Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

Histamine 4 receptor plays an important role in auto-antibody-induced arthritis.

Rheumatoid arthritis is a widespread autoimmune disease. In the murine K/B×N arthritis model, anti-GPI (anti-glucose 6-phosphate isomerase) antibodies lead to the formation of immune complexes. In the course of pathogenesis, these complexes activate the immune system and induce degranulation of mast cells, which are essential in this model of rheumatoid arthritis. A major mediator in mast cell granules is histamine, which is proven to be indispensable for joint inflammation in K/B×N mice. Histamine is known to bind to four different receptors (HR1-4), which have different expression profiles and exert a variety of different functions, including activation of the immune system. To analyze the contribution of the different histamine receptors, we employed histamine receptor antagonists (cetirizine, ranitidine, thioperamide and clozapine) blocking the receptors in C57BL/6 mice. Arthritis was induced via K/B×N serum injection. The results demonstrated that mice treated with all four histamine receptor antagonists simultaneously showed no arthritic symptoms, while positive control mice injected with K/B×N serum and vehicle suffered from severe symptoms. When antagonists specific for HR1-4 were applied individually, only the HR4 antagonist clozapine could protect mice from arthritis, reflecting its expression and functionality in the immune system.

Gut microbiota drives age-related oxidative stress and mitochondrial damage in microglia via the metabolite N6-carboxymethyllysine.

Microglial function declines during aging. The interaction of microglia with the gut microbiota has been well characterized during development and adulthood but not in aging. Here, we compared microglial transcriptomes from young-adult and aged mice housed under germ-free and specific pathogen-free conditions and found that the microbiota influenced aging associated-changes in microglial gene expression. The absence of gut microbiota diminished oxidative stress and ameliorated mitochondrial dysfunction in microglia from the brains of aged mice. Unbiased metabolomic analyses of serum and brain tissue revealed the accumulation of N6-carboxymethyllysine (CML) in the microglia of the aging brain. CML mediated a burst of reactive oxygen species and impeded mitochondrial activity and ATP reservoirs in microglia. We validated the age-dependent rise in CML levels in the sera and brains of humans. Finally, a microbiota-dependent increase in intestinal permeability in aged mice mediated the elevated levels of CML. This study adds insight into how specific features of microglia from aged mice are regulated by the gut microbiota.

Microbiota-dependent increase in δ-valerobetaine alters neuronal function and is responsible for age-related cognitive decline.

Understanding the physiological origins of age-related cognitive decline is of critical importance given the rising age of the world's population1. Previous work in animal models has established a strong link between cognitive performance and the microbiota2-5, and it is known that the microbiome undergoes profound remodeling in older adults6. Despite growing evidence for the association between age-related cognitive decline and changes in the gut microbiome, the mechanisms underlying such interactions between the brain and the gut are poorly understood. Here, using fecal microbiota transplantation (FMT), we demonstrate that age-related remodeling of the gut microbiota leads to decline in cognitive function in mice and that this impairment can be rescued by transplantation of microbiota from young animals. Moreover, using a metabolomic approach, we found elevated concentrations of δ-valerobetaine, a gut microbiota-derived metabolite, in the blood and brain of aged mice and older adults. We then demonstrated that δ-valerobetaine is deleterious to learning and memory processes in mice. At the neuronal level, we showed that δ-valerobetaine modulates inhibitory synaptic transmission and neuronal network activity. Finally, we identified specific bacterial taxa that significantly correlate with δ-valerobetaine levels in the brain. Based on our findings, we propose that δ-valerobetaine contributes to microbiota-driven brain aging and that the associated mechanisms represent a promising target for countering age-related cognitive decline.

CB2 receptor deletion on myeloid cells enhanced mechanical allodynia in a mouse model of neuropathic pain.

Neuropathic pain can develop after nerve injury, leading to a chronic condition with spontaneous pain and hyperalgesia. Pain is typically restricted to the side of the injured nerve, but may occasionally spread to the contralateral side, a condition that is often referred to as mirror-image pain. Mechanisms leading to mirror-image pain are not completely understood, but cannabinoid CB2 receptors have been implicated. In this study, we use genetic mouse models to address the question if CB2 receptors on neurons or on microglia/macrophages are involved. First, we show that a GFP reporter protein under control of the CB2 promoter is induced upon partial sciatic nerve ligation in spinal cord, dorsal root ganglia, and highest in sciatic nerve macrophages, but not in neurons. Mice which lack CB2 receptors specifically on myeloid cells (microglia, macrophages) developed a mirror-image allodynia [treatment F1,48 = 45.69, p < 0.0001] similar to constitutive CB2 receptor knockout mice [treatment F1,70 = 92.41, p < 0.0001]. Such a phenotype was not observed after the deletion of CB2 from neurons [treatment F1,70 = 0.1315, p = 0.7180]. This behavioral pain phenotype was accompanied by an increased staining of microglia in the dorsal horn of the spinal cord, as evidenced by an enhanced Iba 1 expression [CB2KO, p = 0.0175; CB2-LysM, p = 0.0425]. Similarly, myeloid-selective knockouts showed an increased expression of the leptin receptor in the injured ipsilateral sciatic nerve, thus further supporting the notion that leptin signaling contributes to the increased neuropathic pain responses of CB2 receptor knockout mice. We conclude that CB2 receptors on microglia and macrophages, but not on neurons, modulate neuropathic pain responses.

Involvement of leptin signaling in the development of cannabinoid CB2 receptor-dependent mirror image pain.

Neuropathic pain typically appears in a region innervated by an injured or diseased nerve and, in some instances, also on the contralateral side. This so-called mirror image pain is often observed in mice lacking CB2 receptors after sciatic nerve injury, but the underlying mechanisms for this phenotype largely remain unclear. Here we focused on peripheral leptin signaling, which modulates neuropathic pain development and interacts with the endocannabinoid system. Leptin production is induced at the site of nerve injury in CB2-deficient mice (CB2-KO) mice and wild type controls (WT). However, induction of leptin receptor expression was only observed in the injured nerve of CB2-KO mice. This was paralleled by a stimulation of the leptin receptor-downstream STAT3 signaling and an infiltration of F4/80-positive macrophages. Interestingly, an upregulation of leptin receptor expression STAT3 activity and macrophage infiltration was also observed on the non-injured nerve of CB2-KO mice thus reflecting the mirror image pain in CB2-KO animals. Importantly, perineurally-administered leptin-neutralizing antibodies reduced mechanical hyperalgesia, blocked mirror image pain and inhibited the recruitment of F4/80-positive macrophages. These results identify peripheral leptin signaling as an important modulator of CB2 signaling in neuropathic pain.

CB1 receptors modulate affective behaviour induced by neuropathic pain.

Patients suffering from chronic pain are often diagnosed with a psychiatric condition, in particular generalized anxiety and major depression. The underlying pathomechanisms contributing to this comorbidity, however, are not entirely clear. In this manuscript we have focussed on the potential role of the cannabinoid receptor CB1, because it is known to modulate neuronal circuits contributing to chronic pain states and affective behaviours. For this purpose we analysed the consequences of a partial sciatic nerve ligation on anxiety- and depression-related behaviours in mice lacking CB1 receptors. Our results show that the development of mechanical hypersensitivity was similar in CB1 deficient mice and wild type controls. However, CB1 knockouts showed much more pronounced behavioural manifestations of anxiety-related behaviours in the light-dark and zero-maze tests, sucrose anhedonia, and disturbed home-cage activity. These results indicate that the endocannabinoid system affects chronic pain-induced mood changes through CB1 receptors.