Retinal microglia express more MHC class I and promote greater T-cell-driven inflammation than brain microglia.

Introduction: Macrophage function is determined by microenvironment and origin. Brain and retinal microglia are both derived from yolk sac progenitors, yet their microenvironments differ. Utilizing single-cell RNA sequencing (scRNA-seq) data from mice, we tested the hypothesis that retinal and brain microglia exhibit distinct transcriptional profiles due to their unique microenvironments. Methods: Eyes and brains from 2-4 month wildtype mice were combined (20 eyes; 3 brains) to yield one biologically diverse sample per organ. Each tissue was digested into single cell suspensions, enriched for immune cells, and sorted for scRNA-seq. Analysis was performed in Seurat v3 including clustering, integration, and differential expression. Multi-parameter flow cytometry was used for validation of scRNA-seq results. Lymphocytic choriomeningitis virus (LCMV) Clone 13, which produces a systemic, chronic, and neurotropic infection, was used to validate scRNA-seq and flow cytometry results in vivo. Results: Cluster analysis of integrated gene expression data from eye and brain identified 6 Tmem119 + P2ry12 + microglial clusters. Differential expression analysis revealed that eye microglia were enriched for more pro-inflammatory processes including antigen processing via MHC class I (14.0-fold, H2-D1 and H2-K1) and positive regulation of T-cell immunity (8.4-fold) compared to brain microglia. Multi-parameter flow cytometry confirmed that retinal microglia expressed 3.2-fold greater H2-Db and 263.3-fold more H2-Kb than brain microglia. On Day 13 and 29 after LCMV infection, CD8+ T-cell density was greater in the retina than the brain. Discussion: Our data demonstrate that the microenvironment of retina and brain differs, resulting in microglia-specific gene expression changes. Specifically, retinal microglia express greater MHC class I by scRNA-seq and multi-parameter flow cytometry, resulting in a possibly enhanced capability to stimulate CD8+ T-cell inflammation during LCMV infection. These results may explain tissue-specific differences between retina and brain during systemic viral infections and CD8+ T-cell driven autoimmune disease.

A Novel Microglia-Specific Transcriptional Signature Correlates With Behavioral Deficits in Neuropsychiatric Lupus.

Neuropsychiatric symptoms of systemic lupus erythematosus (NP-SLE) affect over one-half of SLE patients, yet underlying mechanisms remain largely unknown. We demonstrate that SLE-prone mice (CReCOM) develop NP-SLE, including behavioral deficits prior to systemic autoimmunity, reduced brain volumes, decreased vascular integrity, and brain-infiltrating leukocytes. NP-SLE microglia exhibit numerical expansion, increased synaptic uptake, and a more metabolically active phenotype. Microglia from multiple SLE-prone models express a "NP-SLE signature" unrelated to type I interferon. Rather, the signature is associated with lipid metabolism, scavenger receptor activity and downregulation of inflammatory and chemotaxis processes, suggesting a more regulatory, anti-inflammatory profile. NP-SLE microglia also express genes associated with disease-associated microglia (DAM), a subset of microglia thought to be instrumental in neurodegenerative diseases. Further, expression of "NP-SLE" and "DAM" signatures correlate with the severity of behavioral deficits in young SLE-prone mice prior to overt systemic disease. Our data are the first to demonstrate the predictive value of our newly identified microglia-specific "NP-SLE" and "DAM" signatures as a surrogate for NP-SLE clinical outcomes and suggests that microglia-intrinsic defects precede contributions from systemic SLE for neuropsychiatric manifestations.

Effect of Granulocyte-Macrophage Colony-Stimulating Factor With or Without Supervised Exercise on Walking Performance in Patients With Peripheral Artery Disease: The PROPEL Randomized Clinical Trial.

Importance: Benefits of granulocyte-macrophage colony-stimulating factor (GM-CSF) for improving walking ability in people with lower extremity peripheral artery disease (PAD) are unclear. Walking exercise may augment the effects of GM-CSF in PAD, since exercise-induced ischemia enhances progenitor cell release and may promote progenitor cell homing to ischemic calf muscle. Objectives: To determine whether GM-CSF combined with supervised treadmill exercise improves 6-minute walk distance, compared with exercise alone and compared with GM-CSF alone; to determine whether GM-CSF alone improves 6-minute walk more than placebo and whether exercise improves 6-minute walk more than an attention control intervention. Design, Setting, and Participants: Randomized clinical trial with 2 × 2 factorial design. Participants were identified from the Chicago metropolitan area and randomized between January 6, 2012, and December 22, 2016, to 1 of 4 groups: supervised exercise + GM-CSF (exercise + GM-CSF) (n = 53), supervised exercise + placebo (exercise alone) (n = 53), attention control + GM-CSF (GM-CSF alone) (n = 53), attention control + placebo (n = 51). The final follow-up visit was on August 15, 2017. Interventions: Supervised exercise consisted of treadmill exercise 3 times weekly for 6 months. The attention control consisted of weekly educational lectures by clinicians for 6 months. GM-CSF (250 µg/m2/d) or placebo were administered subcutaneously (double-blinded) 3 times/wk for the first 2 weeks of the intervention. Main Outcomes and Measures: The primary outcome was change in 6-minute walk distance at 12-week follow-up (minimum clinically important difference, 20 m). P values were adjusted based on the Hochberg step-up method. Results: Of 827 persons evaluated, 210 participants with PAD were randomized (mean age, 67.0 [SD, 8.6] years; 141 [67%] black, 82 [39%] women). One hundred ninety-five (93%) completed 12-week follow-up. At 12-week follow-up, exercise + GM-CSF did not significantly improve 6-minute walk distance more than exercise alone (mean difference, -6.3 m [95% CI, -30.2 to +17.6]; P = .61) or more than GM-CSF alone (mean difference, +28.7 m [95% CI, +5.1 to +52.3]; Hochberg-adjusted P = .052). GM-CSF alone did not improve 6-minute walk more than attention control + placebo (mean difference, -1.4 m [95% CI, -25.2 to +22.4]; P = .91). Exercise alone improved 6-minute walk compared with attention control + placebo (mean difference, +33.6 m [95% CI, +9.4 to +57.7]; Hochberg-adjusted P = .02). Conclusions and Relevance: Among patients with PAD, supervised treadmill exercise significantly improved 6-minute walk distance compared with attention control + placebo, whereas GM-CSF did not significantly improve walking performance, either when used alone or when combined with supervised treadmill exercise. These results confirm the benefits of exercise but do not support using GM-CSF to treat walking impairment in patients with PAD. Trial Registration: clinicaltrials.gov Identifier: NCT01408901.

Bim suppresses the development of SLE by limiting myeloid inflammatory responses.

The Bcl-2 family is considered the guardian of the mitochondrial apoptotic pathway. We demonstrate that Bim acts as a molecular rheostat by controlling macrophage function not only in lymphoid organs but also in end organs, thereby preventing the break in tolerance. Mice lacking Bim in myeloid cells (LysMCreBimfl/fl) develop a systemic lupus erythematosus (SLE)-like disease that mirrors aged Bim-/- mice, including loss of marginal zone macrophages, splenomegaly, lymphadenopathy, autoantibodies (including anti-DNA IgG), and a type I interferon signature. LysMCreBimfl/fl mice exhibit increased mortality attributed to glomerulonephritis (GN). Moreover, the toll-like receptor signaling adaptor protein TRIF (TIR-domain-containing adapter-inducing interferon-ß) is essential for GN, but not systemic autoimmunity in LysMCreBimfl/fl mice. Bim-deleted kidney macrophages exhibit a novel transcriptional lupus signature that is conserved within the gene expression profiles from whole kidney biopsies of patients with SLE. Collectively, these data suggest that the Bim may be a novel therapeutic target in the treatment of SLE.

The caspase-8/RIPK3 signaling axis in antigen presenting cells controls the inflammatory arthritic response.

BACKGROUND: Caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, but maintains functions beyond cell death that involve suppression of receptor-interacting serine-threonine kinases (RIPKs). A genome-wide association study meta-analysis revealed an SNP associated with risk of rheumatoid arthritis (RA) development within the locus containing the gene encoding for caspase-8. Innate immune cells, like macrophages and dendritic cells, are gaining momentum as facilitators of autoimmune disease pathogenesis, and, in particular, RA. Therefore, we examined the involvement of caspase-8 within these antigen-presenting cell populations in the pathogenesis of an arthritis model that resembles the RA effector phase. METHODS: Cre LysM Casp8 flox/flox and Cre CD11c Casp8 flox/flox mice were bred via a cross between Casp8 flox/flox and Cre LysM or Cre CD11c mice. RIPK3 -/- Cre LysM Casp8 flox/flox and RIPK3 -/- Cre CD11c Casp8 flox/flox mice were generated to assess RIPK3 contribution. Mice were subjected to K/BxN serum-transfer-induced arthritis. Luminex-based assays were used to measure cytokines/chemokines. Histological analyses were utilized to examine joint damage. Mixed bone marrow chimeras were generated to assess synovial cell survival. Flow cytometric analysis was employed to characterize cellular distribution. For arthritis, differences between the groups were assessed using two-way analysis of variance (ANOVA) for repeated measurements. All other data were compared by the Mann-Whitney test. RESULTS: We show that intact caspase-8 signaling maintains opposing roles in lysozyme-M- and CD11c-expressing cells in the joint; namely, caspase-8 is crucial in CD11c-expressing cells to delay arthritis induction, while caspase-8 in lysozyme M-expressing cells hinders arthritis resolution. Caspase-8 is also implicated in the maintenance of synovial tissue-resident macrophages that can limit arthritis. Global loss of RIPK3 in both caspase-8 deletion constructs causes the response to arthritis to revert back to control levels via a mechanism potentially independent of cell death. Mixed bone marrow chimeric mice demonstrate that caspase-8 deficiency does not confer preferential expansion of synovial macrophage and dendritic cell populations, nor do caspase-8-deficient synovial populations succumb to RIPK3-mediated necroptotic death. CONCLUSIONS: These data demonstrate that caspase-8 functions in synovial antigen-presenting cells to regulate the response to inflammatory stimuli by controlling RIPK3 action, and this delicate balance maintains homeostasis within the joint.