| Objective:Parkinson's disease(PD)is a common neurodegenerative disorder characterized by a dramatic loss of dopaminergic neurons in the substantia nigra(SN),which is a serious threat to the health of the elderly,and affects their quality of life.PD is a multi factorial disease,although the causes and mechanisms of the disease remain elusive,accumulating clinical and experimental evidences suggest that the inflammation of the CNS plays a critical role in the pathogenesis and progression of PD.The characteristic features of neuroinflammation include the activation of glial cells and of a series of inflammatory mediators including pro-inflammatory cytokines,chemokines,reactive oxygen species and nitric oxide.These elements are considered to be involved in the progression of neuronal degeneration.Despite the protection of the blood brain barrier(BBB),the CNS has been deeply affected by the periphery.It is well established that peripheral inflammation can give rise to neuroinflammation and is linked closely to the etiology and progress of neurodegenerative diseases.One classic study showed that a single intraperitoneal(i.p.)injection of lipopolysaccharide(LPS)resulted in a long lasting increase of pro-inflammatory cytokines and the delayed loss of dopaminergic neurons in the SN.A recent epidemiological study confirmed that there was a significant increase in the incidence of PD after the influenza.On the other hand,the peripheral immunity was considered to be suppressed by the long term use of nonsteroidal anti-inflammatory drugs,and the incidence of PD was decreased by 50%.These results strongly suggest that the neuroinflammation can be strongly influenced by the peripheral inflammation.Microglia,the resident immune cell and the major glial cell in the CNS,plays a crucial role in brain microenvironment homeostasis.Microglia is now recognized as the primary component of the brain immune system,lives with neurons in peace at resting state.Once activated,microglia can change its morphology,proliferate and up-regulate surface molecules,and initiate the inflammatory response by producing pro-inflammatory cytokines and neurotoxic mediators such as nitric oxide,prostaglandin E2,and reactive oxygen speciesn and by cross-talking with other glial cells,BBB cells and neurons.Some of the existing studies have revealed that excessive activation of microglia caused by inflammation may disrupt the microenvironment in the brain,and pose a threat to the survival of neurons,no doubt including dopaminergic neurons.Monocytes,as the promoter and organizer of the peripheral innate immune,play multiple roles in the immune system.One of our recent studies was to exhaust monocytes before peripheral inflammation,which weakened the inflammation that should have occurred.The results revealed the crucial role monocytes played in the formation and progression of peripheral inflammation.As time goes on,a growing body of research is focusing on the role monocytes play in neurological disorders.On the one hand,monocyte and microglia are considered to share the same origin of myeloid lineage,which has the similar antigens expression,function and regulating mechanisms.Because of this,many scholars deem that the behavior of monocytes can reflect the activity of microglia in the brain.On the other hand,mounting evidence reveal that monocytes could infiltrate into the CNS and develop into microglia,or construct a special group of macrophages in the brain.These findings imply that monocyte is crucial medium between the peripheral immune response and the neuroinflammation,its peripheral immune status is of great significance to the prognosis of the neuroinflammation.The initial purpose of inflammation is to protect the body,but everything goes too far,therefore,it is vital to restrict and suppress the immune response in time.In fact,the body has formed a set of strict mechanisms to limit and down-regulate the immune response in the process of evolution,one of the most important segments is the formation of immune tolerance.Immune tolerance refers to the refractoriness of immune system to certain stimulation that exist within the body continuously or repeatedly,accompanied by the decrease of pro-inflammatory cytokines levels,the increase of anti-inflammatory cytokines levels,inhibition of T cells activation,etc.It is a protective mechanism of the body in the face of continuous antigen threat or continuous strike,which avoids over-activation of immune cells and unnecessary by-stander injury.The endotoxin tolerance(ET)of monocytes can be induced by a low dose of endotoxin(LPS),which is considered as the most important component of immune tolerance.Rosenzweig et al.found that LPS preconditioning induced significant neuroprotection against middle cerebral artery occlusion syndrome(MCAO),and decreased infarct size by 40%.Moreover,the neuroprotective effects of LPS preconditioning have also been demonstrated in other studies,such as brain trauma,brain injury resulting from deep hypothermic circulatory arrest,etc.Peripheral inflammation can cause and aggravate inflammation in the CNS,and then,the abnormal activation of microglia induced by neuroinflammation is considered to be the causes to dopaminergic neurons loss and the occurrence of PD.Thus,it is significant to establish the neuroprotective effect by reducing the peripheral immune response.However,the related research is still insufficient.Considering that a low dose of LPS can induce ET in monocytes,meanwhile,monocyte is an important mediator between the brain and the periphery,here,we proposed that the immune activity of monocytes can be down-regulated by ET,then,microglia activation reduced by neuroinflammation will be inhibited and the inflammation in the CNS will be decreased,successively.So that more neurons can survive the innocent damage caused by neuroinflammation,and the neuroprotective effects of ET in monocytes can be seen obviously.Neuroinflammation plays a critical role in the onset and development of neurodegeneration disorders such as Parkinson's Disease.The immune activities of the central nervous system are profoundly affected by peripheral immune activities.Immune tolerance refers to the unresponsiveness of the immune system to continuous or repeated stimulation to avoid excessive inflammation and unnecessary by-stander injury in the face of continuous antigen threat.It has been proved that the immune tolerance could suppress the development of various peripheral inflammation related disease.However,the role of immune tolerance in neuroinflammation and neurodegenerative diseases was not clear.Methods:To investigate whether endotoxic tolerance of PBM could be induced by repeated low-dose LPS intraperitoneal injection,rats were given pre-treatment with repeated i.p.injections of LPS(0.3 mg/kg body weight/single/day)for 4 days.After the peripheral pre-treatment,rats were sacrificed for peripheral blood,and then,peripheral blood monocytes(PBM)were cultured with combined density gradient centrifugation and attachment culture method,then,100ng/ml LPS was given to PBM in order to mimic subsequent inflammation.To test the immunological competence of PBM,the levels of pro-inflammatory cytokines(TNF-? and IL-1?)in the supernatant were measured by enzyme-linked immunosorbent assay(ELISA),while also the expression of toll-like receptor 4(TLR4)in monocytes was measured by Western blot.Meanwhile,to verify that the pre-treatment with repeated low-dose LPS intraperitoneal injection was not sufficient to cause inflammation nor dopaminergic neuronal loss in the SN,we investigated the levels of pro-inflammatory cytokines(TNF-? and IL-1?)and the expression of Iba-1,besides,we carried out immunohistochemical staining of TH and stereological counting of TH-positive neurons in the nonintracranial injected SN of rats at 1,7,14,and 28 days post to the last peripheral LPS injection.To investigate whether striatal injection of LPS could mimic the neuroinflammation-induced PD model,we administered stereotactic injection of 15?g LPS into the right striatum.Then,we carried out immunohistochemical staining of TH and stereological counting of TH-positive neurons in the SN at 1,7,14,and 28 days post to the striatal LPS injection,respectively.Moreover,test for amphetamine-induced rotational behavior was performed at 28 days post to the striatal LPS injection to assess the behavioral impairment.To investigate whether peripheral immune tolerance preconditioning could relieve the subsequent neuroinflammation and exert neuroprotection on dopaminergic neurons,rats were given pre-treatment with repeated i.p.injections of 0.3 mg/kg LPS for 4 days to induce peripheral immune tolerance,then,we administered stereotactic injection of 15?g LPS into the right striatum to mimic the neuroinflammation-induced PD model.At 1,7,14,and 28 days post to the striatal LPS injection,firstly,the levels of pro-inflammatory cytokines(TNF-? and IL-1?)and anti-inflammatory cytokines(IL-10 and TGF-?)in the SN were measured by enzyme-linked immunosorbent assay(ELISA),the expression profiles of TNF-? and IL-1? in the right SN were measured by qRT-PCR,in order to evaluate the inflammation in the brain.Secondly,we observed morphological changes of microglia using immumohistochemical staining of Iba-1,and we measured the expression of ED-1 in the SN by immunofluorescence assay.The expression profiles of ED-1 in the right SN were detected by qRT-PCR to evaluate the microglial activation in brain.Finally,we carried out immunohistochemical staining of TH and stereological counting of TH-positive neurons in the SN at 1,7,14,and 28 days post to the striatal LPS injection,respectively.Moreover,test for amphetamine-induced rotational behavior was performed at 28 days post to the striatal LPS injection to assess the behavioral impairment.Results: 1.Repeated low-dose LPS intraperitoneal injection-induced endotoxic tolerance of PBM.2.Repeated low-dose LPS intraperitoneal injection was not sufficient to cause inflammation in the SN.3.Repeated low-dose LPS intraperitoneal injection was not sufficient to cause dopaminergic neuronal loss in the SN.4.Striatal LPS injection induced dopaminergic neuronal loss in the SN.5.Striatal LPS injection induced behavioral impairment of rats.6.Peripheral immune tolerance preconditioning decreased intracranial LPS injection-induced pro-inflammatory cytokines in the SN.7.Peripheral immune tolerance preconditioning did not alter intracranial LPS injection-induced anti-inflammatory cytokines in the SN.8.Peripheral immune tolerance preconditioning inhibited intracranial LPS injection-induced microglial activation in the SN.9.Peripheral immune tolerance pre-treated PD rats showed decreased intracranial LPS injection-induced dopaminergic neuronal loss in the SN.10.Peripheral immune tolerance pre-treated PD rats showed improved intracranial LPS injection-induced behavioral impairment.Conclusions:Our results showed that repeated low-dose LPS intraperitoneal injection-induced endotoxic tolerance of PBM.Striatal injection of 15?g LPS induced dopaminergic neuronal loss in the SN of rats,which well simulated the neuroinflammation-induced model of PD.Peripheral immune tolerance is an important regulatory mechanism for the immunological activity of PBM.Our results strongly indicated that peripheral immune tolerance attenuated subsequent neuroinflammation and provided neuroprotection from neuroinflammation-induced neurotoxicity,which will provide new therapeutic approaches for neurodegenerative diseases. |
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