Microglia coordinate cellular interactions during spinal cord repair in mice.

Traumatic spinal cord injury (SCI) triggers a neuro-inflammatory response dominated by tissue-resident microglia and monocyte derived macrophages (MDMs). Since activated microglia and MDMs are morphologically identical and express similar phenotypic markers in vivo, identifying injury responses specifically coordinated by microglia has historically been challenging. Here, we pharmacologically depleted microglia and use anatomical, histopathological, tract tracing, bulk and single cell RNA sequencing to reveal the cellular and molecular responses to SCI controlled by microglia. We show that microglia are vital for SCI recovery and coordinate injury responses in CNS-resident glia and infiltrating leukocytes. Depleting microglia exacerbates tissue damage and worsens functional recovery. Conversely, restoring select microglia-dependent signaling axes, identified through sequencing data, in microglia depleted mice prevents secondary damage and promotes recovery. Additional bioinformatics analyses reveal that optimal repair after SCI might be achieved by co-opting key ligand-receptor interactions between microglia, astrocytes and MDMs.

Spinal Cord Injury Impairs Lung Immunity in Mice.

Pulmonary infection is a leading cause of morbidity and mortality after spinal cord injury (SCI). Although SCI causes atrophy and dysfunction in primary and secondary lymphoid tissues with a corresponding decrease in the number and function of circulating leukocytes, it is unknown whether this SCI-dependent systemic immune suppression also affects the unique tissue-specific antimicrobial defense mechanisms that protect the lung. In this study, we tested the hypothesis that SCI directly impairs pulmonary immunity and subsequently increases the risk for developing pneumonia. Using mouse models of severe high-level SCI, we find that recruitment of circulating leukocytes and transcriptional control of immune signaling in the lung is impaired after SCI, creating an environment that is permissive for infection. Specifically, we saw a sustained loss of pulmonary leukocytes, a loss of alveolar macrophages at chronic time points postinjury, and a decrease in immune modulatory genes, especially cytokines, needed to eliminate pulmonary infections. Importantly, this injury-dependent impairment of pulmonary antimicrobial defense is only partially overcome by boosting the recruitment of immune cells to the lung with the drug AMD3100, a Food and Drug Administration-approved drug that mobilizes leukocytes and hematopoietic stem cells from bone marrow. Collectively, these data indicate that the immune-suppressive effects of SCI extend to the lung, a unique site of mucosal immunity. Furthermore, preventing lung infection after SCI will likely require novel strategies, beyond the use of orthodox antibiotics, to reverse or block tissue-specific cellular and molecular determinants of pulmonary immune surveillance.

Genetic deletion of the glucocorticoid receptor in Cx3cr1+ myeloid cells is neuroprotective and improves motor recovery after spinal cord injury.

Glucocorticoid receptors (GRs), part of the nuclear receptor superfamily of transcription factors (TFs), are ubiquitously expressed in all cell types and regulate cellular responses to glucocorticoids (e.g., cortisol in humans; corticosterone in rodents). In myeloid cells, glucocorticoids binding to GRs can enhance or repress gene transcription, thereby imparting distinct and context-dependent functions in macrophages at sites of inflammation. In experimental models and in humans, glucocorticoids are widely used as anti-inflammatory treatments to promote recovery of function after SCI. Thus, we predicted that deleting GR in mouse myeloid lineage cells (i.e., microglia and monocyte-derived macrophages) would enhance inflammation at the site of injury and worsen functional recovery after traumatic spinal cord injury (SCI). Contrary to our prediction, the intraspinal macrophage response to a moderate (75 kdyne) spinal contusion SCI was reduced in Cx3cr1-Cre;GRf/f conditional knockout mice (with GR specifically deleted in myeloid cells). This phenotype was associated with improvements in hindlimb motor recovery, myelin sparing, axon sparing/regeneration, and microvascular protection/plasticity relative to SCI mice with normal myeloid cell GR expression. Further analysis revealed that macrophage GR deletion impaired lipid and myelin phagocytosis and foamy macrophage formation. Together, these data reveal endogenous GR signaling as a key pathway that normally inhibits mechanisms of macrophage-mediated repair after SCI.

Spinal cord injury causes chronic bone marrow failure.

Spinal cord injury (SCI) causes immune dysfunction, increasing the risk of infectious morbidity and mortality. Since bone marrow hematopoiesis is essential for proper immune function, we hypothesize that SCI disrupts bone marrow hematopoiesis. Indeed, SCI causes excessive proliferation of bone marrow hematopoietic stem and progenitor cells (HSPC), but these cells cannot leave the bone marrow, even after challenging the host with a potent inflammatory stimulus. Sequestration of HSPCs in bone marrow after SCI is linked to aberrant chemotactic signaling that can be reversed by post-injury injections of Plerixafor (AMD3100), a small molecule inhibitor of CXCR4. Even though Plerixafor liberates HSPCs and mature immune cells from bone marrow, competitive repopulation assays show that the intrinsic long-term functional capacity of HSPCs is still impaired in SCI mice. Together, our data suggest that SCI causes an acquired bone marrow failure syndrome that may contribute to chronic immune dysfunction.

Human immune cells infiltrate the spinal cord and impair recovery after spinal cord injury in humanized mice.

Humanized mice can be used to better understand how the human immune system responds to central nervous system (CNS) injury and inflammation. The optimal parameters for using humanized mice in preclinical CNS injury models need to be established for appropriate use and interpretation. Here, we show that the developmental age of the human immune system significantly affects anatomical and functional outcome measures in a preclinical model of traumatic spinal cord injury (SCI). Specifically, it takes approximately 3-4 months for a stable and functionally competent human immune system to develop in neonatal immune compromised mice after they are engrafted with human umbilical cord blood stem cells. Humanized mice receiving a SCI before or after stable engraftment exhibit significantly different neuroinflammatory profiles. Importantly, the development of a mature human immune system was associated with worse lesion pathology and neurological recovery after SCI. In these mice, human T cells infiltrate the spinal cord lesion and directly contact human macrophages. Together, data in this report establish an optimal experimental framework for using humanized mice to help translate promising preclinical therapies for CNS injury.

Complement receptor C3aR1 controls neutrophil mobilization following spinal cord injury through physiological antagonism of CXCR2.

Traumatic spinal cord injury (SCI) triggers an acute-phase response that leads to systemic inflammation and rapid mobilization of bone marrow (BM) neutrophils into the blood. These mobilized neutrophils then accumulate in visceral organs and the injured spinal cord where they cause inflammatory tissue damage. The receptor for complement activation product 3a, C3aR1, has been implicated in negatively regulating the BM neutrophil response to tissue injury. However, the mechanism via which C3aR1 controls BM neutrophil mobilization, and also its influence over SCI outcomes, are unknown. Here, we show that the C3a/C3aR1 axis exerts neuroprotection in SCI by acting as a physiological antagonist against neutrophil chemotactic signals. We show that C3aR1 engages phosphatase and tensin homolog (PTEN), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, to restrain C-X-C chemokine receptor type 2-driven BM neutrophil mobilization following trauma. These findings are of direct clinical significance as lower circulating neutrophil numbers at presentation were identified as a marker for improved recovery in human SCI. Our work thus identifies C3aR1 and its downstream intermediary, PTEN, as therapeutic targets to broadly inhibit neutrophil mobilization/recruitment following tissue injury and reduce inflammatory pathology.

The Alternative Receptor for Complement Component 5a, C5aR2, Conveys Neuroprotection in Traumatic Spinal Cord Injury.

This study investigated the role of the alternative receptor for complement activation fragment C5a, C5aR2, in secondary inflammatory pathology after contusive spinal cord injury (SCI) in mice. C5ar2-/- mice exhibited decreased intraparenchymal tumor necrosis factor alpha and interleukin-6 acutely post-injury, but these reductions did not translate into improved outcomes. We show that loss of C5aR2 leads to increased lesion volumes, reduced myelin sparing, and significantly worsened recovery from SCI in C5ar2-/- animals compared to wild-type (WT) controls. Loss of C5aR2 did not alter leukocyte mobilization from the bone marrow in response to SCI, and neutrophil recruitment/presence at the lesion site was also not different between genotypes. Acute treatment of SCI mice with the selective C5aR1 antagonist, PMX205, improved SCI outcomes, compared to vehicle controls, and, importantly, fully alleviated the worsened recovery of C5ar2-/- mice compared to their WT counterparts. Collectively, these findings indicate that C5aR2 is neuroprotective and a novel target to restrain injurious C5a signaling after a major neurotraumatic event.

PI3Kδ inhibition reduces TNF secretion and neuroinflammation in a mouse cerebral stroke model.

Stroke is a major cause of death worldwide and the leading cause of permanent disability. Although reperfusion is currently used as treatment, the restoration of blood flow following ischaemia elicits a profound inflammatory response mediated by proinflammatory cytokines such as tumour necrosis factor (TNF), exacerbating tissue damage and worsening the outcomes for stroke patients. Phosphoinositide 3-kinase delta (PI3Kδ) controls intracellular TNF trafficking in macrophages and therefore represents a prospective target to limit neuroinflammation. Here we show that PI3Kδ inhibition confers protection in ischaemia/reperfusion models of stroke. In vitro, restoration of glucose supply following an episode of glucose deprivation potentiates TNF secretion from primary microglia-an effect that is sensitive to PI3Kδ inhibition. In vivo, transient middle cerebral artery occlusion and reperfusion in kinase-dead PI3Kδ (p110δ(D910A/D910A)) or wild-type mice pre- or post-treated with the PI3Kδ inhibitor CAL-101, leads to reduced TNF levels, decreased leukocyte infiltration, reduced infarct size and improved functional outcome. These data identify PI3Kδ as a potential therapeutic target in ischaemic stroke.

Mobilisation of the splenic monocyte reservoir and peripheral CX3CR1 deficiency adversely affects recovery from spinal cord injury.

Macrophages in the injured spinal cord originate from resident microglia and blood monocytes. Whether this diversity in origins contributes to their seemingly dual role in immunopathology and repair processes has remained poorly understood. Here we took advantage of Cx3cr1(gfp) mice to visualise monocyte-derived macrophages in the injured spinal cord via adoptive cell transfer and bone marrow (BM) chimera approaches. We show that the majority of infiltrating monocytes at 7 days post-injury originate from the spleen and only to a lesser extent from the BM. Prevention of early monocyte infiltration via splenectomy was associated with improved recovery at 42 days post-SCI. In addition, an increased early presence of infiltrating monocytes/macrophages, as a result of CX3CR1 deficiency within the peripheral immune compartment, correlated with worsened injury outcomes. Adoptive transfer of identified Cx3cr1(gfp/+) monocytes confirmed peak infiltration at 7 days post-injury, with inflammatory (Ly6C(high)) monocytes being most efficiently recruited. Focal SCI also changed the composition of the two major monocyte subsets in the blood, with more Ly6C(high) cells present during peak recruitment. Adoptive transfer experiments further suggested high turnover of inflammatory monocytes in the spinal cord at 7 days post-injury. Consistent with this, only a small proportion of infiltrating cells unequivocally expressed polarisation markers for pro-inflammatory (M1) or alternatively activated (M2) macrophages at this time point. Our findings offer new insights into the origins of monocyte-derived macrophages after SCI and their contribution to functional recovery, providing a basis for further scrutiny and selective targeting of Ly6C(high) monocytes to improve outcomes from neurotraumatic events.