Silvia S. Kang

Myelomonocytic Cell Recruitment Causes Fatal Cns Vascular Injury During Acute Viral Meningitis

Lymphocytic choriomeningitis virus infection of the mouse central nervous system (CNS) elicits fatal immunopathology through blood–brain barrier breakdown and convulsive seizures. Although lymphocytic-choriomeningitis-virus-specific cytotoxic T lymphocytes (CTLs) are essential for disease, their mechanism of action is not known. To gain insights into disease pathogenesis, we observed the dynamics of immune cells in the meninges by two-photon microscopy. Here we report visualization of motile CTLs and massive secondary recruitment of pathogenic monocytes and neutrophils that were required for vascular leakage and acute lethality. CTLs expressed multiple chemoattractants capable of recruiting myelomonocytic cells. We conclude that a CD8+ T-cell-dependent disorder can proceed in the absence of direct T-cell effector mechanisms and rely instead on CTL-recruited myelomonocytic cells.

Illuminating viral infections in the nervous system

Viral infections are a major cause of human disease. Although most viruses replicate in peripheral tissues, some have developed unique strategies to move into the nervous system, where they establish acute or persistent infections. Viral infections in the central nervous system (CNS) can alter homeostasis, induce neurological dysfunction and result in serious, potentially life-threatening inflammatory diseases. This Review focuses on the strategies used by neurotropic viruses to cross the barrier systems of the CNS and on how the immune system detects and responds to viral infections in the CNS. A special emphasis is placed on immune surveillance of persistent and latent viral infections and on recent insights gained from imaging both protective and pathogenic antiviral immune responses.

An antibiotic-responsive mouse model of fulminant ulcerative colitis

BACKGROUND The constellation of human inflammatory bowel disease (IBD) includes ulcerative colitis and Crohns disease, which both display a wide spectrum in the severity of pathology. One theory is that multiple genetic hits to the host immune system may contribute to the susceptibility and severity of IBD. However, experimental proof of this concept is still lacking. Several genetic mouse models that each recapitulate some aspects of human IBD have utilized a single gene defect to induce colitis. However, none have produced pathology clearly distinguishable as either ulcerative colitis or Crohns disease, in part because none of them reproduce the most severe forms of disease that are observed in human patients. This lack of severe IBD models has posed a challenge for research into pathogenic mechanisms and development of new treatments. We hypothesized that multiple genetic hits to the regulatory machinery that normally inhibits immune activation in the intestine would generate more severe, reproducible pathology that would mimic either ulcerative colitis or Crohns disease. METHODS AND FINDINGS We generated a novel mouse line (dnKO) that possessed defects in both TGFbetaRII and IL-10R2 signaling. These mice rapidly and reproducibly developed a disease resembling fulminant human ulcerative colitis that was quite distinct from the much longer and more variable course of pathology observed previously in mice possessing only single defects. Pathogenesis was driven by uncontrolled production of proinflammatory cytokines resulting in large part from T cell activation. The disease process could be significantly ameliorated by administration of antibodies against IFNgamma and TNFalpha and was completely inhibited by a combination of broad-spectrum antibiotics. CONCLUSIONS Here, we develop to our knowledge the first mouse model of fulminant ulcerative colitis by combining multiple genetic hits in immune regulation and demonstrate that the resulting disease is sensitive to both anticytokine therapy and broad-spectrum antibiotics. These findings indicated the IL-10 and TGFbeta pathways synergize to inhibit microbially induced production of proinflammatory cytokines, including IFNgamma and TNFalpha, which are known to play a role in the pathogenesis of human ulcerative colitis. Our findings also provide evidence that broad-spectrum antibiotics may have an application in the treatment of patients with ulcerative colitis. This model system will be useful in the future to explore the microbial factors that induce immune activation and characterize how these interactions produce disease.

Lymphocytic choriomeningitis infection of the central nervous system

Viral infection of the central nervous system (CNS) can result in a multitude of responses including pathology, persistence or immune clearance. Lymphocytic choriomeningitis virus (LCMV) is a powerful model system to explore these potential outcomes of CNS infection due to the diversity of responses that can be achieved after viral inoculation. Several factors including tropism, timing, dose and variant of LCMV in combination with the development or suppression of the corresponding immune response dictates whether lethal meningitis, chronic infection or clearance of LCMV in the CNS will occur. Importantly, the functionality and positioning of the LCMV-specific CD8+ T cell response are critical in directing the subsequent outcome of CNS LCMV infection. Although a basic understanding of LCMV and immune interactions in the brain exists, the molecular machinery that shapes the balance between pathogenesis and clearance in the LCMV-infected CNS remains to be elucidated. This review covers the various outcomes of LCMV infection in the CNS and what is currently known about the impact of the virus itself versus the immune response in the development of disease or clearance.

Priming in the Presence of IL-10 Results in Direct Enhancement of CD8+ T Cell Primary Responses and Inhibition of Secondary Responses

Although IL-10 acts as an inhibitory cytokine for APC and CD4+ T cell function, its effects on CD8+ T cells are unclear. Additionally, little is known about whether initial priming in the presence of IL-10 can have long-lasting effects and influence subsequent CD8+ T cell responses that occur in the absence of the cytokine. In the present study, we clarified the role of IL-10 during primary responses and examined whether exposure to IL-10 during initial priming of CD8+ T cells impacted secondary responses. To determine the effect of IL-10 on Ag-specific T cell responses, peptide-pulsed IL-10R2−/− splenic dendritic cells were used to prime T cells from OT-I CD8+ TCR transgenic mice. During the primary response, the presence of IL-10 resulted in enhancement of CD8+ T cell numbers without detectable alterations in the kinetics or percentage of cells that underwent proliferation. A modest increase in survival, not attributable to Bcl-2 or Bcl-xL, was also observed with IL-10 treatment. Other parameters of CD8+ T cell function, including IL-2, IFN-γ, TNF-α, and granzyme production, were unaltered. In contrast, initial exposure to IL-10 during the primary response resulted in decreased OT-I expansion during secondary stimulation. This was accompanied by lowered IL-2 levels and reduced percentages of proliferating BrdU+ cells and OT-I cells that were CD25high. IFN-γ, TNF-α, and granzyme production were unaltered. These data suggest that initial exposure of CD8+ T cells to IL-10 may be temporarily stimulatory; however, programming of the cells may be altered, resulting in diminished overall responses.

Migration of cytotoxic lymphocytes in cell cycle permits local MHC I–dependent control of division at sites of viral infection

Virus-specific cytotoxic CD8+ T cells are in cell cycle as they transit from lymphoid tissues to sites of infection.

CD4-Directed Peptide Vaccination Augments an Antitumor Response, but Efficacy Is Limited by the Number of CD8+ T Cell Precursors

Peptide vaccination is an immunotherapeutic strategy being pursued as a method of enhancing Ag-specific antitumor responses. To date, most studies have focused on the use of MHC class I-restricted peptides, and have not shown a correlation between Ag-specific CD8+ T cell expansion and the generation of protective immune responses. We investigated the effects of CD4-directed peptide vaccination on the ability of CD8+ T cells to mount protective antitumor responses in the DUC18/CMS5 tumor model system. To accomplish this, we extended the amino acid sequence of the known MHC class I-restricted DUC18 rejection epitope from CMS5 to allow binding to MHC class II molecules. Immunization with this peptide (tumor-derived extracellular signal-regulated kinase-II (tERK-II)) induced Ag-specific CD4+ T cell effector function, but did not directly prime CD8+ T cells. Approximately 31% of BALB/c mice immunized with tERK-II were protected from subsequent tumor challenge in a CD40-dependent manner. Priming of endogenous CD8+ T cells in immunized mice was detected only after CMS5 challenge. Heightened CD4+ Th cell function in response to tERK II vaccination allowed a 12-fold reduction in the number of adoptively transferred CD8+ DUC18 T cells needed to protect recipients against tumor challenge as compared with previous studies using unimmunized mice. Furthermore, tERK-II immunization led to a more rapid and transient expansion of transferred DUC18 T cells than was seen in unimmunized mice. These findings illustrate that CD4-directed peptide vaccination augments antitumor immunity, but that the number of tumor-specific precursor CD8+ T cells will ultimately dictate the success of immunotherapy.

Microbial induction of vascular pathology in the CNS

The central nervous system (CNS) is a finely tuned organ that participates in nearly every aspect of our day-to-day function. Neurons lie at the core of this functional unit and maintain an active dialogue with one another as well as their fellow CNS residents (e.g. astrocytes, oligodendrocytes, microglia). Because of this complex dialogue, it is essential that the CNS milieu be tightly regulated in order to permit uninterrupted and efficient neural chemistry. This is accomplished in part by anatomical barriers that segregate vascular components from the cerebral spinal fluid (CSF) and brain parenchyma. These barriers impede entry of noxious materials and enable the CNS to maintain requisite protein and ionic balances for constant electrochemical signaling. Under homeostatic conditions, the CNS is protected by the presence of specialized endothelium/epithelium, the blood brain barrier (BBB), and the blood-CSF barrier. However, following CNS infection these protective barriers can be comprised, sometimes resulting in severe neurological complications triggered by an imbalance or blockage of neural chemistry. In some instances, these disruptions are severe enough to be fatal. This review focuses on a selection of microbes (both viruses and parasites) that compromise vascular barriers and induce neurological complications upon gaining access to the CNS. Emphasis is placed on CNS diseases that result from a pathogenic interplay between host immune defenses and the invading microbe.

Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED)

A benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction‐limited spot. The scanned collection of fluorescence emission is incoherent; that is, no real image needs to be formed on the detector plane. The nearly isotropic emission of fluorescence excited at the focal spot allows for new detection schemes that efficiently funnel all attainable photons to detector(s). We previously showed [Combs, C.A., et al. (2007) Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector. J. Microsc. 228, 330–337] that parabolic mirrors and condensers could be combined to collect the totality of solid angle around the excitation spot for tissue blocks, leading to ∼8‐fold signal gain. Using a similar approach, we have developed an in vivo total emission detection (epiTED) instrument modified to make noncontact images from outside of living tissue. Simulations suggest that a ∼4‐fold enhancement may be possible (much larger with lower NA objectives than the 0.95 NA used here) with this approach, depending on objective characteristics, imaging depth and the characteristics of the sample being imaged. In our initial prototype, 2‐fold improvements were demonstrated in the mouse brain and skeletal muscle as well as the rat kidney, using a variety of fluorophores and no compromise of spatial resolution. These results show this epiTED prototype effectively doubles emission signal in vivo; thus, it will maintain the image signal‐to‐noise ratio at two times the scan rate or enable full scan rate at approximately 30% reduced laser power (to minimize photo‐damage).

Inflammation on the Mind: Visualizing Immunity in the Central Nervous System

The central nervous system (CNS) is a remarkably complex structure that utilizes electrochemical signaling to coordinate activities throughout the entire body. Because the nervous system contains nonreplicative cells, it is postulated that, through evolutionary pressures, this compartment has acquired specialized mechanisms to limit damage. One potential source of damage comes from our immune system, which has the capacity to survey the CNS and periphery for the presence of foreign material. The immune system is equipped with numerous effector mechanisms and can greatly alter the homeostasis and function of the CNS. Degeneration, autoimmunity, and pathogen infection can all result in acute, and sometimes chronic, inflammation within the CNS. Understanding the specialized functionality of innate and adaptive immune cells within the CNS is critical to the design of more efficacious treatments to mitigate CNS inflammatory conditions. Much of our knowledge of CNS-immune interactions stems from seminal studies that have used static and dynamic imaging approaches to visualize inflammatory cells responding to different CNS conditions. This review will focus on how imaging techniques have elevated our understanding of CNS inflammation as well as the exciting prospects that lie ahead as we begin to pursue investigation of the inflamed CNS in real time.