Traumatic brain injury (TBI) represents a major cause of disability and death worldwide with sustained neuroinflammation and autophagy dysfunction contributing to the cellular damage. Stimulator of ...interferon genes (STING)-induced type-I interferon (IFN) signalling is known to be essential in mounting the innate immune response against infections and cell injury in the periphery, but its role in the CNS remains unclear. We previously identified the type-I IFN pathway as a key mediator of neuroinflammation and neuronal cell death in TBI. However, the modulation of the type-I IFN and neuroinflammatory responses by STING and its contribution to autophagy and neuronal cell death after TBI has not been explored.
C57BL/6J wild-type (WT) and STING
mice (8-10-week-old males) were subjected to controlled cortical impact (CCI) surgery and brains analysed by QPCR, Western blot and immunohistochemical analyses at 2 h or 24 h. STING expression was also analysed by QPCR in post-mortem human brain samples.
A significant upregulation in STING expression was identified in late trauma human brain samples that was confirmed in wild-type mice at 2 h and 24 h after CCI. This correlated with an elevated pro-inflammatory cytokine profile with increased TNF-α, IL-6, IL-1β and type-I IFN (IFN-α and IFN-β) levels. This expression was suppressed in the STING
mice with a smaller lesion volume in the knockout animals at 24 h post CCI. Wild-type mice also displayed increased levels of autophagy markers, LC3-II, p62 and LAMP2 after TBI; however, STING
mice showed reduced LAMP2 expression suggesting a role for STING in driving dysfunctional autophagy after TBI.
Our data implicates a detrimental role for STING in mediating the TBI-induced neuroinflammatory response and autophagy dysfunction, potentially identifying a new therapeutic target for reducing cellular damage in TBI.
Type‐1 interferons (IFNs) are pleiotropic cytokines with a critical role in the initiation and regulation of the pro‐inflammatory response. However, the contribution of the type‐1 IFNs to CNS ...disorders, specifically chronic neuropathologies such as Parkinson's disease is still unknown. Here, we report increased type‐1 IFN signaling in both post mortem human Parkinson's disease samples and in the 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) mouse model. In response to MPTP, mice lacking the type‐1 IFN receptor (IFNAR1−/−) displayed decreased type‐1 IFN signaling, an attenuated pro‐inflammatory response and reduced loss of dopaminergic neurons. The neuroprotective potential of targeting the type‐1 IFN pathway was confirmed by reduced neuroinflammation and DA cell death in mice treated with a blocking monoclonal IFNAR1 (MAR‐1) antibody. The MPTP/MAR‐1 treated mice also displayed increased striatal dopamine levels and improved behavioural outcomes compared to their MPTP/IgG controls. These data, implicate for the first time, a deleterious role for the type‐1 IFNs as key modulators of the early neuroinflammatory response and therefore the neuronal cell death in Parkinson's disease. GLIA 2016;64:1590–1604
Main Points
Type‐1 IFNs are upregulated in human post‐mortem Parkinson's disease brains and in the MPTP mouse model.
Genetically or therapeutically targeting type‐1 IFN signaling attenuates neuroinflammation and dopaminergic cell death induced by MPTP.
The chemokine CCL2 (also known as monocyte chemoattractant protein-1, or MCP-1) is upregulated in patients and rodent models of traumatic brain injury (TBI), contributing to post-traumatic ...neuroinflammation and degeneration by directing the infiltration of blood-derived macrophages into the injured brain. Our laboratory has previously reported that Ccl2-/- mice show reduced macrophage accumulation and tissue damage, corresponding to improved motor recovery, following experimental TBI. Surprisingly, Ccl2-deficient mice also exhibited delayed but exacerbated secretion of key proinflammatory cytokines in the injured cortex. Thus we sought to further characterise CCL2's potential ability to modulate immunoactivation of astrocytes in vitro.
Primary astrocytes were isolated from neonatal wild-type and Ccl2-deficient mice. Established astrocyte cultures were stimulated with various concentrations of lipopolysaccharide (LPS) and interleukin (IL)-1β for up to 24 hours. Separate experiments involved pre-incubation with mouse recombinant (r)CCL2 prior to IL-1β stimulation in wild-type cells. Following stimulation, cytokine secretion was measured in culture supernatant by immunoassays, whilst cytokine gene expression was quantified by real-time reverse transcriptase polymerase chain reaction.
LPS (0.1-100 μg/ml; 8 h) induced the significantly greater secretion of five key cytokines and chemokines in Ccl2-/- astrocytes compared to wild-type cells. Consistently, IL-6 mRNA levels were 2-fold higher in Ccl2-deficient cells. IL-1β (10 and 50 ng/ml; 2-24 h) also resulted in exacerbated IL-6 production from Ccl2-/- cultures. Despite this, treatment of wild-type cultures with rCCL2 alone (50-500 ng/ml) did not induce cytokine/chemokine production by astrocytes. However, pre-incubation of wild-type astrocytes with rCCL2 (250 ng/ml, 12 h) prior to stimulation with IL-1β (10 ng/ml, 8 h) significantly reduced IL-6 protein and gene expression.
Our data indicate that astrocytes are likely responsible for the exacerbated cytokine response seen in vivo post-injury in the absence of CCL2. Furthermore, evidence that CCL2 inhibits cytokine production by astrocytes following IL-1β stimulation, suggests a novel, immunomodulatory role for this chemokine in acute neuroinflammation. Further investigation is required to determine the physiological relevance of this phenomenon, which may have implications for therapeutics targeting CCL2-mediated leukocyte infiltration following TBI.
The atypical chemokine receptor ACKR2 promotes resolution of acute inflammation by operating as a scavenger receptor for inflammatory CC chemokines in several experimental models of inflammatory ...disorders, however its role in the brain remains unclear. Based on our previous reports of increased expression of inflammatory chemokines and their corresponding receptors following traumatic brain injury (TBI), we hypothesised that ACKR2 modulates neuroinflammation following brain trauma and that its deletion exacerbates cellular inflammation and chemokine production. We demonstrate increased CCL2 and ACKR2 mRNA expression in post-mortem human brain, whereby ACKR2 mRNA levels correlated with later times post-TBI. This data is consistent with the transient upregulation of ACKR2 observed in mouse brain after closed head injury (CHI). As compared to WT animals, ACKR2-/- mice showed a higher mortality rate after CHI, while the neurological outcome in surviving mice was similar. At day 1 post-injury, ACKR2-/- mice displayed aggravated lesion volume and no differences in CCL2 expression and macrophage recruitment relative to WT mice. Reciprocal regulation of ACKR2 and CCL2 expression was explored in cultured astrocytes, which are recognized as the major source of CCL2 and also express ACKR2. ACKR2 mRNA increased as early as 2 hours after an inflammatory challenge in WT astrocytes. As expected, CCL2 expression also dramatically increased at 4 hours in WT astrocytes but was significantly lower in ACKR2-/- astrocytes, possibly indicating a co-regulation of CCL2 and ACKR2 in these cells. Conversely, in vivo, CCL2 mRNA/protein levels were increased similarly in ACKR2-/- and WT brains at 4 and 12 hours after CHI, in line with the lack of differences in cerebral macrophage recruitment and neurological recovery. In conclusion, ACKR2 is induced after TBI and has a significant impact on mortality and lesion development acutely following CHI, while its role in chemokine expression, macrophage activation, brain pathology, and neurological recovery at later time-points is minor. Concordant to evidence in multiple sclerosis experimental models, our data corroborate a distinct role for ACKR2 in cerebral inflammatory processes compared to its reported functions in peripheral tissues.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recent investigation of the intestine following ischemia and reperfusion (I/R) has revealed that nitric oxide synthase (NOS) neurons are more strongly affected than other neuron types. This implies ...that NO originating from NOS neurons contributes to neuronal damage. However, there is also evidence of the neuroprotective effects of NO. In this study, we compared the effects of I/R on the intestines of neuronal NOS knockout (nNOS−/−) mice and wild-type mice. I/R caused histological damage to the mucosa and muscle and infiltration of neutrophils into the external muscle layers. Damage to the mucosa and muscle was more severe and greater infiltration by neutrophils occurred in the first 24 h in nNOS−/− mice. Immunohistochemistry for the contractile protein, α-smooth muscle actin, was used to evaluate muscle damage. Smooth muscle actin occurred in the majority of smooth muscle cells in the external musculature of normal mice but was absent from most cells and was reduced in the cytoplasm of other cells following I/R. The loss was greater in nNOS−/− mice. Basal contractile activity of the longitudinal muscle and contractile responses to nerve stimulation or a muscarinic agonist were reduced in regions subjected to I/R and the effects were greater in nNOS−/− mice. Reductions in responsiveness also occurred in regions of operated mice not subjected to I/R. This is attributed to post-operative ileus that is not significantly affected by knockout of nNOS. The results indicate that deleterious effects are greater in regions subjected to I/R in mice lacking nNOS compared with normal mice, implying that NO produced by nNOS has protective effects that outweigh any damaging effect of this free radical produced by enteric neurons.
Lysophosphatidic acid (LPA) is involved in physiological and pathological states, including in neural development and inflammation. We assessed the expression pattern of the LPA receptors 1-3 and of ...LPA-producing enzyme autotaxin in post-mortem human brain tissue, both in normal individuals and in individuals who died following traumatic brain injury. We found that LPA receptors and autotaxin are weakly expressed in the normal control adult brain. Quantitative PCR for the LPA receptors and autotaxin mRNA showed an increase of LPAR
2
and a decrease of autotaxin mRNA expression in the cortex following brain injury. Immunohistochemical analysis showed that LPAR
1
colocalized with astrocytes and that LPAR
2
is present on the ependymal cells lining the lateral ventricle in the brain samples from individuals who died following severe head injury. This work shows for the first time that key components of the LPA pathway are modulated following TBI in humans.
Abstract Batten disease (neuronal ceroid lipofuscinoses, NCLs) are a group of inherited childhood diseases that result in severe brain atrophy, blindness and seizures, leading to premature death. To ...date, eight different genes have been identified, each associated with a different form. Linkage analysis indicated a CLN5 form in a colony of affected New Zealand Borderdale sheep. Sequencing studies established the disease-causing mutation to be a substitution at a consensus splice site (c.571 + 1G > A), leading to the excision of exon 3 and a truncated putative protein. A molecular diagnostic test has been developed based on the excision of exon 3. Sequence alignments support the gene product being a soluble lysosomal protein. Western blotting of isolated storage bodies indicates the specific storage of subunit c of mitochondrial ATP synthase. This flock is being expanded as a large animal model for mechanistic studies and trial therapies.
Little is known about the molecular events following severe traumatic brain injury (TBI) in humans and to date there are no efficient therapies for the treatment of patients. In this study, the first ...of its kind in human tissue, a total of 21 post mortem trauma brain samples were analyzed. The inflammatory response within the brain tissue was explored by measuring the expression of various inflammatory cytokines at the mRNA and protein levels. These mediators were interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF). This study shows for the first time in human brain tissue that 1) pro-inflammatory mediator protein levels are significantly increased in situ following acute brain injury while anti-inflammatory cytokines protein levels remain unchanged; 2) the cerebral inflammatory response begins within minutes of acute TBI, much earlier than previously thought; 3) IL-6, IL-8, TNF-alpha, and IL-1beta mRNA levels are significantly increased following injury; 4) the rise in cytokine protein level coincides with increased levels of their mRNAs suggesting that traumatic injury elicits an immediate cerebral inflammatory response. Altogether these data confirm and extend previous observations on the release of cytokines in the cerebrospinal fluid of severe TBI patients. Finally, this study highlights the need to characterize the cell source of cytokines and elucidate their mode of action.
The transforming growth factor-betas (TGF-βs) regulate the induction of dopaminergic neurons and are elevated in the CSF of Parkinson's patients. We report here that mice with TGF-β2 ...haploinsufficiency (TGF-β2
+/−) have subclinical defects in the dopaminergic neurons of their substantia nigra pars compacta. At 6 weeks of age, the TGF-β2
+/− mice had 12% fewer dopaminergic neurons than wild-type littermates. No additional loss of neurons occurred during the next 5 months, although striatal dopamine declined to 70% of normal. The level of 3,4-dihydroxphenylacetic acid was normal in the TGF-β2
+/− mice, indicating that a compensatory mechanism maintains dopamine stimulation of their striatum. The TGF-β2
+/− mice had normal sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tretrahydropyridine, despite having reduced levels of monoamine oxidase-B. These results raise the possibility that people with naturally low levels of TGF-β2 may have less functional reserve in their nigrostriatal pathway, causing them to be at increased risk of developing Parkinson disease.
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