E.S. Wohleb

Imaging Cortical Lesions in Multiple Sclerosis With Ultra–High-Field Magnetic Resonance Imaging

OBJECTIVEnTo determine the sensitivity of T2*-weighted gradient-echo (T2*GRE) and inversion recovery turbo-field-echo (TFE) sequences for cortical multiple sclerosis lesions at 7 T.nnnDESIGN, SETTING, AND PARTICIPANTSnAutopsied brain tissue from individuals with multiple sclerosis was scanned with 3-dimensional T2*GRE and 3-dimensional inversion recovery white matter-attenuated TFE sequences at 7 T. Cortical lesions visible with either sequence were scored for each anatomical lesion type. Imaged brain tissue was then processed for immunohistochemical analysis, and cortical lesions were identified by labeling with antibody against myelin basic protein and CD68 for microglia. Magnetic resonance images were matched with corresponding histological sections and scored retrospectively to determine the sensitivity for each cortical lesion type. Main Outcome Measure Cortical lesion detection by 3-dimensional T2*GRE and white matter-attenuated TFE sequences.nnnRESULTSnThe 3-dimensional T2*GRE and white matter-attenuated TFE sequences retrospectively detected 93% and 82% of all cortical lesions, respectively (with varying sensitivities for different lesion types). Lesion visibility was primarily determined by size as all undetected lesions were smaller than 1.1 mm at their smallest diameter. The T2*GRE images showed hypointense rings in some cortical lesions that corresponded with increased density of activated microglia.nnnCONCLUSIONSnThree-dimensional T2*GRE and white matter-attenuated TFE sequences at a 7-T field strength detect most cortical lesions in postmortem multiple sclerosis tissue. This study indicates the potential of T2*GRE and white matter-attenuated TFE sequences in ultra-high-field magnetic resonance imaging for cortical lesion detection in patients with multiple sclerosis.

Peripheral innate immune challenge exaggerated microglia activation, increased the number of inflammatory CNS macrophages, and prolonged social withdrawal in socially defeated mice

Repeated social defeat (RSD) activates neuroendocrine pathways that have a significant influence on immunity and behavior. Previous studies from our lab indicate that RSD enhances the inflammatory capacity of CD11b⁺ cells in the brain and promotes anxiety-like behavior in an interleukin (IL)-1 and β-adrenergic receptor-dependent manner. The purpose of this study was to determine the degree to which mice subjected to RSD were more responsive to a secondary immune challenge. Therefore, RSD or control (HCC) mice were injected with saline or lipopolysaccharide (LPS) and activation of brain CD11b⁺ cells and behavioral responses were determined. Peripheral LPS (0.5 mg/kg) injection caused an extended sickness response with exaggerated weight loss and prolonged social withdrawal in socially defeated mice. LPS injection also amplified mRNA expression of IL-1β, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), and CD14 in enriched CD11b⁺ cells isolated from socially defeated mice. In addition, IL-1β mRNA levels in enriched CD11b⁺ cells remained elevated in socially defeated mice 24 h and 72 h after LPS. Moreover, microglia and CNS macrophages isolated from socially defeated mice had the highest CD14 expression after LPS injection. Both social defeat and LPS injection increased the percentage of CD11b⁺/CD45(high) macrophages in the brain and the number of inflammatory macrophages (CD11b⁺/CD45(high)/CCR2⁺) was highest in RSD-LPS mice. Anxiety-like behavior was increased by social defeat, but was not exacerbated by the LPS challenge. Nonetheless, reduced locomotor activity and increased social withdrawal were still present in socially defeated mice 72 h after LPS. Last, LPS-induced microglia activation was most evident in the hippocampus of socially defeated mice. Taken together, these findings demonstrate that repeated social defeat enhanced the neuroinflammatory response and caused prolonged sickness following innate immune challenge.

Prolonged Restraint Stress Increases IL-6, Reduces IL-10, and Causes Persistent Depressive-Like Behavior That Is Reversed by Recombinant IL-10

Altered inflammatory cytokine profiles are often observed in individuals suffering from major depression. Recent clinical work reports on elevated IL-6 and decreased IL-10 in depression. Elevated IL-6 has served as a consistent biomarker of depression and IL-10 is proposed to influence depressive behavior through its ability to counterbalance pro-inflammatory cytokine expression. Clinical and animal studies suggest a role for IL-10 in modifying depressive behavior. Murine restraint stress (RST) is regularly employed in the study of behavioral and biological symptoms associated with depressive disorders. While responses to acute RST exposure have been widely characterized, few studies have examined the ongoing and longitudinal effects of extended RST and fewer still have examined the lasting impact during the post-stress period. Consistent with clinical data, we report that a protocol of prolonged murine RST produced altered cytokine profiles similar to those observed in major depressive disorder. Parallel to these changes in circulating cytokines, IL-10 mRNA expression was diminished in the cortex and hippocampus throughout the stress period and following cessation of RST. Moreover, chronic RST promoted depressive-like behavior throughout the 28-day stress period and these depressive-like complications were maintained weeks after cessation of RST. Because of the correlation between IL-10 suppression and depressive behavior and because many successful antidepressant therapies yield increases in IL-10, we examined the effects of IL-10 treatment on RST-induced behavioral changes. Behavioral deficits induced by RST were reversed by exogenous administration of recombinant IL-10. This work provides one of the first reports describing the biological and behavioral impact following prolonged RST and, taken together, this study provides details on the correlation between responses to chronic RST and those seen in depressive disorders.

Sympathetic Release of Splenic Monocytes Promotes Recurring Anxiety Following Repeated Social Defeat.

BACKGROUNDnNeuroinflammatory signaling may contribute to the pathophysiology of chronic anxiety disorders. Previous work showed that repeated social defeat (RSD) in mice promoted stress-sensitization that was characterized by the recurrence of anxiety following subthreshold stress 24 days after RSD. Furthermore, splenectomy following RSD prevented the recurrence of anxiety in stress-sensitized mice. We hypothesize that the spleen of RSD-exposed mice became a reservoir of primed monocytes that were released following neuroendocrine activation by subthreshold stress.nnnMETHODSnMice were subjected to subthreshold stress (i.e., single cycle of social defeat) 24 days after RSD, and immune and behavioral measures were taken.nnnRESULTSnSubthreshold stress 24 days after RSD re-established anxiety-like behavior that was associated with egress of Ly6C(hi) monocytes from the spleen. Moreover, splenectomy before RSD blocked monocyte trafficking to the brain and prevented anxiety-like behavior following subthreshold stress. Splenectomy, however, had no effect on monocyte accumulation or anxiety when determined 14 hours after RSD. In addition, splenocytes cultured 24 days after RSD exhibited a primed inflammatory phenotype. Peripheral sympathetic inhibition before subthreshold stress blocked monocyte trafficking from the spleen to the brain and prevented the re-establishment of anxiety in RSD-sensitized mice. Last, β-adrenergic antagonism also prevented splenic monocyte egress after acute stress.nnnCONCLUSIONSnThe spleen served as a unique reservoir of primed monocytes that were readily released following sympathetic activation by subthreshold stress that promoted the re-establishment of anxiety. Collectively, the long-term storage of primed monocytes in the spleen may have a profound influence on recurring anxiety disorders.

Social defeat promotes a reactive endothelium in a brain region-dependent manner with increased expression of key adhesion molecules, selectins and chemokines associated with the recruitment of myeloid cells to the brain

Repeated social defeat (RSD) in mice causes myeloid cell trafficking to the brain that contributes to the development of prolonged anxiety-like behavior. Myeloid cell recruitment following RSD occurs in regions where neuronal and microglia activation is observed. Thus, we hypothesized that crosstalk between neurons, microglia, and endothelial cells contributes to brain myeloid cell trafficking via chemokine signaling and vascular adhesion molecules. Here we show that social defeat caused an exposure- and brain region-dependent increase in several key adhesion molecules and chemokines involved in the recruitment of myeloid cells. For example, RSD induced distinct patterns of adhesion molecule expression that may explain brain region-dependent myeloid cell trafficking. VCAM-1 and ICAM-1 mRNA expression were increased in an exposure-dependent manner. Furthermore, RSD-induced VCAM-1 and ICAM-1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Next, mRNA expression of additional adhesion molecules (E- and P-selectin, PECAM-1) and chemokines (CXCL1, CXCL2, CXCL12, CCL2) were determined in the brain. Social defeat induced an exposure-dependent increase in mRNA levels of E-selectin, CXCL1, and CXCL2 that increased with additional days of social defeat. While CXCL12 was unaffected by RSD, CCL2 expression was increased by six days of social defeat. Last, comparison between enriched CD11b(+) cells (microglia/macrophages) and enriched GLAST-1(+)/CD11b(-) cells (astrocytes) revealed RSD increased mRNA expression of IL-1β, CCL2, and CXCL2 in microglia/macrophages but not in astrocytes. Collectively, these data indicate that key mediators of leukocyte recruitment were increased in the brain vasculature following RSD in an exposure- and brain region-dependent manner.

Basic aspects of the immunology of neuroinflammation.

Neuroinflammation is used to describe an immune-related process that occurs within the central nervous system (CNS). The objective of this chapter is to introduce basic aspects of neuroinflammation in the context of psychiatric disorders. Inflammatory processes are evident in the CNS with a myriad of stimuli including neurological disease, CNS injury or infection, peripheral infection, and psychological stress. Because the CNS maintains a degree of immune privilege, endothelial cells of the blood-brain barrier (BBB) and resident CNS innate immune cells are integral to the interpretation and propagation of inflammatory signals. For instance, activated CNS immune cells and the BBB coordinate production of cytokines and secondary messengers that act directly to influence neurophysiology. Another key concept is that there is bidirectional communication between the immune system and CNS. Afferent neuro-immune pathways relay the peripheral inflammatory profile to the CNS by secretion of cytokines and direct neuronal activation. Communication through afferent neuro-immune pathways promotes physiological and behavioral responses that are aimed to help clear pathogens from the host. For example, peripheral infection increases circulating IL-1β that induces production of prostaglandins and IL-1β by CNS immune cells that initiate fever and the behavioral symptoms of sickness. Although transient cytokine responses in the brain may be beneficial to the host, prolonged neuroinflammation associated with chronic illness, neurodegenerative disease, stress, and aging impair neuroimmune regulation and negatively affect normal cognitive and behavioral processes. Thus, understanding neuroimmune regulation and mechanisms that mediate neuroinflammation is important because these pathways likely contribute to the pathophysiology of several mental health disorders.

Correction of MFG-E8 Resolves Inflammation and Promotes Cutaneous Wound Healing in Diabetes

Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a peripheral glycoprotein that acts as a bridging molecule between the macrophage and apoptotic cells, thus executing a pivotal role in the scavenging of apoptotic cells from affected tissue. We have previously reported that apoptotic cell clearance activity or efferocytosis is compromised in diabetic wound macrophages. In this work, we test the hypothesis that MFG-E8 helps resolve inflammation, supports angiogenesis, and accelerates wound closure. MFG-E8−/− mice displayed impaired efferocytosis associated with exaggerated inflammatory response, poor angiogenesis, and wound closure. Wound macrophage-derived MFG-E8 was recognized as a critical driver of wound angiogenesis. Transplantation of MFG-E8−/− bone marrow to MFG-E8+/+ mice resulted in impaired wound closure and compromised wound vascularization. In contrast, MFG-E8−/− mice that received wild-type bone marrow showed improved wound closure and improved wound vascularization. Hyperglycemia and exposure to advanced glycated end products inactivated MFG-E8, recognizing a key mechanism that complicates diabetic wound healing. Diabetic db/db mice suffered from impaired efferocytosis accompanied with persistent inflammation and slow wound closure. Topical recombinant MFG-E8 induced resolution of wound inflammation, improvements in angiogenesis, and acceleration of closure, upholding the potential of MFG-E8–directed therapeutics in diabetic wound care.

BAI1 Orchestrates Macrophage Inflammatory Response to HSV Infection—Implications for Oncolytic Viral Therapy

Purpose: Brain angiogenesis inhibitor (BAI1) facilitates phagocytosis and bacterial pathogen clearance by macrophages; however, its role in viral infections is unknown. Here, we examined the role of BAI1, and its N-terminal cleavage fragment (Vstat120) in antiviral macrophage responses to oncolytic herpes simplex virus (oHSV). Experimental Design: Changes in infiltration and activation of monocytic and microglial cells after treatment of glioma-bearing mice brains with a control (rHSVQ1) or Vstat120-expressing (RAMBO) oHSV was analyzed using flow cytometry. Co-culture of infected glioma cells with macrophages or microglia was used to examine antiviral signaling. Cytokine array gene expression and Ingenuity Pathway Analysis (IPA) helped evaluate changes in macrophage signaling in response to viral infection. TNFα-blocking antibodies and macrophages derived from Bai1−/− mice were used. Results: RAMBO treatment of mice reduced recruitment and activation of macrophages/microglia in mice with brain tumors, and showed increased virus replication compared with rHSVQ1. Cytokine gene expression array revealed that RAMBO significantly altered the macrophage inflammatory response to infected glioma cells via altered secretion of TNFα. Furthermore, we showed that BAI1 mediated macrophage TNFα induction in response to oHSV therapy. Intracranial inoculation of wild-type/RAMBO virus in Bai1−/− or wild-type non–tumor-bearing mice revealed the safety of this approach. Conclusions: We have uncovered a new role for BAI1 in facilitating macrophage anti-viral responses. We show that arming oHSV with antiangiogenic Vstat120 also shields them from inflammatory macrophage antiviral response, without reducing safety. Clin Cancer Res; 23(7); 1809–19. ©2016 AACR.

Lumbar Myeloid Cell Trafficking into Locomotor Networks after Thoracic Spinal Cord Injury

Spinal cord injury (SCI) promotes inflammation along the neuroaxis that jeopardizes plasticity, intrinsic repair and recovery. While inflammation at the injury site is well-established, less is known within remote spinal networks. The presence of bone marrow-derived immune (myeloid) cells in these areas may further impede functional recovery. Previously, high levels of the gelatinase, matrix metalloproteinase-9 (MMP-9) occurred within the lumbar enlargement after thoracic SCI and impeded activity-dependent recovery. Since SCI-induced MMP-9 potentially increases vascular permeability, myeloid cell infiltration may drive inflammatory toxicity in locomotor networks. Therefore, we examined neurovascular reactivity and myeloid cell infiltration in the lumbar cord after thoracic SCI. We show evidence of region-specific recruitment of myeloid cells into the lumbar but not cervical region. Myeloid infiltration occurred with concomitant increases in chemoattractants (CCL2) and cell adhesion molecules (ICAM-1) around lumbar vasculature 24h and 7days post injury. Bone marrow GFP chimeric mice established robust infiltration of bone marrow-derived myeloid cells into the lumbar gray matter 24h after SCI. This cell infiltration occurred when the blood-spinal cord barrier was intact, suggesting active recruitment across the endothelium. Myeloid cells persisted as ramified macrophages at 7days post injury in parallel with increased inhibitory GAD67 labeling. Importantly, macrophage infiltration required MMP-9.

The development of a reactive brain endothelium after psychosocial stress

Psychosocial stress is associated with increased inflammation and higher prevalence of mental health disorders like anxiety and depression. Through the activation of several neuroendocrine pathways, psychological stress leads to significant physiological, immunological, and behavioral changes. Repeated social defeat (RSD), a murine model of psychosocial stress, recapitulates many of the behavioral and immunological effects observed in humans, including increased circulating cytokines, immune cell recruitment, and prolonged anxiety-like behavior. The aim of this study was to elucidate the mechanisms underlying stress-induced immune cell trafficking to the brain that leads to the development of a reactive endothelium and behavioral changes. We show that RSD caused an exposure-dependent increase in the gene expression of ICAM1, VCAM1, E-selectin, CXCL1, and CXCL2 in the brain that increased with additional days of stress. RSD-induced ICAM1 and VCAM1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Comparison between enriched CD11b+ cells (microglia/macrophages) and enriched GLAST-1+/CD11b- cells (astrocytes) revealed that RSD increased the gene expression of IL-1 beta, CCL2, and CXCL2 in microglia/macrophages, but not astrocytes. Collectively, these data indicate that critical adhesion mediators are increased in the brain vasculature following RSD. This study begins to establish a mechanism by which the brain facilitates stress-induced immune cell recruitment that may underlie anxiety and mood disorders.