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.

A central nervous system-restricted isoform of the interleukin-1 receptor accessory protein modulates neuronal responses to interleukin-1.

Interleukin-1 (IL-1) has multiple functions in both the periphery and the central nervous system (CNS) and is regulated at many levels. We identified an isoform of the IL-1 receptor (IL-1R) accessory protein (termed AcPb) that is expressed exclusively in the CNS. AcPb interacted with IL-1 and the IL-1R but was unable to mediate canonical IL-1 responses. AcPb expression, however, modulated neuronal gene expression in response to IL-1 treatment in vitro. Animals lacking AcPb demonstrated an intact peripheral IL-1 response and developed experimental autoimmune encephalomyelitis (EAE) similarly to wild-type mice. AcPb-deficient mice were instead more vulnerable to local inflammatory challenge in the CNS and suffered enhanced neuronal degeneration as compared to AcP-deficient or wild-type mice. These findings implicate AcPb as an additional component of the highly regulated IL-1 system and suggest that it may play a role in modulating CNS responses to IL-1 and the interplay between inflammation and neuronal survival.

Conditional genetic deletion of CSF1 receptor in microglia ameliorates the physiopathology of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia in the world. Microglia are the innate immune cells of CNS; their proliferation, activation, and survival in pathologic and healthy brain have previously been shown to be highly dependent on CSF1R. METHODS: Here, we investigate the impact of such receptor on AD etiology and microglia. We deleted CSF1R using Cre/Lox system; the knockout (KO) is restricted to microglia in the APP/PS1 mouse model. We induced the knockout at 3 months old, before plaque formation, and evaluated both 6- and 8-month-old groups of mice. RESULTS: Our findings demonstrated that CSF1R KO did not impair microglial survival and proliferation at 6 and 8 months of age in APP cKO compared to their littermate-control groups APPSwe/PS1. We have also shown that cognitive decline is delayed in CSF1R-deleted mice. Ameliorations of AD etiology are associated with a decrease in plaque volume in the cortex and hippocampus area. A compensating system seems to take place following the knockout, since TREM2/ß-Catenin and IL-34 expression are significantly increased. Such a compensatory mechanism may promote microglial survival and phagocytosis of Aß in the brain. CONCLUSIONS: Our results provide new insights on the role of CSF1R in microglia and how it interacts with the TREM2/ß-Catenin and IL-34 system to clear Aß and ameliorates the physiopathology of AD.

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.

Irradiation does not compromise or exacerbate the innate immune response in the brains of mice that were transplanted with bone marrow stem cells.

Microglia and invading macrophages play key roles in the brain immune response. The contributions of these two populations of cells in health and diseases have yet to be clearly established. The use of chimeric mice receiving bone marrow-derived stem cell grafts from green fluorescent protein (GFP)-expressing mice has provided an invaluable tool to distinguish between local and blood-derived monocytic populations. The validity of the method is questioned because of the possible immune alterations caused by the irradiation of the recipient mouse. In this experiment, we compared the brain expression of innate immune markers Toll-like receptor 2, interleukin-1 beta, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 in C57BL/6, GFP, and chimeric mice following an intracerebral injection of lipopolysaccharide. The endotoxin caused a marked transcriptional activation of all these innate immune genes in microglial cells across the ipsilateral side of injection. The expression patterns and signal intensity were similar in the brains of the three groups of mice. Consequently, the chimera technique is appropriate to study the role of infiltrating and resident immune cells in the brain without having immune compromised hosts. Disclosure of potential conflicts of interest is found at the end of this article.

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.

Streptococcus suis serotype 2, an important swine and human pathogen, induces strong systemic and cerebral inflammatory responses in a mouse model of infection.

Streptococcus suis, an important swine and human pathogen, causes septic shock and meningitis. The pathogenesis of both systemic and CNS infections caused by S. suis is poorly understood. A hematogenous model of infection in CD1 mice was developed to study the systemic release of cytokines during the septic shock phase and the proinflammatory events in the CNS associated with this pathogen. Using a liquid array system, high levels of systemic TNF-alpha, IL-6, IL-12, IFN-gamma, CCL2, CXCL1, and CCL5 were observed 24 h after infection and might be responsible for the sudden death of 20% of animals. Infected mice that survived the early sepsis later developed clinical signs of meningitis and exhibited lesions in the meninges and in numerous regions of the brain, such as the cortex, hippocampus, thalamus, hypothalamus, and corpus callosum. Bacterial Ags were found in association with microglia residing only in the affected zones. In situ hybridization combined with immunocytochemistry showed transcriptional activation of TLR2 and TLR3 as well as CD14, NF-kappaB, IL-1beta, CCL2, and TNF-alpha, mainly in myeloid cells located in affected cerebral structures. Early transcriptional activation of TLR2, CD14, and inflammatory cytokines in the choroid plexus and cells lining the brain endothelium suggests that these structures are potential entry sites for the bacteria into the CNS. Our data indicate an important role of the inflammatory response in the pathogenesis of S. suis infection in mice. This experimental model may be useful for studying the mechanisms underlying sepsis and meningitis during bacterial infection.

Genes involved in the balance between neuronal survival and death during inflammation.

Glucocorticoids are potent regulators of the innate immune response, and alteration in this inhibitory feedback has detrimental consequences for the neural tissue. This study profiled and investigated functionally candidate genes mediating this switch between cell survival and death during an acute inflammatory reaction subsequent to the absence of glucocorticoid signaling. Oligonucleotide microarray analysis revealed that following lipopolysaccharide (LPS) intracerebral administration at striatum level, more modulated genes presented transcription impairment than exacerbation upon glucocorticoid receptor blockage. Among impaired genes we identified ceruloplasmin (Cp), which plays a key role in iron metabolism and is implicated in a neurodegenative disease. Microglial and endothelial induction of Cp is a natural neuroprotective mechanism during inflammation, because Cp-deficient mice exhibited increased iron accumulation and demyelination when exposed to LPS and neurovascular reactivity to pneumococcal meningitis. This study has identified genes that can play a critical role in programming the innate immune response, helping to clarify the mechanisms leading to protection or damage during inflammatory conditions in the CNS.

Family shuffling of a targeted bphA region to engineer biphenyl dioxygenase.

In this work we used a new strategy designed to reduce the size of the library that needs to be explored in family shuffling to evolve new biphenyl dioxygenases (BPDOs). Instead of shuffling the whole gene, we have targeted a fragment of bphA that is critical for enzyme specificity. We also describe a new protocol to screen for more potent BPDOs that is based on the detection of catechol metabolites from chlorobiphenyls. Several BphA variants with extended potency to degrade polychlorinated biphenyls (PCBs) were obtained by shuffling critical segments of bphA genes from Burkholderia sp. strain LB400, Comamonas testosteroni B-356, and Rhodococcus globerulus P6. Unlike all parents, these variants exhibited high activity toward 2,2'-, 3,3'-, and 4,4'-dichlorobiphenyls and were able to oxygenate the very persistent 2,6-dichlorobiphenyl. The data showed that the replacement of a short segment (335TFNNIRI341) of LB400 BphA by the corresponding segment (333GINTIRT339) of B-356 BphA or P6 BphA contributes to relax the enzyme toward PCB substrates.