection; all of this will help physicians formulate more precise therapeutic strategies for their patients and support better patients outcome from this potentially devastating and increasingly prevalent disease.
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