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.

Tissue-resident, extravascular Ly6c- monocytes are critical for inflammation in the synovium.

Monocytes are abundant immune cells that infiltrate inflamed organs. However, the majority of monocyte studies focus on circulating cells, rather than those in tissue. Here, we identify and characterize an intravascular synovial monocyte population resembling circulating non-classical monocytes and an extravascular tissue-resident monocyte-lineage cell (TR-MC) population distinct in surface marker and transcriptional profile from circulating monocytes, dendritic cells, and tissue macrophages that are conserved in rheumatoid arthritis (RA) patients. TR-MCs are independent of NR4A1 and CCR2, long lived, and embryonically derived. TR-MCs undergo increased proliferation and reverse diapedesis dependent on LFA1 in response to arthrogenic stimuli and are required for the development of RA-like disease. Moreover, pathways that are activated in TR-MCs at the peak of arthritis overlap with those that are downregulated in LFA1-/- TR-MCs. These findings show a facet of mononuclear cell biology that could be imperative to understanding tissue-resident myeloid cell function in RA.

Three Distinct Transcriptional Profiles of Monocytes Associate with Disease Activity in Scleroderma Patients.

OBJECTIVE: Patients with diffuse cutaneous systemic sclerosis (dcSSc) display a complex clinical phenotype. Transcriptional profiling of whole blood or tissue from patients are affected by changes in cellular composition that drive gene expression and an inability to detect minority cell populations. We undertook this study to focus on the 2 main subtypes of circulating monocytes, classical monocytes (CMs) and nonclassical monocytes (NCMs) as a biomarker of SSc disease severity. METHODS: SSc patients were recruited from the Prospective Registry for Early Systemic Sclerosis. Clinical data were collected, as well as peripheral blood for isolation of CMs and NCMs. Age-, sex-, and race-matched healthy volunteers were recruited as controls. Bulk macrophages were isolated from the skin in a separate cohort. All samples were assayed by RNA sequencing (RNA-seq). RESULTS: We used an unbiased approach to cluster patients into 3 groups (groups A-C) based on the transcriptional signatures of CMs relative to controls. Each group maintained their characteristic transcriptional signature in NCMs. Genes up-regulated in group C demonstrated the highest expression compared to the other groups in SSc skin macrophages, relative to controls. Patients from groups B and C exhibited worse lung function than group A, although there was no difference in SSc skin disease at baseline, relative to controls. We validated our approach by applying our group classifications to published bulk monocyte RNA-seq data from SSc patients, and we found that patients without skin disease were most likely to be classified as group A. CONCLUSION: We are the first to show that transcriptional signatures of CMs and NCMs can be used to unbiasedly stratify SSc patients and correlate with disease activity outcome measures.

Ocular macrophage origin and heterogeneity during steady state and experimental choroidal neovascularization.

BACKGROUND: Neovascular age-related macular degeneration (nAMD) commonly causes vision loss from aberrant angiogenesis, termed choroidal neovascularization (CNV). Macrophages are heterogeneous cells that are necessary for experimental CNV, present in human CNV samples, and can display diverse functions, which are dependent upon both their origin and tissue microenvironment. Despite these associations, choroidal macrophage heterogeneity remains unexplored. METHODS: We performed multi-parameter flow cytometry on wildtype (WT) and Ccr2-/- mice after laser injury to identify macrophage subtypes, and determine which subsets originate from classical monocytes. To fate map tissue resident macrophages at steady state and after laser injury, we used the Cx3cr1CreER/+ ; Rosa26zsGFP/+ mouse model. We reanalyzed previously published single-cell RNA-seq of human choroid samples from healthy and nAMD patients to investigate human macrophage heterogeneity, disease association, and function. RESULTS: We identified 4 macrophage subsets in mice: microglia, MHCII+CD11c-, MHCII+CD11c+, and MHCII-. Microglia are tissue resident macrophages at steady state and unaffected by laser injury. At steady state, MHCII- macrophages are long lived, tissue resident macrophages, while MHCII+CD11c- and MHCII+CD11c+ macrophages are partially replenished from blood monocytes. After laser injury, MHCII+CD11c- macrophages are entirely derived from classical monocytes, MHCII- macrophages originate from classical monocytes (90%) and an expansion of tissue resident macrophages (10%), and MHCII+CD11c+ macrophages are derived from classical monocytes (70%), non-classical monocytes (10%), and an expansion of tissue resident macrophages (20%). Single-cell RNA-seq analysis of human choroid found 5 macrophage subsets: two MHCII+CD11C- and three MHCII+CD11C+ populations. One MHCII+CD11C+ subset was 78% derived from a patient with nAMD. Differential expression analysis identified up-regulation of pro-angiogenic gene expression in one MHCII+CD11C- and two MHCII+CD11C+ subsets, including the disease-associated cluster. The upregulated MHCII+CD11C- pro-angiogenic genes were unique compared to the increased MHCII+CD11C+ angiogenesis genes. CONCLUSIONS: Macrophage origin impacts heterogeneity at steady state and after laser injury in mice. Both mice and human patients demonstrate similar macrophage subtypes. Two discrete pro-angiogenic macrophage populations exist in the human choroid. Targeting specific, pro-angiogenic macrophage subsets is a potential novel therapeutic for nAMD.

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.

Microglia Adopt Longitudinal Transcriptional Changes After Traumatic Brain Injury.

BACKGROUND: Traumatic brain injury (TBI) is an under-recognized public health threat. Even mild brain injuries can lead to long-term neurologic impairment. Microglia play a fundamental role in the development and progression of this ensuing neurologic impairment. Despite this, a microglia-specific injury signature has yet to be identified. We hypothesized that TBI would lead to long-term changes in the transcriptional profile of microglial pathways associated with the development of subsequent neurologic impairment. MATERIALS AND METHODS: Male C57BL/6 mice underwent TBI via a controlled cortical impact and were followed longitudinally. FACSorted microglia from TBI mice were subjected to Quantiseq 3'-biased RNA sequencing at 7, 30, and 90 d after TBI. K-means clustering on 396 differentially expressed genes was performed, and gene ontology enrichment analysis was used to determine corresponding enriched processes. RESULTS: Differentially expressed genes in microglia exhibited four main patterns of expression over the course of TBI. In particular, we identified four gene clusters which corresponded to the host defense response, synaptic plasticity, lipid remodeling, and membrane polarization. CONCLUSIONS: Transcriptional profiling within individual populations of microglia after TBI remains a critical unmet research need within the field of TBI. This focused study identified several physiologic processes within microglia that may be associated with development of long-term neurologic impairment after TBI. These data demonstrate the capability of longitudinal transcriptional profiling to uncover potential cell-specific targets for the treatment of TBI.

Lipocalin-2 is a pathogenic determinant and biomarker of neuropsychiatric lupus.

Neuropsychiatric manifestations in lupus (NPSLE) affect ∼20-40% of patients. In the central nervous system, lipocalin-2 (LCN2) can promote injury through mechanisms directly linked to NPSLE, including brain barrier disruption, neurotoxicity, and glial activation. Since LCN2 is elevated in lupus and has been implicated in neuroinflammation, we investigated whether LCN2 is required for the pathogenesis of NPSLE. Here, we investigated the effects of LCN2 deficiency on the development of neurobehavioral deficits in the B6.Sle1.Sle3 (Sle1,3) mouse lupus model. Sle1,3 mice exhibited depression-like behavior and impaired spatial and recognition memory, and these deficits were attenuated in Sle1,3-LCN2KO mice. Whole-brain flow cytometry showed a significant increase in brain infiltrating leukocytes in Sle1,3 mice that was not reduced by LCN2 deficiency. RNA sequencing on sorted microglia revealed that several genes differentially expressed between B6 and Sle1,3 mice were regulated by LCN2, and that these genes are key mediators of the neuroinflammatory cascade. Importantly, LCN2 is upregulated in the cerebrospinal fluid of NPSLE patients across 2 different ethnicities. Our findings establish the Sle1,3 strain as an NPSLE model, demonstrate that LCN2 is a major regulator of the detrimental neuroimmune response in NPSLE, and identify CSF LCN2 as a novel biomarker for NPSLE.

Monocyte depletion attenuates the development of posttraumatic hydrocephalus and preserves white matter integrity after traumatic brain injury.

Monocytes are amongst the first cells recruited into the brain after traumatic brain injury (TBI). We have shown monocyte depletion 24 hours prior to TBI reduces brain edema, decreases neutrophil infiltration and improves behavioral outcomes. Additionally, both lesion and ventricle size correlate with poor neurologic outcome after TBI. Therefore, we aimed to determine the association between monocyte infiltration, lesion size, and ventricle volume. We hypothesized that monocyte depletion would attenuate lesion size, decrease ventricle enlargement, and preserve white matter in mice after TBI. C57BL/6 mice underwent pan monocyte depletion via intravenous injection of liposome-encapsulated clodronate. Control mice were injected with liposome-encapsulated PBS. TBI was induced via an open-head, controlled cortical impact. Mice were imaged using magnetic resonance imaging (MRI) at 1, 7, and 14 days post-injury to evaluate progression of lesion and to detect morphological changes associated with injury (3D T1-weighted MRI) including regional alterations in white matter patterns (multi-direction diffusion MRI). Lesion size and ventricle volume were measured using semi-automatic segmentation and active contour methods with the software program ITK-SNAP. Data was analyzed with the statistical software program PRISM. No significant effect of monocyte depletion on lesion size was detected using MRI following TBI (p = 0.4). However, progressive ventricle enlargement following TBI was observed to be attenuated in the monocyte-depleted cohort (5.3 ± 0.9mm3) as compared to the sham-depleted cohort (13.2 ± 3.1mm3; p = 0.02). Global white matter integrity and regional patterns were evaluated and quantified for each mouse after extracting fractional anisotropy maps from the multi-direction diffusion-MRI data using Siemens Syngo DTI analysis package. Fractional anisotropy (FA) values were preserved in the monocyte-depleted cohort (123.0 ± 4.4mm3) as compared to sham-depleted mice (94.9 ± 4.6mm3; p = 0.025) by 14 days post-TBI. All TBI mice exhibited FA values lower than those from a representative naïve control group with intact white matter tracts and FA~200 mm3). The MRI derived assessment of injury progression suggests that monocyte depletion at the time of injury may be a novel therapeutic strategy in the treatment of TBI. Furthermore, non-invasive longitudinal imaging allows for the evaluation of both TBI progression as well as therapeutic response over the course of injury.

Neuropsychiatric Systemic Lupus Erythematosus Is Dependent on Sphingosine-1-Phosphate Signaling.

About 40% of patients with systemic lupus erythematosus experience diffuse neuropsychiatric manifestations, including impaired cognition and depression. Although the pathogenesis of diffuse neuropsychiatric SLE (NPSLE) is not fully understood, loss of brain barrier integrity, autoreactive antibodies, and pro-inflammatory cytokines are major contributors to disease development. Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator, prevents lymphocyte egress from lymphoid organs through functional antagonism of S1P receptors. In addition to reducing the circulation of autoreactive lymphocytes, fingolimod has direct neuroprotective effects such as preserving brain barrier integrity and decreasing pro-inflammatory cytokine secretion by astrocytes and microglia. Given these effects, we hypothesized that fingolimod would attenuate neurobehavioral deficits in MRL-lpr/lpr (MRL/lpr) mice, a validated neuropsychiatric lupus model. Fingolimod treatment was initiated after the onset of disease, and mice were assessed for alterations in cognitive function and emotionality. We found that fingolimod significantly attenuated spatial memory deficits and depression-like behavior in MRL/lpr mice. Immunofluorescent staining demonstrated a dramatic lessening of brain T cell and macrophage infiltration, and a significant reduction in cortical leakage of serum albumin, in fingolimod treated mice. Astrocytes and endothelial cells from treated mice exhibited reduced expression of inflammatory genes, while microglia showed differential regulation of key immune pathways. Notably, cytokine levels within the cortex and hippocampus were not appreciably decreased with fingolimod despite the improved neurobehavioral profile. Furthermore, despite a reduction in splenomegaly, lymphadenopathy, and circulating autoantibody titers, IgG deposition within the brain was unaffected by treatment. These findings suggest that fingolimod mediates attenuation of NPSLE through a mechanism that is not dependent on reduction of autoantibodies or cytokines, and highlight modulation of the S1P signaling pathway as a novel therapeutic target in lupus involving the central nervous system.

Highly selective inhibition of Bruton's tyrosine kinase attenuates skin and brain disease in murine lupus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects different end organs, including skin and brain. We and others have previously shown the importance of macrophages in the pathogenesis of cutaneous and neuropsychiatric lupus. Additionally, autoantibodies produced by autoreactive B cells are thought to play a role in both the skin and central nervous system pathologies associated with SLE. METHODS: We used a novel inhibitor of Bruton's tyrosine kinase (BTK), BI-BTK-1, to target both macrophage and B cell function in the MRL-lpr/lpr murine model of SLE, and examined the effect of treatment on skin and brain disease. RESULTS: We found that treatment with BI-BTK-1 significantly attenuated the lupus associated cutaneous and neuropsychiatric disease phenotypes in MRL/lpr mice. Specifically, BI-BTK-1 treated mice had fewer macroscopic and microscopic skin lesions, reduced cutaneous cellular infiltration, and diminished inflammatory cytokine expression compared to control mice. BTK inhibition also significantly improved cognitive function, and decreased accumulation of T cells, B cells, and macrophages within the central nervous system, specifically the choroid plexus. CONCLUSIONS: Directed therapies may improve the response rate in lupus-driven target organ involvement, and decrease the dangerous side effects associated with global immunosuppression. Overall, our results suggest that inhibition of BTK may be a promising therapeutic option for cutaneous and neuropsychiatric disease associated with SLE.

Nonclassical Monocytes Mediate Secondary Injury, Neurocognitive Outcome, and Neutrophil Infiltration after Traumatic Brain Injury.

Traumatic brain injury (TBI) results in rapid recruitment of leukocytes into the injured brain. Monocytes constitute a significant proportion of the initial infiltrate and have the potential to propagate secondary brain injury or generate an environment of repair and regeneration. Monocytes are a diverse population of cells (classical, intermediate, and nonclassical) with distinct functions, however, the recruitment order of these subpopulations to the injured brain largely remains unknown. Thus, we examined which monocyte subpopulations are required for the generation of early inflammatory infiltrate within the injured brain, and whether their depletion attenuates secondary injury or neurocognitive outcome. Global monocyte depletion correlated with significant improvements in brain edema, motor coordination, and working memory, and abrogated neutrophil infiltration into the injured brain. However, targeted depletion of classical monocytes alone had no effect on neutrophil recruitment to the site of injury, implicating the nonclassical monocyte in this process. In contrast, mice that have markedly reduced numbers of nonclassical monocytes (CX3CR1-/-) exhibited a significant reduction in neutrophil infiltration into the brain after TBI as compared with control mice. Our data suggest a critical role for nonclassical monocytes in the pathology of TBI in mice, including important clinical outcomes associated with mortality in this injury process.

The Role of Microglia in the Etiology and Evolution of Chronic Traumatic Encephalopathy.

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disease that presents as a late sequela from traumatic brain injury (TBI). TBI is a growing and under-recognized public health concern with a high degree of morbidity and large associated global costs. While the immune response to TBI is complex, its contribution to the development of CTE remains largely unknown. In this review, we summarize the current understanding of the link between CTE and the resident innate immune system of the brain-microglia. We discuss the neuropathology underlying CTE including the creation and aggregation of phosphorylated tau protein into neurofibrillary tangles and the formation of amyloid beta deposits. We also present how microglia, the resident innate immune cells of the brain, drive the continuous low-level inflammation associated with the insidious onset of CTE. In this review, we conclude that the latency period between the index brain injury and the long-term development of CTE presents an opportunity for therapeutic intervention. Encouraging advances with microtubule stabilizers, cis p-tau antibodies, and the ability to therapeutically alter the inflammatory state of microglia have shown positive results in both animal and human trials. Looking forward, recent advancements in next-generation sequencing technology for the study of genomic, transcriptomic, and epigenetic information will provide an opportunity for significant advancement in our understanding of prorepair and pro-injury gene signatures allowing for targeted intervention in this highly morbid injury process.

Characterization of IL-22 and IL-17 Expressing Leukocytes in the Cervix.

PROBLEM: Identification of the types of cells that produce IL-17 and IL-22 in the genital tract can clarify the roles that these cytokines play in responses to pathogens. METHOD OF STUDY: We isolated and stimulated cells from cervical tissue to identify and characterize cytokine-producing cells. RESULTS: Upon stimulation of CD3+ CD4+ endocervical cells, 1.6, 3.4, and 1.5% were induced to produce IL-22, IL-17, and both cytokines, respectively. Stimulation of CD3+ CD4+ ectocervical cells resulted in 3.3% IL-22+, 5.5% IL-17(+) and 2.6% IL-22(+) IL17+ cells. CD45+ CD3- cells had relatively high endogenous levels of cytokine expression that did not increase upon stimulation. Innate lymphoid cells (ILCs) made up 5.7-8% of CD45+ cervical cells and stimulation caused increases in IL-17 and IL-22. CONCLUSION: These studies show that the majority of the CD45+ leukocytes that can be induced to produce IL-22 and IL-17 in cervix are CD3+ CD4+, but ILCs are also present and can make both cytokines.

Effect of pH on Cleavage of Glycogen by Vaginal Enzymes.

Glycogen expressed by the lower genital tract epithelium is believed to support Lactobacillus growth in vivo, although most genital isolates of Lactobacillus are not able to use glycogen as an energy source in vitro. We recently reported that α-amylase is present in the genital fluid of women and that it breaks down glycogen into small carbohydrates that support growth of lactobacilli. Since the pH of the lower genital tract can be very low, we determined how low pH affects glycogen processing by α-amylase. α-amylase in saliva degraded glycogen similarly at pH 6 and 7, but activity was reduced by 52% at pH 4. The glycogen degrading activity in nine genital samples from seven women showed a similar profile with an average reduction of more than 50% at pH 4. However, two samples collected from one woman at different times had a strikingly different pH profile with increased glycogen degradation at pH 4, 5 and 6 compared to pH 7. This second pH profile did not correlate with levels of human α-acid glucosidase or human intestinal maltase glucoamylase. High-performance anion-exchange chromatography showed that mostly maltose was produced from glycogen by samples with the second pH profile in contrast to genital α-amylase that yielded maltose, maltotriose and maltotetraose. These studies show that at low pH, α-amylase activity is reduced to low but detectable levels, which we speculate helps maintain Lactobacillus growth at a limited but sustained rate. Additionally, some women have a genital enzyme distinct from α-amylase with higher activity at low pH. Further studies are needed to determine the identity and distribution of this second enzyme, and whether its presence influences the makeup of genital microbiota.

IL-22 levels are associated with Trichomonas vaginalis infection in the lower genital tract.

PROBLEM: IL-22 has important functions at mucosal surfaces, including the induction of antimicrobial peptides and maintenance of epithelium. However, IL-22 has not been investigated in the genital tract during TV infection. METHODS OF STUDY: Women who visited an STD clinic and women from a cohort with frequent Trichomoniasis were studied. IL-22, IL-17, and antimicrobial peptides were measured in cervicovaginal lavage by ELISA. RESULTS: In women visiting the STD clinic, those without STDs (n = 10) had a median IL-22 of 0 pg/mL, while women with infections (n = 30) had 27 pg/mL (P = 0.04). In the cohort, women with Trichomoniasis (n = 19) had significantly higher IL-22 than women with no infections (n = 21, 74 versus 0 pg/mL, P = 0.0001). IL-17 was also significantly increased in Trichomoniasis, and there was a correlation between IL-22 and IL-17 (P = 0.001). CONCLUSION: IL-22 is increased in STDs generally and in Trichomoniasis specifically suggesting an antimicrobial response of the mucosa and an epithelial repair process induced by the STDs.

Short-chain fatty acids induce pro-inflammatory cytokine production alone and in combination with toll-like receptor ligands.

PROBLEM: Short-chain fatty acids (SCFAs), produced at relatively high levels by anaerobic bacteria in bacterial vaginosis (BV), are believed to be anti-inflammatory. BV, a common alteration in the genital microbiota associated with increased susceptibility to HIV infection, is characterized by increased levels of both pro-inflammatory cytokines and SCFAs. We investigated how SCFAs alone or together with Toll-like receptor (TLR) ligands affected pro-inflammatory cytokine secretion. METHOD OF STUDY: Cytokines were measured by ELISA. Flow was used for phenotyping and reactive oxygen species (ROS) measurement. RESULTS: Short-chain fatty acids, at 20 mM, induced interleukin (IL)-8, IL-6, and IL-1ß release, while lower levels (0.02-2 mM) did not induce cytokine secretion. Levels >20 mM were toxic to cells. Interestingly, lower levels of SCFAs significantly enhanced TLR2 ligand- and TLR7 ligand-induced production of IL-8 and TNFα in a time- and dose-dependent manner, but had little effect on lipopolysaccharide-induced cytokine release. SCFAs mediated their effects on pro-inflammatory cytokine production at least in part by inducing the generation of ROS. CONCLUSION: Our data suggest that SCFAs, especially when combined with specific TLR ligands, contribute to a pro-inflammatory milieu in the lower genital tract and help further our understanding of how BV affects susceptibility to microbial infections.