Early monocyte modulation by the non-erythropoietic peptide ARA 290 decelerates AD-like pathology progression.

Alzheimer's disease (AD) pathology is characterized by amyloid-ß (Aß) deposition and tau hyper-phosphorylation, accompanied by a progressive cognitive decline. Monocytes have been recently shown to play a major role in modulating Aß pathology, and thereby have been pointed as potential therapeutic targets. However, the main challenge remains in identifying clinically relevant interventions that could modulate monocyte immune functions in absence of undesired off-target effects. Erythropoietin (EPO), a key regulator of erythrocyte production, has been shown to possess immunomodulatory potential and to provide beneficial effects in preclinical models of AD. However, the transition to use recombinant human EPO in clinical trials was hindered by unwanted erythropoietic effects that could lead to thrombosis. Here, we used a recently identified non-erythropoietic analogue of EPO, ARA 290, to evaluate its therapeutic potential in AD therapy. We first evaluated the effects of early systemic ARA 290 administration on AD-like pathology in an early-onset model, represented by young APP/PS1 mice. Our findings indicate that ARA 290 early treatment decelerated Aß pathology progression in APP/PS1 mice while improving cognitive functions. ARA 290 potently increased the levels of total monocytes by specifically stimulating the generation of Ly6CLow patrolling subset, which are implicated in clearing Aß from the cerebral vasculature, and subsequently reducing overall Aß burden in the brain. Moreover, ARA 290 increased the levels of monocyte progenitors in the bone marrow. Using chimeric APP/PS1 mice in which Ly6CLow patrolling subset are selectively depleted, ARA 290 was inefficient in attenuating Aß pathology and ameliorating cognitive functions in young animals. Interestingly, ARA 290 effects were compromised when delivered in a late-onset model, represented by aged APP1/PS1. In aged APP/PS1 mice in which AD-like pathology is at advanced stages, ARA 290 failed to reverse Aß pathology and to increase the levels of circulating monocytes. Our study suggests that ARA 290 early systemic treatment could prevent AD-like progression via modulation of monocyte functions by specifically increasing the ratio of patrolling monocytes.

Muramyl dipeptide-mediated immunomodulation on monocyte subsets exerts therapeutic effects in a mouse model of Alzheimer's disease.

BACKGROUND: Muramyl dipeptide (MDP) is a component derived from minimal peptidoglycan motif from bacteria, and it is a ligand for the NOD2 receptor. Peripheral administration of MDP converts Ly6Chigh into Ly6Clow monocytes. Previously, we have shown that Ly6Clow monocytes play crucial roles in the pathology of a mouse model of Alzheimer's disease (AD). However, medications with mild immunomodulatory effects that solely target specific monocyte subsets, without triggering microglial activation, are rare. METHODS: Three months old APPswe/PS1 transgenic male mice and age-matched C57BL/6 J mice were used for high frequency (2 times/week) over 6 months and low frequency (once a week) over 3 months of intraperitoneally MDP (10 mg/kg) administrations. Flow cytometry analysis of monocyte subsets in blood, and behavioral and postmortem analyses were performed. RESULTS: Memory tests showed mild to a strong improvement in memory function, increased expression levels of postsynaptic density protein 95 (PSD95), and low-density lipoprotein receptor-related protein 1 (LRP1), which are involved in synaptic plasticity and amyloid-beta (Aß) elimination, respectively. In addition, we found monocyte chemoattractant protein-1(MCP-1) levels significantly increased, whereas intercellular adhesion molecule-1(ICAM-1) significantly decreased, and microglial marker (Iba1) did not change in the treatment group compared to the control. In parallel, we discovered elevated cyclooxygenase-2 (COX2) expression levels in the treated group, which might be a positive factor for synaptic activity. CONCLUSIONS: Our results demonstrate that MDP is beneficial in both the early phase and, to some extent, later phases of the pathology in the mouse model of AD. These data open the way for potential MDP-based medications for AD.

Role of the chemokine receptors CCR2 and CX3CR1 in an experimental model of thrombotic stroke.

Stroke is the second cause of mortality worldwide and occurs following the interruption of cerebral blood circulation by cerebral vessel burst or subsequent to a local thrombus formation. Ischemic lesion triggers an important inflammatory response, characterized by massive infiltration of leukocytes, activation of glial cells and neurovascular reorganization. Chemokines and their receptors, such as CCR2 and CX3CR1, play an important role in leukocyte recruitment in the damaged area. Mice genetically depleted for the two receptors CCR2 and CX3CR1 underwent focal cerebral ischemia, based on the topical application of ferric chloride to truncate the distal middle cerebral artery. The infarct, limited only to the cortical area, remained stable in WT mice, while it is reduced overtime in the transgenic mice. Moreover, we did not observe any significant changes in the level of the inflammatory response in the infarcted areas while immune cell infiltration and neurovascularization are modulated according to genotype. Our results show that the genetic deletion of both CCR2 and CX3CR1 receptors has neuroprotective effects in response to a cerebral permanent ischemia. This study underlines a key role of CCR2- and CX3CR1-expressing immune cells in the neuropathology associated with ischemic injuries.

Toll-like receptor 4 stimulation with the detoxified ligand monophosphoryl lipid A improves Alzheimer's disease-related pathology.

Alzheimer's disease (AD) is the most common cause of dementia worldwide. The pathogenesis of this neurodegenerative disease, currently without curative treatment, is associated with the accumulation of amyloid ß (Aß) in brain parenchyma and cerebral vasculature. AD patients are unable to clear this toxic peptide, leading to Aß accumulation in their brains and, presumably, the pathology associated with this devastating disease. Compounds that stimulate the immune system to clear Aß may therefore have great therapeutic potential in AD patients. Monophosphoryl lipid A (MPL) is an LPS-derived Toll-like receptor 4 agonist that exhibits unique immunomodulatory properties at doses that are nonpyrogenic. We show here that repeated systemic injections of MPL, but not LPS, significantly improved AD-related pathology in APP(swe)/PS1 mice. MPL treatment led to a significant reduction in Aß load in the brain of these mice, as well as enhanced cognitive function. MPL induced a potent phagocytic response by microglia while triggering a moderate inflammatory reaction. Our data suggest that the Toll-like receptor 4 agonist MPL may be a treatment for AD.

Herpes Simplex Virus 1 Infection Does Not Increase Amyloid-ß Pathology in APP/PS1 Mice.

Using APP/PS1 mice that overproduce amyloid-ß (Aß) peptides, we investigated whether intranasal infection with a neurovirulent clinical strain of herpes simplex virus 1 (HSV-1) before Aß deposition could accelerate or increase Alzheimer's disease-like pathology. After HSV-1 infection, APP/PS1 mice presented a similar disease as wild type animals based on body weight changes, clinical symptoms, and survival rates. The number and volume of Aß plaques, the number of microglia, and the percentages of circulating monocyte subsets were similar in APP/PS1 mice infected or not with HSV-1. Thus, intranasal infection with HSV-1 does not alter Aß pathology in this mouse model.

Muramyl Dipeptide Administration Delays Alzheimer's Disease Physiopathology via NOD2 Receptors.

Alzheimer's disease (AD) is the most common form of dementia in the world. The prevalence is steadily increasing due to an aging population and the lack of effective treatments. However, modulation of innate immune cells is a new therapeutic avenue, which is quite effective at delaying disease onset and improving cognitive decline. METHODS: We studied the effect of the NOD2 receptor ligand muramyl dipeptide (MDP) on the modulation of the innate immune cells, namely patrolling monocytes and microglia. We administrated MDP once a week for 3 months in an APPswe/PS1 mouse model in both sexes. We started the treatment at 3 months before plaque formation and evaluated its effects at 6 months. RESULTS: We showed that the MDP injections delay cognitive decline in both sexes via different mechanisms and protect the blood brain barrier (BBB). In males, MDP triggers the sink effect from the BBB, leading to a diminution in the amyloid load in the brain. This phenomenon is underlined by the increased expression of phagocytosis markers such as TREM2, CD68, and LAMP2 and a higher expression of ABCB1 and LRP1 at the BBB level. The beneficial effect seems more restricted to the brain in females treated with MDP, where microglia surround amyloid plaques and prevent the spreading of amyloid peptides. This phenomenon is also associated with an increase in TREM2 expression. Interestingly, both treated groups showed an increase in Arg-1 expression compared to controls, suggesting that MDP modulates the inflammatory response. CONCLUSION: These results indicate that stimulation of the NOD2 receptor in innate immune cells is a promising therapeutic avenue with potential different mechanisms between males and females.

Selective Immunomodulatory and Neuroprotective Effects of a NOD2 Receptor Agonist on Mouse Models of Multiple Sclerosis.

The significance of monocytes has been demonstrated in multiple sclerosis (MS). One of the therapeutic challenges is developing medications that induce mild immunomodulation that is solely targeting specific monocyte subsets without affecting microglia. Muramyl dipeptide (MDP) activates the NOD2 receptor, and systemic MDP administrations convert Ly6Chigh into Ly6Clow monocytes. Here, we report selective immunomodulatory and therapeutic effects of MDP on cuprizone and experimental autoimmune encephalomyelitis (EAE) mouse models of MS. MDP treatment exerted various therapeutic effects in EAE, including delaying EAE onset and reducing infiltration of leukocytes into the CNS before EAE onset. Of great interest is the robust beneficial effect of the MDP treatment in mice already developing the disease several days after EAE onset. Finally, we found that the NOD2 receptor plays a critical role in MDP-mediated EAE resistance. Our results demonstrate that MDP is beneficial in both early and progressive phases of EAE. Based on these results, and upon comprehensive basic and clinical research, we anticipate developing NOD2 agonist-based medications for MS in the future.

Role of Macrophage Colony-Stimulating Factor Receptor on the Proliferation and Survival of Microglia Following Systemic Nerve and Cuprizone-Induced Injuries.

Microglia are the innate immune cells of the CNS and their proliferation, activation, and survival have previously been shown to be highly dependent on macrophage colony-stimulating factor receptor (CSF1R). Here we investigated the impact of the receptor in such processes using two different models of nerve injuries, namely hypoglossal axotomy and cuprizone-induced demyelination. Both models are associated with a robust microgliosis. The role of CSF1R was investigated using the gene deletion Cre/Lox system, which allows the conditional knock-out following tamoxifen administration. We found that after 5 weeks of cuprizone diet that CSF1R suppression caused a significant impairment of microglia function. A reduced microgliosis was detected in the corpus collosum of CSF1R knock-out mice compared to controls. In contrast to cuprizone model, the overall number of Iba1 cells was unchanged at all the times evaluated following hypoglossal axotomy in WT and cKO conditions. After nerve lesion, a tremendous proliferation was noticed in the ipsilateral hypoglossal nucleus to a similar level in both knock-out and wild-type groups. We also observed infiltration of bone-marrow derived cells specifically in CSF1R-deficient mice, these cells tend to compensate the CSF1R signaling pathway suppression in resident microglia. Taking together our results suggest a different role of CSF1R in microglia depending on the model. In the pathologic context of cuprizone-induced demyelination CSF1R signaling pathway is essential to trigger proliferation and survival of microglia, while this is not the case in a model of systemic nerve injury. M-CSF/CSF1R is consequently not the unique system involved in microgliosis following nerve damages.

Toll-like receptor 2 acts as a natural innate immune receptor to clear amyloid beta 1-42 and delay the cognitive decline in a mouse model of Alzheimer's disease.

Microglia are the immune cells of the brain, they are activated in the brain of Alzheimer's disease (AD) patients and mouse models of AD, and they express the innate immune receptor toll-like receptor 2 (TLR2). The present study investigated role of this receptor in the progression of AD-like pathologies. Here we show that amyloid beta (A beta) stimulates TLR2 expression in a small proportion of microglia. We then generated triple transgenic mice that are deficient in TLR2 from mice that harbor a mutant human presenelin 1 and a chimeric mouse/human amyloid precursor protein (APP) genes. TLR2 deficiency accelerated spatial and contextual memory impairments, which correlated with increased levels of A beta(1-42) and transforming growth factor beta1 in the brain. NMDA receptors 1 and 2A expression levels were also lower in the hippocampus of APP-TLR2(-/-) mice. Gene therapy in cells of the bone marrow using lentivirus constructs expressing TLR2 rescued the cognitive impairment of APP-TLR2(-/-) mice. Indeed, lenti-green fluorescent protein/TLR2 treatment had beneficial effects by restoring the memory consolidation process disrupted by TLR2 deficiency in APP mice. These data suggest that TLR2 acts as an endogenous receptor for the clearance of toxic A beta by bone-marrow-derived immune cells. The cognitive decline is markedly accelerated in a context of TLR2 deficiency. Upregulating this innate immune receptor may then be considered as a potential new powerful therapeutic approach for AD.

A comprehensive joint replacement program for total knee arthroplasty: a descriptive study.

BACKGROUND: Total knee arthroplasty (TKA) is a commonly performed surgical procedure in the US. It is important to have a comprehensive inpatient TKA program which maximizes outcomes while minimizing adverse events. The purpose of this study was to describe a TKA program - the Joint Replacement Program (JRP) - and report post-surgical outcomes. METHODS: 74 candidates for a primary TKA were enrolled in the JRP. The JRP was designed to minimize complications and optimize patient-centered outcomes using a team approach including the patient, patient's family, and a multidisciplinary team of health professionals. The JRP consisted of a pre-operative class, standard pathways for medical care, comprehensive peri-operative pain management, aggressive physical therapy (PT), and proactive discharge planning. Measures included functional tests, knee range of motion (ROM), and medical record abstraction of patient demographics, length of stay, discharge disposition, and complications over a 6-month follow-up period. RESULTS: All patients achieved medical criteria for hospital discharge. The patients achieved the knee flexion ROM goal of 90 degrees (91.7 +/- 5.4 degrees ), but did not achieve the knee extension ROM goal of 0 degrees (2.4 +/- 2.6 degrees ). The length of hospital stay was two days for 53% of the patients, with 39% and 7% discharged in three and four days, respectively. All but three patients were discharged home with functional independence. 68% of these received outpatient physical therapy compared with 32% who received home physical therapy immediately after discharge. Two patients (< 3%) had medical complications during the inpatient hospital stay, and 9 patients (12%) had complications during the 6-month follow-up period. CONCLUSION: The comprehensive JRP for TKA was associated with satisfactory clinical outcomes, short lengths of stay, a high percentage of patients discharged home with outpatient PT, and minimal complications. This JRP may represent an efficient, effective and safe protocol for providing care after a TKA.

Bone Marrow Chimeras to Study Neuroinflammation.

Bone marrow transplantation is the standard of care for a host of diseases such as leukemia and multiple myeloma, as well as genetically inherited metabolic diseases affecting the central nervous system. In mouse models, bone marrow transplantation has proven a valuable tool for understanding the hematopoietic system and the homing of hematopoietic cells to their target organs. Many techniques have been developed to create chimeric mice, animals with a hematopoietic system derived from a genetic background that differs from the rest of the body. Current genetic tools allow for virtually limitless possibilities in the choice of donor mice. This protocol describes methods of bone marrow transplantation in mouse models for studies of the brain under basal and pathological conditions. Specific points to be addressed include the preparation of recipient mice by irradiation or chemotherapy; the choice, isolation, and injection of donor cells; and analytical methods such as fluorescence-activated cell sorting and immunostaining. © 2018 by John Wiley & Sons, Inc.

mCSF-Induced Microglial Activation Prevents Myelin Loss and Promotes Its Repair in a Mouse Model of Multiple Sclerosis.

A pathological hallmark of multiple sclerosis (MS) is myelin loss in brain white matter accompanied by compromised remyelination. Demyelinated lesions are deeply associated with oligodendrocyte apoptosis and a robust inflammatory response. Although various studies point towards a noxious role of inflammation in MS, others emphasize a positive role for the innate immune cells in disease progression. A cytokine well-known to stimulate cell survival, proliferation and differentiation of myeloid cells, macrophage colony-stimulating factor (mCSF), was administered to mice during a 5 week-long cuprizone diet. Treated mice exhibited reduced myelin loss during the demyelination phase, together with an increased number of microglia and oligodendrocyte precursor cells in lesion sites. Tamoxifen-induced conditional deletion of the mCSF receptor in microglia from cuprizone-fed mice caused aberrant myelin debris accumulation in the corpus callosum and reduced microglial phagocytic response. mCSF therefore plays a key role in stimulating myelin clearance by the brain innate immune cells, which is a prerequisite for proper remyelination and myelin repair processes.

Triggering of NOD2 Receptor Converts Inflammatory Ly6Chigh into Ly6Clow Monocytes with Patrolling Properties.

The signals that regulate the fate of circulating monocytes remain unknown. In the present study, we demonstrate that triggering of the NOD2 receptor by muramyl dipeptide (MDP) converts inflammatory Ly6Chigh monocytes into patrolling Ly6Clow monocytes. Administration of MDP to Nr4a1-/- mice, which lack Ly6Clow monocytes, or to Ly6Clow-depleted mice led to the emergence of blood-patrolling monocytes with a profile similar to that of Ly6Clow monocytes, including high expression of CX3CR1 and LFA1. Using intravital microscopy in animal models of inflammatory diseases, we also found that converted Ly6Chigh monocytes patrol the endothelium of blood vessels and that their presence contributes to a reduction in the inflammatory response following MDP injection. Our results demonstrate that NOD2 contributes to the regulation of blood monocytes and suggest that it could be therapeutically targeted to treat inflammatory diseases.

A Comparative Study to Determine Functional and Clinical Outcome Differences Between Patients Receiving Outpatient Direct Physical Therapy Versus Home Physical Therapy Followed by Outpatient Physical Therapy After Total Knee Arthroplasty.

BACKGROUND: Rehabilitation outcomes for patients with total knee arthroplasty (TKA) after hospital discharge are not well understood. PURPOSE: The purpose of this retrospective cohort study was to describe outpatient physical therapy (PT) after TKA and compare short-term (2 months) functional and clinical outcomes of patients following TKA who were discharged from the hospital to home and received (a) outpatient PT immediately (OP) or (b) home health PT before outpatient PT (HH). METHODS: The medical records of 109 men and women postoperative TKA discharged home were abstracted for the 6-minute walk test (6MWT), Knee Osteoarthritis and Outcome Score (KOOS), and knee range of motion (ROM) preoperatively and after discharge from all postoperative PT. Patients received outpatient clinic-based PT immediately after discharge from the hospital (OP) (n = 87) or home health PT before continued rehabilitation in an outpatient setting (HH) (n = 22). RESULTS: Despite demographic differences between OP and HH preoperatively, adjusted models revealed no significant differences among KOOS, 6MWT, or knee ROM between OP and HH upon completion of postoperative PT. Patients in OP completed PT on average 20 days sooner (p = .0007), although the amount of time in outpatient PT (p = .55) and the number of outpatient PT visits (p = .68) were similar between groups. CONCLUSION: Gains in function were achieved by patients in OP and HH independent of the postoperative PT setting, although OP achieved gains sooner.

Inefficient clearance of myelin debris by microglia impairs remyelinating processes.

An imbalance between remyelinating and demyelinating rates underlies degenerative processes in demyelinating diseases such as multiple sclerosis. An optimal therapeutic strategy would be to stimulate remyelination while limiting demyelination. Although accumulation of myelin debris impairs remyelination, the mechanisms regulating the clearance of such debris by mononuclear phagocytic cells are poorly understood. We demonstrate that after cuprizone intoxication, CCR2-dependent infiltration of mouse bone marrow-derived cells is abundant in demyelinating areas, but that these cells do not impact demyelination. However, in CX3CR1-deficient mice, the clearance of myelin debris by microglia was blocked greatly, affecting the integrity of the axon and myelin sheaths and thus preventing proper remyelination. These results highlight the crucial role played by CX3CR1 in myelin removal and show that there can be no efficient remyelination after a primary demyelinating insult if myelin clearance by microglia is impaired.

Real-time in vivo imaging reveals the ability of monocytes to clear vascular amyloid beta.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid beta (Aß) that is assumed to result from impaired elimination of this neurotoxic peptide. Most patients with AD also exhibit cerebral amyloid angiopathy, which consists of Aß deposition within the cerebral vasculature. The contribution of monocytes in AD has so far been limited to macrophage precursors. In this study, we aimed to investigate whether circulating monocytes could play a role in the elimination of Aß. With live intravital two-photon microscopy, we demonstrate that patrolling monocytes are attracted to and crawl onto the luminal walls of Aß-positive veins, but not on Aß-positive arteries or Aß-free blood vessels. Additionally, we report the presence of crawling monocytes carrying Aß in veins and their ability to circulate back into the bloodstream. Selective removal of Ly6C(lo) monocytes in APP/PS1 mice induced a significant increase of Aß load in the cortex and hippocampus. These data uncover the ability of Ly6C(lo) monocytes to naturally target and eliminate Aß within the lumen of veins and constitute a potential therapeutic target in AD.

Mild chronic cerebral hypoperfusion induces neurovascular dysfunction, triggering peripheral beta-amyloid brain entry and aggregation.

BACKGROUND: The Blood-brain barrier (BBB) controls brain supply with oxygen and nutrients, and protects the brain from toxic metabolites, such as beta-amyloid (Aß) peptides. The neurovascular unit (NVU) couples vascular and neuronal functions by controlling BBB parameters based on brain needs. As such, NVU/BBB dysfunction, associated to irregularities in cerebral blood flow (CBF), has been proposed to contribute in the pathogenesis of Alzheimer's disease (AD), mainly by impairing cerebral Aß clearance. However, the spatiotemporal contribution of the NVU/BBB in the neurodegenerative cascades remains elusive. RESULTS: By using C57BL/6J mice subjected to right common carotid artery (rCCA) permanent ligation in order to induce mild chronic cerebral hypoperfusion, we show here that cerebral hypoperfusion induced NVU dysfunction by reducing ABCB1 protein expression in brain capillaries. ABCB1 reduction was mainly triggered by an enhanced Glycogen Synthase Kinase 3 (GSK3ß) activation, which decreased ß-catenin nuclear abundance. Moreover, cerebral hypoperfusion triggered early vascular deposition of peripherally applied human Aß1-42 peptides, which has shifted from highly vascular to the parenchyma 6 weeks later, forming small stable Aß deposits. Hypoperfusion induced a deregulation in glucose metabolism, as brain reperfusion, or the administration of a high dose of glucose, diminished GSK3ß activation, recuperated ß-catenin nuclear abundance, reestablished ABCB1 protein expression, and prevented Aß vascular early deposition. These results demonstrate that mild chronic cerebral hypoperfusion creates a metabolically deregulated microenvironment, thus triggering the brain entry and aggregation of peripherally applied human Aß1-42 peptides. CONCLUSION: Our study offers new insights on the initiation of the neurodegenerative cascades observed in AD, which could be valuable in developing adequate treatment strategies.