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Monocyte-derived and tissue-resident macrophages are ontogenetically distinct components of the innate immune system. Assessment of their respective functions in pathology is complicated by changes to the macrophage phenotype during inflammation. Here we find that Cxcr4-CreER enables permanent genetic labeling of hematopoietic stem cells (HSCs) and distinguishes HSC-derived monocytes from microglia and other tissue-resident macrophages. By combining Cxcr4-CreER-mediated lineage tracing with Cxcr4 inhibition or conditional Cxcr4 ablation in photothrombotic stroke, we find that Cxcr4 promotes initial monocyte infiltration and subsequent territorial restriction of monocyte-derived macrophages to infarct tissue. After transient focal ischemia, Cxcr4 deficiency reduces monocyte infiltration and blunts the expression of pattern recognition and defense response genes in monocyte-derived macrophages. This is associated with an altered microglial response and deteriorated outcomes. Thus, Cxcr4 is essential for an innate-immune-system-mediated defense response after cerebral ischemia. We further propose Cxcr4-CreER as a universal tool to study functions of HSC-derived cells.The authors establish inducible Cxcr4-CreER-based fate mapping as a universal means to identify bone-marrow-derived myeloid cells in the injured brain and demonstrate that Cxcr4 deficiency affects the innate immune response and outcome after stroke.

Inflammation triggers secondary brain damage after stroke. The meninges and other CNS border compartments serve as invasion sites for leukocyte influx into the brain thus promoting tissue damage after stroke. However, the post-ischemic immune response of border compartments compared to brain parenchyma remains poorly characterized. Here, we deeply characterize tissue-resident leukocytes in meninges and brain parenchyma and discover that leukocytes respond differently to stroke depending on their site of residence. We thereby discover a unique phenotype of myeloid cells exclusive to the brain after stroke. These stroke-associated myeloid cells partially resemble neurodegenerative disease-associated microglia. They are mainly of resident microglial origin, partially conserved in humans and exhibit a lipid-phagocytosing phenotype. Blocking markers specific for these cells partially ameliorates stroke outcome thus providing a potential therapeutic target. The injury-response of myeloid cells in the CNS is thus compartmentalized, adjusted to the type of injury and may represent a therapeutic target. How ischaemic stroke affects the brain borders is not fully understood. Here the authors show that a stroke-associated myeloid cell population occurs exclusively in brain parenchyma that shares features with neurodegenerative microglia and blockade of proteins on these cells can ameliorate stroke symptoms.

While CNS microglia have been extensively studied, relatively little is known about macrophages populating the peripheral nervous system. Here we performed ontogenic, transcriptomic and spatial characterization of sciatic nerve macrophages (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and become replaced by bone-marrow-derived macrophages over time. Using single-cell transcriptomics, we identified a tissue-specific core signature of snMacs and two spatially separated snMacs: Relmα+Mgl1+ snMacs in the epineurium and Relmα–Mgl1– snMacs in the endoneurium. Globally, snMacs lack most of the core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. In response to nerve injury, the two resident snMac populations respond differently. Moreover, and unlike in the CNS, monocyte-derived macrophages that develop during injury can engraft efficiently in the pool of resident peripheral nervous system macrophages.The authors identify two subsets of peripheral nerve macrophages residing in the endoneurium and the epineurium and displaying a distinct transcriptome and response to injury. These cells lack the main microglia identity and have a distinct origin.

Conventional dendritic cells (cDC) are key activators of naive T cells, and can be targeted in adults to induce adaptive immunity, but in early life are considered under-developed or functionally immature. Here we show that, in early life, when the immune system develops, cDC2 exhibit a dual hematopoietic origin and, like other myeloid and lymphoid cells, develop in waves. Developmentally distinct cDC2 in early life, despite being distinguishable by fate mapping, are transcriptionally and functionally similar. cDC2 in early and adult life, however, are exposed to distinct cytokine environments that shape their transcriptional profile and alter their ability to sense pathogens, secrete cytokines and polarize T cells. We further show that cDC2 in early life, despite being distinct from cDC2 in adult life, are functionally competent and can induce T cell responses. Our results thus highlight the potential of harnessing cDC2 for boosting immunity in early life. Type 2 conventional dendritic cells (cDC2) are important immune activators in adults, but their development and functions at the neonatal stage remain unclear. Here the authors show, using fate-mapping and single-cell RNA sequencing, that neonatal cDC2 come from multiple origins, but converge functionally as potent immune activators upon proper stimuli.

Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion. ACKR3 is a critical regulator of platelet-mediated thrombosis and organ injury following ischemia/reperfusion. Platelet ACKR3 surface expression is independently associated with all-cause mortality in patients with cardiovascular diseases.

Background The innate lymphoid cell (ILC) family consists of NK cells, ILC type 1, 2, 3 and lymphoid tissue inducer cells. They have been shown to play important roles in homeostasis and immune responses and are generally considered tissue resident. Not much is known about the presence of ILC members within the central nervous system and whether they are tissue resident in this organ too. Therefore, we studied the presence of all ILC members within the central nervous system and after ischemic brain insult. Methods We used the photothrombotic ischemic lesion method to induce ischemic lesions within the mouse brain. Using whole-mount immunofluorescence imaging, we established that the ILCs were present at the rim of the lesion. We quantified the increase of all ILC members at different time-points after the ischemic lesion induction by flow cytometry. Their migration route via chemokine CXCL12 was studied by using different genetic mouse models, in which we induced deletion of Cxcl12 within the blood–brain barrier endothelium, or its receptor, Cxcr4 , in the ILCs. The functional role of the ILCs was subsequently established using the beam-walk sensorimotor test. Results Here, we report that ILCs are not resident within the mouse brain parenchyma during steady-state conditions, but are attracted towards the ischemic stroke. Specifically, we identify NK cells, ILC1s, ILC2s and ILC3s within the lesion, the highest influx being observed for NK cells and ILC1s. We further show that CXCL12 expressed at the blood–brain barrier is essential for NK cells and NKp46 + ILC3s to migrate toward the lesion. Complementary, Cxcr4 -deficiency in NK cells prevents NK cells from entering the infarct area. Lack of NK cell migration results in a higher neurological deficit in the beam-walk sensorimotor test. Conclusions This study establishes the lack of ILCs in the mouse central nervous system at steady-state and their migration towards an ischemic brain lesion. Our data show a role for blood–brain barrier-derived CXCL12 in attracting protective NK cells to ischemic brain lesions and identifies a new CXCL12/CXCR4-mediated component of the innate immune response to stroke.

The atypical chemokine receptor 3 (ACKR3) plays a pivotal role in directing the migration of various cellular populations and its over-expression in tumors promotes cell proliferation and invasiveness. The intracellular signaling pathways transducing ACKR3-dependent effects remain poorly characterized, an issue we addressed by identifying the interactome of ACKR3. Here, we report that recombinant ACKR3 expressed in HEK293T cells recruits the gap junction protein Connexin 43 (Cx43). Cx43 and ACKR3 are co-expressed in mouse brain astrocytes and human glioblastoma cells and form a complex in embryonic mouse brain. Functional in vitro studies show enhanced ACKR3 interaction with Cx43 upon ACKR3 agonist stimulation. Furthermore, ACKR3 activation promotes β-arrestin2- and dynamin-dependent Cx43 internalization to inhibit gap junctional intercellular communication in primary astrocytes. These results demonstrate a functional link between ACKR3 and gap junctions that might be of pathophysiological relevance. The atypical chemokine receptor 3 (ACKR3) is known to regulate cell migration, but the underlying mechanisms are unclear. Here, the authors show, from an interactome analysis, ACKR3 association with the gap junction protein Connexin 43 in vivo and ACKR3-mediated inhibition of astrocyte gap junctional communication.

The chemokine receptor CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. Upon stimulation by the endogenous ligand CXCL12, CXCR4 becomes phosphorylated at multiple sites in its C-terminal domain. Mutations in the CXCR4 gene affecting C-terminal phosphorylation sites are a hallmark of WHIM syndrome, a genetic disorder characterized by a gain-of-CXCR4-function. To better understand how multi-site phosphorylation of CXCR4 is organized and how perturbed phosphorylation might affect CXCR4 function, we developed novel phosphosite-specific CXCR4 antibodies and studied the differential regulation and interaction of three C-terminal phosphorylation sites in human embryonic kidney cells (HEK293). CXCL12 promoted a robust phosphorylation at S346/347 which preceded phosphorylation at S324/325 and S338/339. After CXCL12 washout, the phosphosites S338/339 and S324/325 were rapidly dephosphorylated whereas phosphorylation at S346/347 was long-lasting. CXCL12-induced phosphorylation at S346/347 was staurosporine-insensitive and mediated by GRK2/3. WHIM syndrome-associated CXCR4 truncation mutants lacking the S346/347 phosphosite and the recently identified E343K WHIM mutant displayed strongly impaired phosphorylation at S324/325 and S338/339 as well as reduced CXCL12-induced receptor internalization. Relevance of the S346-S348 site was confirmed by a S346-348A mutant showing strongly impaired CXCL12-promoted phosphorylation at S324/325 and S338/339, defective internalization, gain of calcium mobilization, and reduced desensitization. Thus, the triple serine motif S346-S348 contains a major initial CXCR4 phosphorylation site and is required for efficient subsequent multi-site phosphorylation and receptor regulation. Hierarchical organization of CXCR4 phosphorylation explains why small deletions at the extreme CXCR4 C terminus typically associated with WHIM syndrome severely alter CXCR4 function.

Biallelic mutations in DONSON , an essential gene encoding for a replication fork protection factor, were linked to skeletal abnormalities and microcephaly. To better understand DONSON function in corticogenesis, we characterized Donson expression and consequences of conditional Donson deletion in the mouse telencephalon. Donson was widely expressed in the proliferation and differentiation zones of the embryonic dorsal and ventral telencephalon, which was followed by a postnatal expression decrease. Emx1 -Cre-mediated Donson deletion in progenitors of cortical glutamatergic neurons caused extensive apoptosis in the early dorsomedial neuroepithelium, thus preventing formation of the neocortex and hippocampus. At the place of the missing lateral neocortex, these mutants exhibited a dorsal extension of an early-generated paleocortex. Targeting cortical neurons at the intermediate progenitor stage using Tbr2 -Cre evoked no apparent malformations, whereas Nkx2 . 1 -Cre-mediated Donson deletion in subpallial progenitors ablated 75% of Nkx2 . 1 -derived cortical GABAergic neurons. Thus, the early telencephalic neuroepithelium depends critically on Donson function. Our findings help explain why the neocortex is most severely affected in individuals with DONSON mutations and suggest that DONSON-dependent microcephaly might be associated with so far unrecognized defects in cortical GABAergic neurons. Targeting Donson using an appropriate recombinase is proposed as a feasible strategy to ablate proliferating and nascent cells in experimental research.

Olfaction depends on lifelong production of sensory neurons from CXCR4 expressing neurogenic stem cells. Signaling by CXCR4 depends on the concentration of CXCL12, CXCR4’s principal ligand. Here, we use several genetic models to investigate how regulation of CXCL12 in the olfactory stem cell niche adjusts neurogenesis. We identify subepithelial tissue and sustentacular cells, the olfactory glia, as main CXCL12 sources. Lamina propria-derived CXCL12 accumulates on quiescent gliogenic stem cells via heparan sulfate. Additionally, CXCL12 is secreted within the olfactory epithelium by sustentacular cells. Both sustentacular-cell-derived and lamina propria-derived CXCL12 are required for CXCR4 activation. ACKR3, a high-affinity CXCL12 scavenger, is expressed by mature glial cells and titrates CXCL12. The accurate adjustment of CXCL12 by ACKR3 is critical for CXCR4-dependent proliferation of neuronal stem cells and for proper lineage progression. Overall, these findings establish precise regulation of CXCL12 by glia cells as a prerequisite for CXCR4-dependent neurogenesis and identify ACKR3 as a scavenger influencing tissue homeostasis beyond embryonic development.