Regulatory T Cells Contribute to Resistance against Lyme Arthritis.

The symptoms of Lyme disease are caused by inflammation induced by species of the Borrelia burgdorferisensu lato complex. The various presentations of Lyme disease in the population suggest that differences exist in the intensity and regulation of the host response to the spirochete. Previous work has described correlations between the presence of regulatory T cells and recovery from Lyme arthritis. However, the effects of Foxp3-expressing CD4+ T cells existing prior to, and during, B. burgdorferi infection have not been well characterized. Here, we used C57BL/6 "depletion of regulatory T cell" mice to assess the effects these cells have on the arthritis-resistant phenotype characteristic of this mouse strain. We showed that depletion of regulatory T cells prior to infection with B. burgdorferi resulted in sustained swelling, as well as histopathological changes, of the tibiotarsal joints that were not observed in infected control mice. Additionally, in vitro stimulation of splenocytes from these regulatory T cell-depleted mice resulted in increases in gamma interferon and interleukin-17 production and decreases in interleukin-10 production that were not evident among splenocytes of infected mice in which Treg cells were not depleted. Depletion of regulatory T cells at various times after infection also induced rapid joint swelling. Collectively, these findings provide evidence that regulatory T cells existing at the time of, and possibly after, B. burgdorferi infection may play an important role in limiting the development of arthritis.

Interleukin-10 (IL-10) inhibits Borrelia burgdorferi-induced IL-17 production and attenuates IL-17-mediated Lyme arthritis.

Previous studies have shown that cells and cytokines associated with interleukin-17 (IL-17)-driven inflammation are involved in the arthritic response to Borrelia burgdorferi infection. Here, we report that IL-17 is a contributing factor in the development of Lyme arthritis and show that its production and histopathological effects are regulated by interleukin-10 (IL-10). Spleen cells obtained from B. burgdorferi-infected, "arthritis-resistant" wild-type C57BL/6 mice produced low levels of IL-17 following stimulation with the spirochete. In contrast, spleen cells obtained from infected, IL-10-deficient C57BL/6 mice produced a significant amount of IL-17 following stimulation with B. burgdorferi. These mice developed significant arthritis, including erosion of the bones in the ankle joints. We further show that treatment with antibody to IL-17 partially inhibited the significant hind paw swelling and histopathological changes observed in B. burgdorferi-infected, IL-10-deficient mice. Taken together, these findings provide additional evidence of a role for IL-17 in Lyme arthritis and reveal an additional regulatory target of IL-10 following borrelial infection.

Effects of Regulatory T Cell Depletion in BALB/c Mice Infected with Low Doses of Borrelia burgdorferi.

We previously demonstrated that a depletion of regulatory T (Treg) cells in Lyme arthritis-resistant C57BL/6 mice leads to pathological changes in the tibiotarsal joints following infection with Borrelia burgdorferi. Here, we assessed the effects of Treg cells on the response to B. burgdorferi infection in BALB/c mice, which exhibit infection-dose-dependent disease and a different sequence of immune events than C57BL/6 mice. The depletion of Treg cells prior to infection with 1 × 102, but not 5 × 103, organisms led to increased swelling of the tibiotarsal joints. However, Treg cell depletion did not significantly affect the development of histopathology at these low doses of infection. BALB/c mice depleted of Treg cells before infection with 1 × 103 spirochetes harbored a higher borrelial load in the hearts and exhibited higher levels of serum interleukin-10 five weeks later. These results indicate that Treg cells regulate certain aspects of the response to B. burgdorferi in a mouse strain that may display a range of disease severities. As the presentation of Lyme disease may vary among humans, it is necessary to consider multiple animal models to obtain a complete picture of the various means by which Treg cells affect the host response to B. burgdorferi.

Hamster and murine models of severe destructive Lyme arthritis.

Arthritis is a frequent complication of infection in humans with Borrelia burgdorferi. Weeks to months following the onset of Lyme borreliosis, a histopathological reaction characteristic of synovitis including bone, joint, muscle, or tendon pain may occur. A subpopulation of patients may progress to a chronic, debilitating arthritis months to years after infection which has been classified as severe destructive Lyme arthritis. This arthritis involves focal bone erosion and destruction of articular cartilage. Hamsters and mice are animal models that have been utilized to study articular manifestations of Lyme borreliosis. Infection of immunocompetent LSH hamsters or C3H mice results in a transient synovitis. However, severe destructive Lyme arthritis can be induced by infecting irradiated hamsters or mice and immunocompetent Borrelia-vaccinated hamsters, mice, and interferon-gamma- (IFN-γ-) deficient mice with viable B. burgdorferi. The hamster model of severe destructive Lyme arthritis facilitates easy assessment of Lyme borreliosis vaccine preparations for deleterious effects while murine models of severe destructive Lyme arthritis allow for investigation of mechanisms of immunopathology.

Role of IL-17, transforming growth factor-beta, and IL-6 in the development of arthritis and production of anti-outer surface protein A borreliacidal antibodies in Borrelia-vaccinated and -challenged mice.

We showed recently that the adaptive immune events leading to the development of arthritis in Borrelia burgdorferi isolate 297-vaccinated and Borrelia bissettii-challenged mice involve IL-17. Here, we show in Borrelia-vaccinated and -challenged mice that two cytokines known to induce the production of IL-17, IL-6 and transforming growth factor (TGF)-beta, are also involved in the development of arthritis. Vaccinated and challenged mice administered either anti-TGF-beta or anti-IL-6 antibodies developed histopathologic changes of the hind paws similar to or greater than untreated control mice. By contrast, simultaneous blockage of these cytokines reduced the severity of arthritis in Borrelia-vaccinated and -challenged mice. Moreover, administration of anti-IL-17 antibodies to these dual-antibody-treated mice completely prevented the development of histopathologic changes of the ankle joints, significantly reduced edema of the hind paws, and prevented the production of anti-outer surface protein A borreliacidal antibodies. These findings demonstrate a role for the combined effects of IL-17, IL-6, and TGF-beta in the adaptive immune events leading to the development of Borrelia-induced arthritis.

CD4+ cell-derived interleukin-17 in a model of dysregulated, Borrelia-induced arthritis.

Lyme borreliosis, which is caused in the United States by the spirochete Borrelia burgdorferi, may manifest as different arrays of signs, symptoms and severities between infected individuals. Recent studies have indicated that particularly severe forms of Lyme borreliosis in humans are associated with an increased Th17 response. Here, we hypothesized that a murine model combining the dysregulated immune response of an environment lacking interleukin-10 (IL-10) with a robust T-cell-driven inflammatory response would reflect arthritis associated with the production of IL-17 by CD4+ cells. We demonstrate that IL-10 regulates the production of IL-17 by Borrelia-primed CD4+ cells early after interaction with Lyme spirochetes in vitro and that infection of Borrelia-primed mice with B. burgdorferi leads to significant production of IL-17 that contributes to the development of severe arthritis. These results extend our previous findings by demonstrating that a dysregulated adaptive immune response to Lyme spirochetes can contribute to severe, Th17-associated arthritis. These findings may lead to therapeutic measures for individuals with particularly severe symptoms of Lyme borreliosis.

Arthritis is developed in Borrelia-primed and -infected mice deficient of interleukin-17.

Interleukin-17 (IL-17) has been shown to participate in the development of Lyme arthritis in experimental mice. For example, neutralization of IL-17 with antibodies inhibits induction of arthritis in Borrelia-primed and -infected C57BL/6 wild-type mice. We hypothesized that mice lacking IL-17 would fail to develop Borrelia-induced arthritis. IL-17-deficient and wild-type C57BL/6 mice were primed with heat-inactivated Borrelia and then infected with viable spirochetes 3 weeks later. No swelling or major histopathological changes of the hind paws were detected in IL-17-deficient or wild-type mice that were primed with Borrelia or infected with viable spirochetes. By contrast, IL-17-deficient and wild-type mice that were primed and subsequently infected with heterologous Borrelia developed severe swelling and histopathological changes of the hind paws. In addition, Borrelia-primed and -infected IL-17-deficient mice exhibited elevated gamma-interferon (IFN-γ) levels in sera and increased frequencies of IFN-γ-expressing lymphocytes in popliteal lymph nodes compared to Borrelia-primed and -infected wild-type mice. These results demonstrate that IL-17 is not required for development of severe pathology in response to infection with Borrelia burgdorferi, but may contribute to disease through an interaction with IFN-γ.

Interleukin-35 enhances Lyme arthritis in Borrelia-vaccinated and -infected mice.

Interleukin-35 (IL-35) has been reported to inhibit the production of interleukin-17 (IL-17) as a means of preventing arthritis and other inflammatory diseases. We previously showed that treatment of Borrelia-vaccinated and -infected mice with anti-IL-17 antibody at the time of infection prevented the development of arthritis. The anti-IL-17 antibody-treated mice lacked the extensive tissue damage, such as bone and cartilage erosion, that occurred in the tibiotarsal joints of untreated Borrelia-vaccinated and -infected control mice. We hypothesized that IL-35 would reduce the severity of arthritis by suppressing the production of IL-17 in Borrelia-vaccinated and -infected mice. Here, we show that administration of recombinant IL-35 (rIL-35) to Borrelia-vaccinated and -infected mice augments the development of severe arthritis compared to the results seen with untreated control mice. Borrelia-vaccinated and -infected mice treated with rIL-35 had significantly (P < 0.05) greater hind paw swelling and histopathological changes from day 4 through day 10 than non-rIL-35-treated Borrelia-vaccinated and -infected mice. In addition, the treatment with IL-35 only slightly decreased the production of IL-17 in Borrelia-primed immune cells and did not prevent the development of borreliacidal antibody. Our data do not support a role for IL-35 as a potential therapeutic agent to reduce inflammation in Lyme arthritis.

Significant differences between the Borrelia-infection and Borrelia-vaccination and -infection models of Lyme arthritis in C3H/HeN mice.

The immunological events leading to the development of Lyme arthritis in humans are partially understood. Much of this information has been gained by studying the course of infection of naïve or vaccinated mice with Borrelia burgdorferi. However, the Borrelia-vaccination and -infection model has not been described using the organismal parameters commonly used in the widely accepted Borrelia-infection model. This is the first comparison between the Borrelia-infection and the Borrelia-vaccination and -infection models of arthritis. Borrelia-vaccinated and -infected C3H/HeN mice develop acute inflammation comparable to that of nonvaccinated, Borrelia-infected C3H/HeN mice. The duration and severity of arthritis in Borrelia-vaccinated and -infected mice was slightly increased compared with Borrelia-infected mice. Significantly, Borrelia-vaccinated and -infected C3H/HeN mice produce interleukin-17 (IL-17), while Borrelia-infected mice that had not been previously vaccinated do not. Neutralization of IL-17 in Borrelia-vaccinated and -infected C3H/HeN mice decreased the severity of arthritis, although not to the degree we observed previously in C57BL/6 mice. Collectively, these findings show that the Borrelia-vaccination and -infection model of Lyme arthritis incorporates elements of adaptive immunity that likely have relevance to human disease, but may not be observed in Borrelia-infected C3H/HeN mice.

Human Lyme disease vaccines: past and future concerns.

The development of a vaccine for Lyme disease was intensely pursued in the 1990s. However, citing a lack of demand, the first human Lyme disease vaccine was withdrawn from the market less than 5 years after its approval. The public's concerns about the vaccine's safety also likely contributed to the withdrawal of the vaccine. Nearly a decade later, no vaccine for human Lyme disease exists. The expansion of Lyme disease's endemic range, as well as the difficulty of diagnosing infection and the disease's steady increase in incidence in the face of proven preventative measures, make the pursuit of a Lyme disease vaccine a worthwhile endeavor. Many believe that the negative public perception of the Lyme disease vaccine will have tarnished any future endeavors towards its development. Importantly, many of the drawbacks of the Lyme disease vaccine were apparent or foreseeable prior to its approval. These pitfalls must be confronted before the construction of a new, effective and safe human Lyme disease vaccine.

Interleukin-23 is required for development of arthritis in mice vaccinated and challenged with Borrelia species.

We recently hypothesized that T helper 17 (Th17) cells and their associated cytokines are involved in the development of arthritis following infection with Borrelia burgdorferi. Here, we show that interleukin-23 (IL-23), a survival factor for Th17 cells, is required for the induction of arthritis in mice vaccinated with B. burgdorferi strain 297 and challenged with "Borrelia bissettii." When Borrelia-vaccinated and -challenged mice were given antibodies to the p19 subunit of IL-23, they failed to develop the histopathological changes observed in untreated vaccinated and challenged mice. In addition, viable B. bissettii organisms stimulated the secretion of IL-17 from Borrelia-immune lymph node cells during in vitro culture. When anti-IL-23 p19 antibody was included in cultures of B. bissettii organisms and Borrelia-immune lymph node cells, the production of IL-17 was reduced to levels observed in cultures containing immune cells alone. Taken together, these results support the hypothesis that Th17 cell-associated cytokines are involved in the development of Borrelia-mediated arthritis. These findings provide insight into previously overlooked immune mechanisms responsible for the development of Lyme arthritis.

Anti-p19 antibody treatment exacerbates lyme arthritis and enhances borreliacidal activity.

Considerable effort has been made to elucidate the mechanism of Lyme arthritis. We focused on p19, a cell cycle-regulating molecule, because it is known to inhibit cell cycle division of T lymphocytes which may be responsible for the induction of arthritis. We show that anti-p19 antibody treatment enhances the inflammatory response normally detected at the tibiotarsal joints of Borrelia burgdorferi-vaccinated and Borrelia bissettii-challenged mice. Specifically, anti-p19 antibody treatment augmented the severity of inflammation within the synovial and subsynovial tissue. Moreover, treatment with anti-p19 antibody caused severe erosion of cartilage and bone with ankle joint destruction. In addition, anti-p19 antibody treatment of Borrelia-vaccinated and -challenged mice enhanced the borreliacidal antibody response, especially against the vaccine isolate. The novel activities of anti-p19 antibody show that p19 may be an important therapeutic site for the treatment of Lyme arthritis.

Anti-CD25 antibody treatment of mice vaccinated and challenged with Borrelia spp. does not exacerbate arthritis but inhibits borreliacidal antibody production.

CD4(+) CD25(+) T cells are a population of regulatory T cells responsible for the modulation of the immune response in several autoimmune and infectious disease models. We previously showed that adoptive transfer of enriched CD4(+) CD25(+) T cells also plays a major role in the prevention of arthritis in Borrelia-vaccinated (Borrelia burgdorferi isolate 297) and -challenged (B. bissettii) mice. Here, we present evidence that administration of anti-CD25 antibody at the time of challenge or at later intervals fails to enhance the development of severe destructive osteoarthropathy in Borrelia-vaccinated C57BL mice. However, Borrelia-vaccinated and -challenged mice receiving anti-CD25 antibody developed decreased borreliacidal antibody titers compared to vaccinated and challenged controls. These findings suggest that additional mechanisms besides CD4(+) CD25(+) T cells are involved in the regulation of the immune response to Borrelia infection following vaccination.

Interleukin-6 promotes anti-OspA borreliacidal antibody production in vitro.

Determination of the immunological mediators responsible for promoting the production of borreliacidal antibody may facilitate the development of an improved borreliosis vaccine for human and veterinary use. Previously, we developed an in vitro assay to determine if borreliacidal antibody production could be augmented by treatment with different cytokines. In this study, in vitro treatment of lymph node cells producing borreliacidal antibody with recombinant interleukin-6 (rIL-6) resulted in a fourfold enhancement of anti-OspA borreliacidal antibody. Moreover, rIL-6 enhanced Western immunoblot titers and increased the number of B lymphocytes. In contrast, treatment of anti-OspA borreliacidal antibody-producing cells with anti-IL-6 resulted in a fourfold reduction in borreliacidal activity. Treatment with anti-IL-6 also inhibited enhanced borreliacidal antibody production induced by anti-gamma interferon. These data suggest that IL-6 plays a significant role in the production of anti-OspA borreliacidal antibodies.

Anti-interleukin-15 prevents arthritis in Borrelia-vaccinated and -infected mice.

We showed previously that interleukin-17 (IL-17) plays a significant role in the induction of arthritis associated with Borrelia vaccination and challenge. Little information, however, is available about the chain of immunologic events that leads to the release of IL-17. The production of IL-17 has been linked to stimulation of memory cells by IL-15. Therefore, we hypothesized that IL-15 is involved in the induction of arthritis associated with Borrelia vaccination and infection of mice. Here we present evidence that treatment of Borrelia-vaccinated and -infected mice with anti-IL-15 antibody prevents swelling of the hind paws. More importantly, both anti-IL-15 antibody- and recombinant IL-15 receptor alpha-treated Borrelia-vaccinated and -infected mice were free of major histopathologic indications of arthritis, including hyperplasia, hypertrophy, and vilus formation of the synovium. Similarly, the synovial space and perisynovium were free of inflammatory cells. By contrast, the synovium of nontreated Borrelia-vaccinated and -infected mice had overt hyperplasia, hypertrophy, and vilus formation. Moreover, the synovial space and perisynovium were infiltrated with neutrophils, macrophages, and lymphocytes. Finally, we show that recombinant IL-15 stimulates the release of IL-17 from lymph node cells obtained near the arthritic site. These results suggest that IL-15 plays a major role in orchestrating IL-17 induction of arthritis associated with Borrelia-vaccinated and -infected mice.

CD4(+) CD25(+) T cells prevent arthritis associated with Borrelia vaccination and infection.

CD4(+) CD25(+) T cells are a population of regulatory T cells associated with control of arthritis in anti-interleukin-17 antibody-treated Borrelia-vaccinated and challenged gamma interferon-deficient mice. Here, we present direct evidence that adoptive transfer of enriched CD4(+) CD25(+) T cells from these mice can prevent the development of arthritis in Borrelia-vaccinated and challenged mice. These findings establish a major role for CD4(+) CD25(+) T cells in the prevention of arthritis in Borrelia-vaccinated and challenged animals.

Association of CD4+ CD25+ T cells with prevention of severe destructive arthritis in Borrelia burgdorferi-vaccinated and challenged gamma interferon-deficient mice treated with anti-interleukin-17 antibody.

CD4+ CD25+ T cells are a population of regulatory T cells responsible for active suppression of autoimmunity. Specifically, CD4+ CD25+ T cells have been shown to prevent insulin-dependent diabetes mellitus, inflammatory bowel disease, and pancreatitis. Here, we present evidence that CD4+ CD25+ T cells also play a major role in controlling the severity of arthritis detected in Borrelia burgdorferi-vaccinated gamma interferon-deficient (IFN-gamma degrees ) C57BL/6 mice challenged with the Lyme spirochete. When B. burgdorferi-vaccinated and challenged IFN-gamma degrees mice were treated with anti-interleukin-17 (IL-17) antibody, the number of CD4+ CD25+ T cells increased in the local lymph nodes. Furthermore, histopathologic examination showed the mice to be free of destructive arthritis. When these anti-IL-17-treated B. burgdorferi-vaccinated and challenged mice were also administered anti-CD25 antibody, the number of CD4+ CD25+ T cells in the local lymph nodes decreased. More importantly, severe destructive arthropathy was induced. In addition, delayed administration of anti-CD25 antibody decreased the severity of the arthritis. These results suggest that CD4+ CD25+ T cells are involved in regulation of a severe destructive arthritis induced with an experimental model of vaccination and challenge with B. burgdorferi.

Neutralization of gamma interferon augments borreliacidal antibody production and severe destructive Lyme arthritis in C3H/HeJ mice.

Development of a high level of sustained borreliacidal antibody is paramount for maintaining protection against infection with Borrelia burgdorferi. We show that production of borreliacidal antibody can be enhanced by preventing the effects of gamma interferon (IFN-gamma). When lymph node cells capable of producing borreliacidal antibody were cultured with anti-murine IFN-gamma, an eightfold increase in borreliacidal antibody production was obtained. However, anti-IFN-gamma treatment of these cells also enhanced their ability to adaptively induce arthritis. When anti-IFN-gamma-treated lymph node cells producing borreliacidal antibody were infused into C3H/HeJ mice and the mice were then challenged with B. burgdorferi, the mice developed severe destructive Lyme arthritis. Additional studies are needed to delineate the immune response responsible for the induction of arthritis and production of borreliacidal antibody. These studies are needed to ensure an effective and safe vaccine against infection with B. burgdorferi.

Mycobacterium tuberculosis susceptibility testing by flow cytometry.

Here is another unit with clinical relevance. Tuberculosis remains a major health problem throughout the world, with approximately one-quarter of the population being infected. Rapid and accurate susceptibility testing for the tubercle bacillus is essential for control of the disease. Such testing can be accomplished by flow cytometry within twenty-four hours, instead of the days to weeks required by traditional methods. The use of flow cytometry both improves the quality of susceptibility testing and advances public health measures for the prevention and control of this ancient scourge.