• Genetic Ablation of CD68 Results in Mice with Increased Bone and Dysfunctional Osteoclasts

      Ashley, Jason W.; Shi, Zhenqi; Zhao, Haibo; Li, Xingsheng; Kesterson, Robert A.; Feng, Xu; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2011-10-3)
      CD68 is a member of the lysosome associated membrane protein (LAMP) family that is restricted in its expression to cells of the monocyte/macrophage lineage. This lineage restriction includes osteoclasts, and, while previous studies of CD68 in macrophages and dendritic cells have proposed roles in lipid metabolism, phagocytosis, and antigen presentation, the expression and function of CD68 in osteoclasts have not been explored. In this study, we investigated the expression and localization of CD68 in macrophages and osteoclasts in response to the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). We found that M-CSF stimulates CD68 expression and RANKL alters the apparent molecular weight of CD68 as measured by Western immunoblotting. In addition, we explored the significance of CD68 expression in osteoclasts by generating mice that lack expression of CD68. These mice have increased trabecular bone, and in vitro assessment of CD68â /â osteoclasts revealed that, in the absence of CD68, osteoclasts demonstrate an accumulation of intracellular vesicle-like structures, and do not efficiently resorb bone. These findings demonstrate a role for CD68 in the function of osteoclasts, and future studies will determine the mechanistic nature of the defects seen in CD68â /â osteoclasts.
    • Infiltrating Cells, Interferon-gamma and Intraocular Spread of HSV-1 after Anterior Chamber Injection

      Cathcart, Heather M.; Department of Cellular Biology and Anatomy (2009-12)
      Following uniocular anterior chamber (AC) inoculation of HSV-1 (KOS), the anterior segment of the injected eye becomes inflamed and infected; however, virus does not spread from the anterior segment to infect the retina of the injected eye. The overall goal of this study was to identify interferons (IFNs) and early infiltrating cells which may play a role in protecting the retina of the ipsilateral (injected) eye. Female BALB/c, IFNy-/- and macrophage depleted (clodronate, CI2MBP treated) mice were injected in one AC with 3*104 - 6x104 PFU of HSV-1 (KOS). Mice were killed at various time points ranging from 12 to 120 hours post injection (p.i.). The injected eyes were enucleated, snap frozen and frozen sections were stained with antibodies specific for HSV-1, IFNy, Mac-1 (CD11b), Gr-1, CD49b, F4/80, CD4, CD8 and CD11c. The same antibodies were also used to stain freshly isolated single-cell suspensions from the eye or spleen for flow cytometry. Additionally, whole injected eyes were used to determine gene expression levels of IFNs and IFN associated genes. In the anterior segment of the injected eye, the ciliary body and iris were virus infected and inflamed, and infiltrating cells increased during the period of observation. Mac-1 + and F4/80+ cells colocalized with IFNy in the anterior segment and Mac-1 + cells increased in the injected eye beginning at 24 hours p.i. and continuing through 72 hours p.i. Although virus staining was increased in the ciliary body of macrophage depleted mice at 48 and 72 hours p.i., destructive retinitis was not observed in the injected eye of these animals. IFNy gene expression was up regulated in injected eyes of BALB/c mice from 48 to 120 hours p.i., and while HSV-1 infection of IFNy-/- mice resulted in increased virus staining in the ciliary body, destructive retinitis was rarely observed in IFNy-/- mice. Microglia and IFNy play important roles in the immune response to virus infection, but depletion of single cell types or cytokines did not result in early panretinal HSV-1 infection in the injected eye. Taken together, these findings support the idea that the timing and appearance of different cell types and cytokines is critical to protection of the retina of the injected eye from infection due to direct spread of virus; however, it is likely that during the innate immune response in the eye, other cell types and cytokines can compensate for the absence of a single cell type or of a single cytokine.
    • The Innate Immune System Regulates Stem Cell Responsiveness During Zebrafish Retinal Regeneration

      White, David Thomas; Department of Neuroscience and Regenerative Medicine (2015-10)
      Zebrafish replace lost retinal cells via activation of a potentially conserved vertebrate retinal stem cell type, Müller glia. We hypothesize that the innate immune system plays a key role in regulating Müller glia responsiveness to retinal cell death, as occurs during degenerative disease, thereby impacting the regenerative potential of retinal stem cells. To test this, we visualized immune cell subtypes via intravital imaging following induction of selective rod photoreceptor loss. Time-lapse imaging and immunolabeling showed that macrophages and microglia showed immune cell hallmarks consistent with reactivity to rod cell death. However, whereas microglia acted within the retina directly, macrophages were restricted to the extraocular space. Microglia activation was characterized by translocation toward the rod cell layer, proliferation, and phagocytosis of dying rod cells. To test the role of microglia during regeneration, we co-ablated microglia/rod cells or applied immune suppression, and characterized the kinetics of: (1) rod cell clearance, (2) stem cell proliferation, and (3) rod cell regeneration. The data revealed that the rate of stem cell proliferation and rod cell replacement were dependent on the presence of microglia, establishing a role for this innate immune cell subtype in regulating retinal regeneration. Additionally, characterization of the retinal milieu following rod cell ablation indicated a complex inflammatory response. Determining how innate immune cells shape retinal stem cell responsiveness will help to inform therapeutic strategies—e.g., modulating cytokine signaling to promote stem cell proliferation—aimed at reversing vision loss caused by degenerative retinal conditions.
    • Mechanisms Driving Innate Regulation Of Immunological Tolerance To Apoptotic Cells Preventing Autoimmunity

      Shinde, Rahul; Department of Neuroscience and Regenerative Medicine (2015-08)
      Innate immune responses to apoptosis are crucial for self-tolerance. Although upstream signals promoting recognition and processing of apoptotic cells have been extensively studied, downstream molecular mechanisms driving innate regulation of apoptotic cell responses are less understood. Here we report an unsuspected discovery that the ligand dependent transcription factor aryl hydrocarbon receptor (AhR) initiates tolerogenic signaling to apoptotic cells and prevents systemic autoimmunity. AhR is known to control xenobiotic stress responses and recently has been linked to modulation of T cell and DC function. In this study, we found that apoptotic cells induced AhR signals in tissueresident MΦs and activation was dependent on DNA from apoptotic cells. AhR was required for apoptotic cell driven immune suppression as deletion of AhR abrogated IL-10, promoting the inflammatory cytokines IL-6 and IL-12, while supplementing IL-10 restored the regulatory phenotype of MΦs. Moreover, inhibition of the AhR pathway fundamentally altered immune responses to apoptotic cells resulting in proinflammatory cytokine production, increased effector T cell responses and abrogation of long-term allograft tolerance to apoptotic cell associated antigens. Further, mice lacking AhR developed spontaneous autoimmunity characterized by excessive macrophage and lymphocyte activation associated with renal pathology. Deficiency of AhR led to breakdown in tolerance with rapid increases in anti-dsDNA and anti-histone antibody responses after chronic challenge with apoptotic cells. Similarly, when SLE-prone mice were treated with AhR antagonist they exhibited significantly elevated humoral auto-reactivity, augmented inflammatory cytokine production in MΦs, intensified autoreactive B and T cells, renal pathology, and mortality; while AhR agonist treatment resulted in significant reduction of autoimmune disease parameters compared to control mice. Collectively, the data demonstrate apoptotic cell activation of AhR is a key mechanism suppressing anti-apoptotic cell inflammatory responses preventing autoimmunity.