Shawn F. Sorrells

The Stressed CNS: When Glucocorticoids Aggravate Inflammation

Glucocorticoids (GCs) are hormones released during the stress response that are well known for their immunosuppressive and anti-inflammatory properties; however, recent advances have uncovered situations wherein they have effects in the opposite direction. The CNS is a particularly interesting example, both because of its unique immune environment, and because GCs affect immune responses differently in different brain regions. In this minireview we discuss the contexts wherein GCs increase CNS inflammation and point out directions for future investigation.

Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus. This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease. In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day, whereas other studies find many fewer putative new neurons. Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal development. We also find that the number of proliferating progenitors and young neurons in the dentate gyrus declines sharply during the first year of life and only a few isolated young neurons are observed by 7 and 13 years of age. In adult patients with epilepsy and healthy adults (18–77 years; n = 17 post-mortem samples from controls; n = 12 surgical resection samples from patients with epilepsy), young neurons were not detected in the dentate gyrus. In the monkey (Macaca mulatta) hippocampus, proliferation of neurons in the subgranular zone was found in early postnatal life, but this diminished during juvenile development as neurogenesis decreased. We conclude that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the dentate gyrus does not continue, or is extremely rare, in adult humans. The early decline in hippocampal neurogenesis raises questions about how the function of the dentate gyrus differs between humans and other species in which adult hippocampal neurogenesis is preserved.

Glucocorticoids Exacerbate Lipopolysaccharide-Induced Signaling in the Frontal Cortex and Hippocampus in a Dose-Dependent Manner

Although the anti-inflammatory actions of glucocorticoids (GCs) are well established, evidence has accumulated showing that proinflammatory GC effects can occur in the brain, in a poorly understood manner. Using electrophoretic mobility shift assay, real-time PCR, and immunoblotting, we investigated the ability of varying concentrations of corticosterone (CORT, the GC of rats) to modulate lipopolysaccharide (LPS)-induced activation of NF-κB (nuclear factor κB), expression of anti- and proinflammatory factors and of the MAP (mitogen-activated protein) kinase family [ERK (extracellular signal-regulated kinase), p38, and JNK/SAPK (c-Jun N-terminal protein kinase/stress-activated protein kinase)], and AKT. In the frontal cortex, elevated CORT levels were proinflammatory, exacerbating LPS effects on NF-κB, MAP kinases, and proinflammatory gene expression. Milder proinflammatory GCs effects occurred in the hippocampus. In the absence of LPS, elevated CORT levels increased basal activation of ERK1/2, p38, SAPK/JNK, and AKT in both regions. These findings suggest that GCs do not uniformly suppress neuroinflammation and can even enhance it at multiple levels in the pathway linking LPS exposure to inflammation.

Brain size and limits to adult neurogenesis.

The walls of the cerebral ventricles in the developing embryo harbor the primary neural stem cells from which most neurons and glia derive. In many vertebrates, neurogenesis continues postnatally and into adulthood in this region. Adult neurogenesis at the ventricle has been most extensively studied in organisms with small brains, such as reptiles, birds, and rodents. In reptiles and birds, these progenitor cells give rise to young neurons that migrate into many regions of the forebrain. Neurogenesis in adult rodents is also relatively widespread along the lateral ventricles, but migration is largely restricted to the rostral migratory stream into the olfactory bulb. Recent work indicates that the wall of the lateral ventricle is highly regionalized, with progenitor cells giving rise to different types of neurons depending on their location. In species with larger brains, young neurons born in these spatially specified domains become dramatically separated from potential final destinations. Here we hypothesize that the increase in size and topographical complexity (e.g., intervening white matter tracts) in larger brains may severely limit the long‐term contribution of new neurons born close to, or in, the ventricular wall. We compare the process of adult neuronal birth, migration, and integration across species with different brain sizes, and discuss how early regional specification of progenitor cells may interact with brain size and affect where and when new neurons are added. J. Comp. Neurol. 524:646–664, 2016.

Glucocorticoids increase excitotoxic injury and inflammation in the hippocampus of adult male rats.

Background/Aims: Stress exacerbates neuron loss in many CNS injuries via the actions of adrenal glucocorticoid (GC) hormones. For some injuries, this GC endangerment of neurons is accompanied by greater immune cell activation in the CNS, a surprising outcome given the potent immunosuppressive properties of GCs. Methods: To determine whether the effects of GCs on inflammation contribute to neuron death or result from it, we tested whether nonsteroidal anti-inflammatory drugs could protect neurons from GCs during kainic acid excitotoxicity in adrenalectomized male rats. We next measured GC effects on (1) chemokine production (CCL2 and CINC-1), (2) signals that suppress immune activation (CX3CL1, CD22, CD200, and TGF-ß), and (3) NF-κB activity. Results: Concurrent treatment with minocycline, but not indomethacin, prevented GC endangerment. GCs did not substantially affect CCL2, CINC-1, or baseline NF-κB activity, but they did suppress CX3CL1, CX3CR1, and CD22 expression in the hippocampus - factors that normally restrain inflammatory responses. Conclusions: These findings demonstrate that cellular inflammation is not necessarily suppressed by GCs in the injured hippocampus; instead, GCs may worsen hippocampal neuron death, at least in part by increasing the neurotoxicity of CNS inflammation.

Glucocorticoid signaling in myeloid cells worsens acute CNS injury and inflammation.

Glucocorticoid stress hormones (GCs) are well known for being anti-inflammatory, but some reports suggest that GCs can also augment aspects of inflammation during acute brain injury. Because the GC receptor (GR) is ubiquitously expressed throughout the brain, it is difficult to know which cell types might mediate these unusual “proinflammatory” GC actions. We examined this with cell type-specific deletion or overexpression of GR in mice experiencing seizure or ischemia. Counter to their classical anti-inflammatory actions, GR signaling in myeloid cells increased Iba-1 and CD68 staining as well as nuclear p65 levels in the injured tissue. GCs also reduced levels of occludin, claudin 5, and caveolin 1, proteins central to blood–brain-barrier integrity; these effects required GR in endothelial cells. Finally, GCs compromised neuron survival, an effect mediated by GR in myeloid and endothelial cells to a greater extent than by neuronal GR.

Mechanistic insights into corticosteroids in multiple sclerosis: War horse or chameleon?☆

OBJECTIVES Relapse management is a crucial component of multiple sclerosis (MS) care. High-dose corticosteroids (CSs) are used to dampen inflammation, which is thought to hasten the recovery of MS relapse. A diversity of mechanisms drive the heterogeneous clinical response to exogenous CSs in patients with MS. Preclinical research is beginning to provide important insights into how CSs work, both in terms of intended and unintended effects. In this article we discuss cellular, systemic, and clinical characteristics that might contribute to intended and unintended CS effects when utilizing supraphysiological doses in clinical practice. The goal of this article is to consider recent insights about CS mechanisms of action in the context of MS. METHODS We reviewed relevant preclinical and clinical studies on the desirable and undesirable effects of high-dose corticosteroids used in MS care. RESULTS Preclinical studies reviewed suggest that corticosteroids may act in unpredictable ways in the context of autoimmune conditions. The precise timing, dosage, duration, cellular exposure, and background CS milieu likely contribute to their clinical heterogeneity. CONCLUSION It is difficult to predict when patients will respond favorably to CSs, both in terms of therapeutic response and tolerability profile. There are specific cellular, systemic, and clinical characteristics that might merit further consideration when utilizing CSs in clinical practice, and these should be explored in a translational setting.

Glucocorticoids can arm macrophages for innate immune battle

Despite the drama of the paradigm shift, science is more the pointillist process of adding or replacing facts, pixel by pixel. This replacement can be an uphill battle, basically consisting of saying, “Well yes, this is how things work. . .but not exactly.” And with enough of those replacements, the overall picture changes. Such a change is occurring now with regulators of neuroinflammation, and the work of Frank et al., on page XXX (Frank et al., 2009) is an important contribution to this changing picture. The revisionism concerns the effects of glucocorticoids (GCs) on inflammation. GCs are steroids released from the adrenal gland. The endogenous GCs, cortisol and corticosterone (CORT), are near to the hearts of stress physiologists. These GCs are secreted during stress and are central to the bodys adaptive response to short-term stressors as well as to the pathogenicity of chronic stress. Meanwhile, synthetic steroids such as prednisone and dexamethasone, are near to the hearts of physicians (Sapolsky et al., 2000) The reason, of course, is the ability of GCs to inhibit inflammation. These effects cover the gamut from tightening of the blood brain barrier, to induction of apoptosis in lymphocytes and to inhibition of NF-κB activity. These actions are the foundation for the vast use of synthetic GCs in medicine, including neurology (McEwen et al., 1997). The “this is how things work. . ..but not exactly” scenario has been emerging for many years, insofar as GCs are not uniformly anti-inflammatory, including in the case of post-stroke edema. This fact has prompted several accomplished neurologists to warn against the indiscriminate use of GCs (often to little effect) (Gomes et al., 2005). It has become clear recently that the “. . .but not exactly” element of the story is even more dramatic. Specifically, in some circumstances, GCs can have pro-inflammatory effects, worsening the outcome of necrotic neurological insults. These pro-inflammatory GC effects occur at multiple levels, with GCs: (a) increasing migration of microglia, neutrophils and macrophages to an injury site; (b) increasing and potentiating production and release of pro-inflammatory cytokines; and, (c) NF-κB activity (de Pablos et al., 2006; Madrigal et al., 2002; Munhoz et al., 2006). They occur in the context of both excitotoxic (i.e., kainic acid or hypoxia–ischemia) and inflammatory challenges (i.e., LPS), and are induced by stress itself. Moreover, these are mediated by the glucocorticoid receptor (GR), which is the receptor most involved in the stress response. Finally, and intriguingly, these pro-inflammatory GC actions occur in the cortex and, to a lesser extent, the hippocampus, which are the two major GC targets in the brain. Making this picture more complex, those effects occur while GCs, simultaneously, are anti-inflammatory in the hypothalamus (Sorrells et al., 2009). A question then becomes when are GCs pro-inflammatory. The latest in a series of excellent papers from the laboratory of Steven Maier at the University of Colorado (Frank et al., 2009) explore the issue of time course of GC exposure. A stripped down summary: rats, challenged with LPS, had either basal circulating CORT levels or levels raised with exogenous CORT into the stress range. Critically, CORT levels were raised beginning either 24 or 2 h pre-LPS, or 1-h post. And as the key finding, CORT elevation pre-LPS potentiated the inflammatory response in the hippocampus and liver (i.e., levels of TNFα, IL-1b and IL-6). In contrast, CORT elevation commencing post-LPS inhibited these inflammatory endpoints, as would be predicted. The authors provide a key mechanism that might explain these effects, in that CORT pre-treatment up-regulated TLR2. The fact that these pro-inflammatory effects also occurred outside the nervous system in the liver suggests a role for macrophages. The results are convincing, the magnitude large, and the conclusion clear, namely that stress levels of GCs can augment neuroinflammation. These findings fit well with the old, often underappreciated concept of “permissive” endocrine effects, where a hormone has no effect on X, but ‘permits” Y to have a stronger effect than usual on X (Sapolsky et al., 2000). Permissive effects are traditionally considered to arise from basal hormone levels. In contrast, in the present study, it was stress levels of GCs that primed subsequent inflammation. Neuroinflammatory insults trigger considerable GC secretion; such secretion might worsen the consequences of subsequent inflammatory challenges. Naturally, more work is needed. One issue is how GCs cause the change in TLR2; the standard mechanism of action of steroid hormones suggests that it will be a genomic GC action. The authors have previously reported that stress itself has priming effects (Johnson et al., 2002), and it is important to know whether GCs partially or entirely explain stress effects. Perhaps the most pressing question spurred by this excellent paper is whether synthetic GCs have similar priming effects. If so, this would have disquieting neurological implications for the huge numbers of people treated chronically with GCs.

Derivation of Injury-Responsive Dendritic Cells for Acute Brain Targeting and Therapeutic Protein Delivery in the Stroke-Injured Rat

Research with experimental stroke models has identified a wide range of therapeutic proteins that can prevent the brain damage caused by this form of acute neurological injury. Despite this, we do not yet have safe and effective ways to deliver therapeutic proteins to the injured brain, and this remains a major obstacle for clinical translation. Current targeted strategies typically involve invasive neurosurgery, whereas systemic approaches produce the undesirable outcome of non-specific protein delivery to the entire brain, rather than solely to the injury site. As a potential way to address this, we developed a protein delivery system modeled after the endogenous immune cell response to brain injury. Using ex-vivo-engineered dendritic cells (DCs), we find that these cells can transiently home to brain injury in a rat model of stroke with both temporal and spatial selectivity. We present a standardized method to derive injury-responsive DCs from bone marrow and show that injury targeting is dependent on culture conditions that maintain an immature DC phenotype. Further, we find evidence that when loaded with therapeutic cargo, cultured DCs can suppress initial neuron death caused by an ischemic injury. These results demonstrate a non-invasive method to target ischemic brain injury and may ultimately provide a way to selectively deliver therapeutic compounds to the injured brain.

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies.

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically diverse. Inroads have been made in understanding the roles of distinct cortical interneuron subgroups, however, there are still many mechanisms to be worked out that may contribute to the development and maturation of different types of GABAergic cells. Moreover, altered GABAergic signaling may contribute to phenotypes of autism, schizophrenia and epilepsy. Specific Cre-driver lines have begun to parcel out the functions of unique interneuron subgroups. Despite the advances in mouse models, it is often difficult to efficiently study GABAergic cortical interneuron progenitors with molecular approaches in vivo. One important technique used to study the cell autonomous programming of these cells is transplantation of MGE cells into host cortices. These transplanted cells migrate extensively, differentiate, and functionally integrate. In addition, MGE cells can be efficiently transduced with lentivirus immediately prior to transplantation, allowing for a multitude of molecular approaches. Here we detail a protocol to efficiently transduce MGE cells before transplantation for in vivo analysis, using available Cre-driver lines and Cre-dependent expression vectors. This approach is advantageous because it combines precise genetic manipulation with the ability of these cells to disperse after transplantation, permitting greater cell-type specific resolution in vivo.