In amyotrophic lateral sclerosis (ALS), immune cells and glia contribute to motor neuron (MN) degeneration. We report the presence of NK cells in post-mortem ALS motor cortex and spinal cord tissues, and the expression of NKG2D ligands on MNs. Using a mouse model of familial-ALS, hSOD1^G93A, we demonstrate NK cell accumulation in the motor cortex and spinal cord, with an early CCL2-dependent peak. NK cell depletion reduces the pace of MN degeneration, delays motor impairment and increases survival. This is confirmed in another ALS mouse model, TDP43^A315T. NK cells are neurotoxic to hSOD1^G93AMNs which express NKG2D ligands, while IFNγ produced by NK cells instructs microglia toward an inflammatory phenotype, and impairs FOXP3⁺/Treg cell infiltration in the spinal cord of hSOD1^G93Amice. Together, these data suggest a role of NK cells in determining the onset and progression of MN degeneration in ALS, and in modulating Treg recruitment and microglia phenotype.

Microglia cells are active players in regulating synaptic development and plasticity in the brain. However, how they influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological microglia depletion, achieved by administration of PLX5622, on hippocampal CA3‐CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features and higher levels of plasticity. Microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. These events were accompanied by hippocampal astrogliosis, although in the absence ofneuroinflammatory condition. PLX‐induced synaptic changes were absent in Cx3cr1^−/− mice, highlighting the role of CX3CL1/CX3CR1 axis in microglia control of synaptic functioning. Remarkably, microglia repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in organization and activity of glutamatergic synapses.

Microglia, the brain's resident macrophages, actively contribute to the homeostasis of cerebral parenchyma by sensing neuronal activity and supporting synaptic remodeling and plasticity. While several studies demonstrated different roles for astrocytes in sleep, the contribution of microglia in the regulation of sleep/wake cycle and in the modulation of synaptic activity in the different day phases has not been deeply investigated. Using light as a zeitgeber cue, we studied the effects of microglial depletion with the colony stimulating factor‐1 receptor antagonist PLX5622 on the sleep/wake cycle and on hippocampal synaptic transmission in male mice. Our data demonstrate that almost complete microglial depletion increases the duration of NREM sleep and reduces the hippocampal excitatory neurotransmission. The fractalkine receptor CX3CR1 plays a relevant role in these effects, because cx3cr1^GFP/GFP mice recapitulate what found in PLX5622‐treated mice. Furthermore, during the light phase, microglia express lower levels of cx3cr1 and a reduction of cx3cr1 expression is also observed when cultured microglial cells are stimulated by ATP, a purinergic molecule released during sleep. Our findings suggest that microglia participate in the regulation of sleep, adapting their cx3cr1 expression in response to the light/dark phase, and modulating synaptic activity in a phase‐dependent manner.

Neuroinflammation is one of the main hallmarks of amyotrophic lateral sclerosis (ALS). Recently, peripheral immune cells were discovered as pivotal players that promptly participate in this process, speeding up neurodegeneration during progression of the disease. In particular, infiltrating T cells and natural killer cells release inflammatory cytokines that switch glial cells toward a pro-inflammatory/detrimental phenotype, and directly attack motor neurons with specific ligand-receptor signals. Here, we assessed the presence of lymphocytes in the spinal cord of sporadic ALS patients. Furthermore, we demonstrate that blocking the extravasation of immune cells in the central nervous system using Natalizumab (NAT), an antibody for the α4 integrin, reduces the level of interferon-γ in the spinal cord of ALS mouse models, such as the hSOD1^G93A and TDP43^A315T mice, modifying microglia and astrocytes phenotype, increasing motor neuron number and prolonging the survival time. Taken together, our results establish a central role for the immune cells as drivers of inflammation in ALS.

The antimetastatic activity of NK cells is well established in several cancer types, but the mechanisms underlying NK cell metastasis infiltration and acquisition of antitumor characteristics remain unclear. Herein, we investigated the cellular and molecular factors required to facilitate the generation of an ILC1-like CD49a ⁺ NK cell population within the liver metastasis (LM) environment of colorectal cancer (CRC). We show that CD49a ⁺ NK cells had the highest cytotoxic capacity among metastasis-infiltrating NK cells in the MC38 mouse model. Furthermore, the chemokine receptor CXCR3 promoted CD49a ⁺ NK cell accumulation and persistence in metastasis where NK cells colocalize with macrophages in CXCL9- and CXCL10-rich areas. By mining a published scRNA-seq dataset of a cohort of patients with CRC who were treatment naive, we confirmed the accumulation of CXCR3 ⁺ NK cells in metastatic samples. Conditional deletion of Cxcr3 in NKp46 ⁺ cells and antibody-mediated depletion of metastasis-associated macrophages impaired CD49a ⁺ NK cell development, indicating that CXCR3 and macrophages contribute to efficient NK cell localization and polarization in LM. Conversely, CXCR3 ^neg NK cells maintained a CD49a ^– phenotype in metastasis with reduced parenchymal infiltration and tumor killing capacity. Furthermore, CD49a ⁺ NK cell accumulation was impaired in an independent SL4-induced CRC metastasis model, which fails to accumulate CXCL9 ⁺ macrophages. Together, our results highlight a role for CXCR3/ligand axis in promoting macrophage-dependent NK cell accumulation and functional sustenance in CRC LM.

In glioma, microglia and infiltrating macrophages are exposed to factors that force them to produce cytokines and chemokines, which contribute to tumor growth and to maintaining a pro-tumorigenic, immunosuppressed microenvironment. We demonstrate that housing glioma-bearing mice in enriched environment (EE) reverts the immunosuppressive phenotype of infiltrating myeloid cells, by modulating inflammatory gene expression. Under these conditions, the branching and patrolling activity of myeloid cells is increased, and their phagocytic activity is promoted. Modulation of gene expression depends on interferon-(IFN)-γ produced by natural killer (NK) cells. This modulation disappears in mice depleted of NK cells or lacking IFN-γ, and was mimicked by exogenous interleukin-15 (IL-15). Further, we describe a key role for brain-derived neurotrophic factor (BDNF) that is produced in the brain of mice housed in EE, in mediating the expression of IL-15 in CD11b⁺ cells. These data define novel mechanisms linking environmental cues to the acquisition of a pro-inflammatory, anti-tumor microenvironment in mouse brain.