Frank Brand’s research while affiliated with University of Miami and other places

Background Interleukin (IL)-1β is involved in the pathology of intervertebral disc degeneration. Under normal conditions, IL-1β is present in cells in an inactive form (pro-IL-1β). However, under pathological conditions, pro-IL-1β is turned into its active form (IL-1β) by the inflammasome, a multi-protein complex of the innate immune response that activates caspase-1. Under conditions of degeneration, the disc experiences an environment of increased acidification. However, the implications of acidification on the innate immune response remain poorly explored. Methods Here we have studied how pH changes in human nucleus pulposus cells affect inflammasome activation by immunoblot analysis of protein lysates obtained from nucleus pulposus cells that were exposed to different pH levels in culture. ResultsIn this study, we have found that in nucleus pulposus cells, with increased acidification, there was a decrease in inflammasome activation consistent with lower levels of active IL-1β. However, this effect at a pH of 6.5, the lowest pH level tested, was abrogated when cells were treated with IL-1β. Conclusions Taken together, these findings suggest that the inflammatory response through IL-1β experienced by the human disc is not initiated in nucleus pulposus cells when the stimulus is acidification.

Background Focal cerebral ischemia induces an inflammatory response that when exacerbated contributes to deleterious outcomes. The molecular basis regarding the regulation of the innate immune response after focal cerebral ischemia remains poorly understood. Methods In this study we examined the expression of retinoic acid-inducible gene (RIG)-like receptor-I (RIG-I) and its involvement in regulating inflammation after ischemia in the brain of rats subjected to middle cerebral artery occlusion (MCAO). In addition, we studied the regulation of RIG-I after oxygen glucose deprivation (OGD) in astrocytes in culture. Results In this study we show that in the hippocampus of rats, RIG-I and IFN-α are elevated after MCAO. Consistent with these results was an increased in RIG-I and IFN-α after OGD in astrocytes in culture. These data are consistent with immunohistochemical analysis of hippocampal sections, indicating that in GFAP-positive cells there was an increase in RIG-I after MCAO. In addition, in this study we have identified n-propyl gallate as an inhibitor of IFN-α signaling in astrocytes. Conclusion Our findings suggest a role for RIG-I in contributing to the innate immune response after focal cerebral ischemia.

Periodic treatments with estrogen receptor subtype‐β ( ER ‐β) agonist reduce post‐ischemic hippocampal injury in ovariectomized rats. However, the underlying mechanism of how ER ‐β agonists protect the brain remains unknown. Global cerebral ischemia activates the innate immune response, and a key component of the innate immune response is the inflammasome. This study tests the hypothesis that ER ‐β regulates inflammasome activation in the hippocampus, thus reducing ischemic hippocampal damage in reproductively senescent female rats that received periodic ER ‐β agonist treatments. First, we determined the effect of hippocampal ER ‐β silencing on the expression of the inflammasome proteins caspase 1, apoptosis‐associated speck‐like protein containing a CARD ( ASC ), and interleukin ( IL )‐1β. Silencing of ER ‐β attenuated 17β‐estradiol mediated decrease in caspase 1, ASC , and IL ‐1β. Next, we tested the hypothesis that periodic ER ‐β agonist treatment reduces inflammasome activation and ischemic damage in reproductively senescent female rats. Periodic ER ‐β agonist treatments significantly decreased inflammasome activation and increased post‐ischemic live neuronal counts by 32% ( p < 0.05) as compared to the vehicle‐treated, reproductively senescent rats. Current findings demonstrated that ER ‐β activation regulates inflammasome activation and protects the brain from global ischemic damage in reproductively senescent female rats. Further investigation on the role of a periodic ER ‐β agonist regimen to reduce the innate immune response in the brain could help reduce the incidence and the impact of global cerebral ischemia in post‐menopausal women. image We propose that estrogen receptor subtype‐β (ER‐β) activation regulates inflammasome activation and protects the brain from global ischemic damage in reproductively senescent female rats.

Stroke is associated with an inflammatory response that contributes to deleterious outcomes. However, the molecular basis in the regulation of inflammation remains poorly understood. Here, we examine the activation of retinoic acid inducible gene-like (RIG) receptors (RLRs) and the involvement of RLR signaling in the regulation of type I IFNs following mid-cerebral artery occlusion (MCAO). In this study we show that in the rat hippocampus RIG-I and IFN-α expression are increased after MCAO. Similarly, we show that in astrocytes in culture, oxygen glucose deprivation (OGD) resulted in increased RIG-I and IFN-α expression. These data are consistent with immunohistochemical analysis of hippocampal sections, indicating that in glial fibrillary acidic protein (GFAP) positive cells there was an increase in RIG-I immunoreactivity after MCAO. Moreover, n-propyl gallate (n-PG) can be used to inhibit RIG-I/IFN-α signaling in astrocytes. In conclusion, these data indicate that RIG-I is involved in the inflammatory response after stroke.

Neuroinflammation is a response against harmful effects of diverse stimuli and participates in the pathogenesis of brain and spinal cord injury (SCI). The innate immune response plays a role in neuroinflammation following central nervous system (CNS) injury via activation of multi-protein complexes termed inflammasomes that regulate the activation of caspase-1 and the processing of the pro-inflammatory cytokines IL-1β and IL-18. We report here that the expression of components of the nucleotide-binding-and-oligomerization domain (NOD)-like receptor protein-1 (NLRP-1) inflammasome, apoptosis speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 are significantly elevated in spinal cord motor neurons and cortical neurons after CNS trauma. Moreover, NLRP1 inflammasome proteins are present in exosomes derived from cerebrospinal fluid (CSF) of SCI and traumatic brain- injured patients following trauma. To investigate whether exosomes could be used to therapeutically block inflammasome activation in the CNS, exosomes were isolated from embryonic cortical neuronal cultures and loaded with short-interfering RNA (siRNA) against ASC and administered to spinal cord-injured animals. Neuronal-derived exosomes crossed the injured blood-spinal cord barrier, and delivered their cargo in vivo, resulting in knock down of ASC protein levels by approximately 76% when compared to SCI rats treated with scrambled siRNA. Surprisingly, siRNA silencing of ASC also led to a significant decrease in caspase-1 activation and processing of IL-1β after SCI. These findings indicate that exosome-mediated siRNA delivery may be a strong candidate to block inflammasome activation following CNS injury.This article is protected by copyright. All rights reserved.

Neuroinflammation plays a critical role in the pathogenesis of Alzheimer's disease (AD) and involves activation of the innate immune response via recognition of diverse stimuli by pattern recognition receptors (PRRs). The inflammatory inducers and precise innate signaling pathway contributing to AD pathology remain largely undefined. In the present study we analyzed expression levels of innate immune proteins in temporal and occipital cortices from preclinical (no cognitive impairment, NCI, N = 22) to mild cognitive impairment (MCI, N = 20) associated with AD pathology (N = 20) and AD patients (N = 23). We found that retinoic acid-inducible gene-I (RIG-1) is significantly elevated in the temporal cortex and plasma in patients with MCI. In addition, primary human astrocytes stimulated with the RIG-1 ligand 5[prime]ppp RNA showed increased expression of amyloid precursor protein (APP) and amyloid-beta (Abeta), supporting the idea that RIG-1 is involved in the pathology of MCI associated with early progression to AD. These findings suggest that RIG-1 may play a critical role in incipient AD.

Inflammasomes that activate caspase-1 govern the innate immune inflammatory response. Whether hair loss associated with androgenetic alopecia (AGA) involves caspase-1 activation is not known. Immunohistochemical staining for caspase-1 was performed on scalp tissue sections, and protein lysates were analyzed from individuals with AGA (no treatment), and individuals with AGA taking finasteride with apparent hair growth, individuals with AGA taking finasteride without noted hair growth, and normal controls. In vitro studies of human keratinocytes were conducted to establish effects of finasteride, dihydrotestosterone (DHT), and testosterone on caspase-1 levels using immunoblot analysis. Caspase-1 is expressed in normal human adult epidermal keratinocytes. Caspase-1 expression is greater in men with AGA. In contrast, in men taking finasteride, caspase-1 levels were lower and were similar to those in normal controls. In vitro studies showed that keratinocytes treated with finasteride in combination with testosterone or DHT resulted in a significant decrease in caspase-1 expression. In vivo and in vitro finasteride treatment resulted in lower caspase-1 expression, supporting the idea that androgens influence innate immunity involved in the hair cycle in AGA. These findings may provide a basis for development of novel treatments for inflammatory skin and hair diseases.