| Abstract: | CXCR6-GFP+ cells, which encompass 70% invariant natural killer T cells (iNKT cells), have been found primarily patrolling inside blood vessels in the liver. Although the iNKT cells fail to interact with live pathogens, they do respond to bacterial glycolipids presented by CD1d on liver macrophage that have caught the microbe. In contrast, in this study using dual laser multichannel spinning-disk intravital microscopy of joints, the CXCR6-GFP, which also made up 60–70% iNKT cells, were not found in the vasculature but rather closely apposed to and surrounding the outside of blood vessels, and to a lesser extent throughout the extravascular space. These iNKT cells also differed in behavior, responding rapidly and directly to joint-homing pathogens like Borrelia burgdorferi, which causes Lyme disease. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted dissemination attempts by these microbes into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway, an observation also made in vitro for iNKT cells from joint but not liver or spleen. These results suggest a novel, critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier and explains a large increase in pathogen burden of B. burgdorferi in the joint of iNKT cell-deficient mice, and perhaps the greater susceptibility of humans to this pathogen because of fewer iNKT cells in human joints.An essential component of homeostasis is the delivery of oxygen and nutrients to tissues via an extensive vascular network. This process, however, also creates a portal for pathogens to exploit the dissemination of invading bacteria. Intravascular immunity is an emerging concept that suggests that the host immune system remain vigilant and proactive within the vasculature, limiting or preventing pathogen dissemination (1). As such, it is not surprising that numerous cell types have been discovered patrolling the vasculature, including rolling neutrophils in places like skin, crawling monocytes (2), and invariant natural killer T cells (iNKT cells) (3) in skin, brain, muscle, and lung, and immobilized macrophages, including Kupffer cells and splenic macrophage in liver and spleen, respectively (4). Although macrophage have been demonstrated to directly catch, phagocytose, and destroy various pathogens, iNKT cells have to date been suggested to receive signals via antigen presentation and produce potent cytokines that can enhance immunity to infections (4–6), but their direct role in killing pathogens has not been reported. However, it is worth mentioning a recent publication suggesting that iNKT cells can produce granzyme B (7), a molecule known to be used by other immune cells to kill tumor cells as well as various fungi.The natural killer T cells, a subpopulation of T lymphocytes, express a T-cell receptor (TCR) with an invariant variable α-segment 14-joining α-segment 18 (Vα14-Jα18) TCR-α chain that is paired with a restricted subset of TCR Vβ chains in mice (Vα24-Jα18 or Vβ11 in humans) (5, 6). This highly restricted repertoire of TCRs expressed by iNKT cells allows them to recognize lipid antigens presented by CD1d (8, 9), a nonclassical MHC class I-like molecule. The most potent iNKT antigen identified was α-galactosylceramide (αGC) (10), and in vivo administration of this molecule leads to rapid stopping of patrolling iNKT cells with subsequent production of various cytokines, including IL-4 and IFN-γ (5, 6). More recently, several lipid antigens from pathogens have been reported, including α-galactosyl diacylglycerolipid from Borrelia burgdorferi, a spirochete responsible for human Lyme disease (8, 9, 11–13). Imaging of the intravascular immune response in vivo to αGC, which would exclusively bind CD1d, has shown a cessation of iNKT cell crawling and the production of cytokines. Unlike a specific CD1d ligand, an intact pathogen has many activating molecules, ranging from Toll-like receptor (TLR) ligands to complement to chemoattractants, and its direct interaction with iNKT cells is not fully elucidated.B. burgdorferi is arguably the prototype emerging pathogen, becoming a global public health concern estimated at as many as 300,000 patients a year in North America (www.cdc.gov/media/releases/2013/p0819-lyme-disease.html). Lyme disease is one of the most common vector-borne diseases and the number of infected patients is continuing to increase (14). A variety of symptoms have been identified, the most common late-stage manifestation being joint inflammation, known as Lyme arthritis (14). In most cases, treatment with antibiotics leads to resolution of symptoms, but in the absence of antibiotic therapy, intermittent or chronic synovial inflammation can occur (14, 15). Although iNKT cells have not been reported in joints of mice, mice lacking iNKT cells have a joint-specific 25-fold increase (PCR product) in pathogen burden in Lyme borreliosis (4, 13). Although it is well appreciated that iNKT cells receive signals from antigen-presenting cells to induce a systemic increase in IFN-γ, an important cytokine in the fight against infection, it is unclear why the absence of this response would favor localization of B. burgdorferi only in joints (4, 13). We hypothesized that the iNKT cells were also exerting their protective effects directly in the knee microvasculature. Indeed, we have identified a population of CXCR6-GFP+ cells, 60–70% of which were iNKT cells that resided in the tissues and preferentially surrounding the joint vasculature. These cells performed direct immune surveillance in joints that function as a cytotoxic barrier, killing pathogens via a granzyme-dependent mechanism. These cells appear to be less prevalent in human joints and may explain the greater susceptibility to Lyme borreliosis in these human tissues. |
