Publications by authors named "Carla M Cuda"

35 Publications

Transcriptional profiling of pediatric cholestatic livers identifies three distinct macrophage populations.

PLoS One 2021 7;16(1):e0244743. Epub 2021 Jan 7.

Division of Rheumatology, Department of Medicine, Northwestern University, Chicago, Illinois, United States of America.

Background & Aims: Limited understanding of the role for specific macrophage subsets in the pathogenesis of cholestatic liver injury is a barrier to advancing medical therapy. Macrophages have previously been implicated in both the mal-adaptive and protective responses in obstructive cholestasis. Recently two macrophage subsets were identified in non-diseased human liver; however, no studies to date fully define the heterogeneous macrophage subsets during the pathogenesis of cholestasis. Here, we aim to further characterize the transcriptional profile of macrophages in pediatric cholestatic liver disease.

Methods: We isolated live hepatic immune cells from patients with biliary atresia (BA), Alagille syndrome (ALGS), and non-cholestatic pediatric liver by fluorescence activated cell sorting. Through single-cell RNA sequencing analysis and immunofluorescence, we characterized cholestatic macrophages. We next compared the transcriptional profile of pediatric cholestatic and non-cholestatic macrophage populations to previously published data on normal adult hepatic macrophages.

Results: We identified 3 distinct macrophage populations across cholestatic liver samples and annotated them as lipid-associated macrophages, monocyte-like macrophages, and adaptive macrophages based on their transcriptional profile. Immunofluorescence of liver tissue using markers for each subset confirmed their presence across BA (n = 6) and ALGS (n = 6) patients. Cholestatic macrophages demonstrated reduced expression of immune regulatory genes as compared to normal hepatic macrophages and were distinct from macrophage populations defined in either healthy adult or pediatric non-cholestatic liver.

Conclusions: We are the first to perform single-cell RNA sequencing on human pediatric cholestatic liver and identified three macrophage subsets with distinct transcriptional signatures from healthy liver macrophages. Further analyses will identify similarities and differences in these macrophage sub-populations across etiologies of cholestatic liver disease. Taken together, these findings may allow for future development of targeted therapeutic strategies to reprogram macrophages to an immune regulatory phenotype and reduce cholestatic liver injury.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0244743PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790256PMC
May 2021

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

J Neuroinflammation 2020 Nov 13;17(1):341. Epub 2020 Nov 13.

Department of Ophthalmology, Feinberg School of Medicine, Northwestern University, 240 E Huron St, McGaw M343, Chicago, IL, 60611, USA.

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 Cx3cr1 ; Rosa26 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, MHCIICD11c, MHCIICD11c, 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 MHCIICD11c and MHCIICD11c macrophages are partially replenished from blood monocytes. After laser injury, MHCIICD11c macrophages are entirely derived from classical monocytes, MHCII macrophages originate from classical monocytes (90%) and an expansion of tissue resident macrophages (10%), and MHCIICD11c 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 MHCIICD11C and three MHCIICD11C populations. One MHCIICD11C subset was 78% derived from a patient with nAMD. Differential expression analysis identified up-regulation of pro-angiogenic gene expression in one MHCIICD11C and two MHCIICD11C subsets, including the disease-associated cluster. The upregulated MHCIICD11C pro-angiogenic genes were unique compared to the increased MHCIICD11C 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.
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http://dx.doi.org/10.1186/s12974-020-02010-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666512PMC
November 2020

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes.

J Vis Exp 2020 06 17(160). Epub 2020 Jun 17.

Department of Ophthalmology, Feinberg School of Medicine, Northwestern University;

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.
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http://dx.doi.org/10.3791/61348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757749PMC
June 2020

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

Front Immunol 2020 26;11:230. Epub 2020 Feb 26.

Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States.

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.
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http://dx.doi.org/10.3389/fimmu.2020.00230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055359PMC
March 2021

Monocyte-Derived Macrophages Are Necessary for Beta-Adrenergic Receptor-Driven Choroidal Neovascularization Inhibition.

Invest Ophthalmol Vis Sci 2019 12;60(15):5059-5069

Department of Ophthalmology, Feinberg School of Medicine, Northwestern University, Chicago, Ilinois, United States.

Purpose: Beta-adrenergic receptor (AR) antagonists, like propranolol, inhibit angiogenesis in multiple ocular conditions through an unknown mechanism. We previously showed that propranolol reduces choroidal neovascularization (CNV) by decreasing interleukin-6 levels. Since macrophages are one of the central producers of interleukin-6, we examined whether macrophages are required for propranolol-driven inhibition of choroidal angiogenesis.

Methods: We tested the anti-angiogenic properties of propranolol in the choroidal sprouting assay and the laser-induced CNV model. Bone marrow-derived monocytes (BMDMs) were added to the choroidal sprouting assay and Ccr2-/- mice were subjected to laser-induced CNV. Multi-parameter flow cytometry was performed to characterize the ocular mononuclear phagocyte populations after laser injury and during propranolol treatment.

Results: Propranolol reduced choroidal angiogenesis by 41% (P < 0.001) in the choroidal sprouting assay. Similarly, propranolol decreased laser-induced CNV by 50% (P < 0.05) in female mice, with no change in males. BMDMs increased choroidal sprouting by 146% (P < 0.0001), and this effect was ablated by propranolol. Beta-AR inhibition had no effect upon laser-induced CNV area in female Ccr2-/- mice. MHCII+ and MHCII- macrophages increased 20-fold following laser treatment in wildtype mice as compared to untreated mice, and this effect was completely attenuated in lasered Ccr2-/- mice. Moreover, propranolol increased the numbers of MHCII+ and MHCII- macrophages by 1.9 (P = 0.07) and 3.1 (P < 0.05) fold in lasered female mice with no change in macrophage numbers in males.

Conclusions: Our data suggest that propranolol inhibits angiogenesis through recruitment of monocyte-derived macrophages in female mice only. These data show the anti-angiogenic nature of beta-AR blocker-recruited monocyte-derived macrophages in CNV.
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http://dx.doi.org/10.1167/iovs.19-28165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894922PMC
December 2019

A spatially restricted fibrotic niche in pulmonary fibrosis is sustained by M-CSF/M-CSFR signalling in monocyte-derived alveolar macrophages.

Eur Respir J 2020 01 16;55(1). Epub 2020 Jan 16.

Division of Pulmonary and Critical Care Medicine, Dept of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.
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http://dx.doi.org/10.1183/13993003.00646-2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962769PMC
January 2020

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

PLoS One 2018 1;13(11):e0202722. Epub 2018 Nov 1.

Department of Surgery, Division of Trauma and Critical Care, Northwestern University, Chicago, Illinois, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202722PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211627PMC
April 2019

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

Front Immunol 2018 26;9:2189. Epub 2018 Sep 26.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, United States.

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.
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http://dx.doi.org/10.3389/fimmu.2018.02189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168636PMC
October 2019

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

J Autoimmun 2019 01 30;96:59-73. Epub 2018 Aug 30.

Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY, USA; Division of Rheumatology, Albert Einstein College of Medicine, Bronx, NY, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jaut.2018.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310639PMC
January 2019

Transcriptional Profiling of Synovial Macrophages Using Minimally Invasive Ultrasound-Guided Synovial Biopsies in Rheumatoid Arthritis.

Arthritis Rheumatol 2018 06 3;70(6):841-854. Epub 2018 May 3.

Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Objective: Currently, there are no reliable biomarkers for predicting therapeutic response in patients with rheumatoid arthritis (RA). The synovium may unlock critical information for determining efficacy, since a reduction in the numbers of sublining synovial macrophages remains the most reproducible biomarker. Thus, a clinically actionable method for the collection of synovial tissue, which can be analyzed using high-throughput strategies, must become a reality. This study was undertaken to assess the feasibility of utilizing synovial biopsies as a precision medicine-based approach for patients with RA.

Methods: Rheumatologists at 6 US academic sites were trained in minimally invasive ultrasound-guided synovial tissue biopsy. Biopsy specimens obtained from patients with RA and synovial tissue from patients with osteoarthritis (OA) were subjected to histologic analysis, fluorescence-activated cell sorting, and RNA sequencing (RNA-seq). An optimized protocol for digesting synovial tissue was developed to generate high-quality RNA-seq libraries from isolated macrophage populations. Associations were determined between macrophage transcriptional profiles and clinical parameters in RA patients.

Results: Patients with RA reported minimal adverse effects in response to synovial biopsy. Comparable RNA quality was observed from synovial tissue and isolated macrophages between patients with RA and patients with OA. Whole tissue samples from patients with RA demonstrated a high degree of transcriptional heterogeneity. In contrast, the transcriptional profile of isolated RA synovial macrophages highlighted different subpopulations of patients and identified 6 novel transcriptional modules that were associated with disease activity and therapy.

Conclusion: Performance of synovial tissue biopsies by rheumatologists in the US is feasible and generates high-quality samples for research. Through the use of cutting-edge technologies to analyze synovial biopsy specimens in conjunction with corresponding clinical information, a precision medicine-based approach for patients with RA is attainable.
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http://dx.doi.org/10.1002/art.40453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984677PMC
June 2018

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

Arthritis Res Ther 2018 01 25;20(1):10. Epub 2018 Jan 25.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA.

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.
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http://dx.doi.org/10.1186/s13075-017-1500-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785891PMC
January 2018

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

J Exp Med 2017 Dec 7;214(12):3753-3773. Epub 2017 Nov 7.

Division of Rheumatology, Feinberg School of Medicine, Northwestern University, Chicago, IL

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 (LysMBim) 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. LysMBim mice exhibit increased mortality attributed to glomerulonephritis (GN). Moreover, the toll-like receptor signaling adaptor protein TRIF (TIR-domain-containing adapter-inducing interferon-β) is essential for GN, but not systemic autoimmunity in LysMBim 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.
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http://dx.doi.org/10.1084/jem.20170479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716039PMC
December 2017

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

J Immunol 2017 11 9;199(10):3583-3591. Epub 2017 Oct 9.

Division of Trauma and Critical Care, Department of Surgery, Northwestern University, Chicago, IL 60611; and

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.
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http://dx.doi.org/10.4049/jimmunol.1700896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679470PMC
November 2017

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

Arthritis Res Ther 2017 Oct 4;19(1):224. Epub 2017 Oct 4.

Department of Medicine, Division of Rheumatology, Northwestern University, Feinberg School of Medicine, 240 East Huron Street, M300, Chicago, IL, 60611, USA.

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 Casp8 and Cre Casp8 mice were bred via a cross between Casp8 and Cre or Cre mice. RIPK3 Cre Casp8 and RIPK3 Cre Casp8 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.
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http://dx.doi.org/10.1186/s13075-017-1436-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628498PMC
October 2017

Genetic deficiency of Wnt5a diminishes disease severity in a murine model of rheumatoid arthritis.

Arthritis Res Ther 2017 07 19;19(1):166. Epub 2017 Jul 19.

Molecular Cardiology, Whitaker Cardiovascular Institute, Boston University School of Medicine, 700 Albany Street, W-611, Boston, MA, 02118, USA.

Background: Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic inflammation of the joints, leading to bone erosion and joint dysfunction. Despite the recent successes of disease-modifying anti-rheumatic drugs (DMARDs), there is still clinical need for understanding the development and molecular etiology of RA. Wnts are developmental morphogens whose roles in adult pathology are poorly characterized. Wnt5a is a member of the non-canonical family of Wnts that modulates a wide range of cell processes, including differentiation, migration, and inflammation. Wnt5a has been implicated as a possible contributor to arthritis and it is upregulated in synovial fibroblasts from RA patients.

Methods: We investigated the role of endogenous Wnt5a in RA. Tamoxifen-inducible, Wnt5a knockout (Wnt5a cKO) mice and littermate controls were monitored for arthritis development and joint pathology using the K/BxN serum transfer-induced arthritis (STIA) model. To explore a role of Wnt5a in osteoclast fusion, bone marrow-derived monocytes (BMDMs) were differentiated in vitro.

Results: Wnt5a cKO mice were resistant to arthritis development compared to control littermates as assessed by ankle thickness and histologic measurements. Some parameters of inflammation were reduced in the Wnt5a cKO mice, including the extent of polymononuclear cell infiltration and extra-articular inflammation. Wnt5a cKO mice also exhibited less cartilage destruction and a reduction in osteoclast activity with concomitant reduction in tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), macrophage colony-stimulating factor (MCSF), matrix metalloproteinase (MMP)2 and MMP9 in the arthritic joints. Treatment of BMDMs with Wnt5a enhanced osteoclast fusion and increased the expression of dendrocyte-expressed seven transmembrane protein (DCSTAMP) and MMP9, that are necessary for osteoclast formation and activity.

Conclusions: These data suggest that Wnt5a modulates the development of arthritis by promoting inflammation and osteoclast fusion, and provide the first mouse genetic evidence of a role for endogenous Wnt5a in autoimmune disease.
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http://dx.doi.org/10.1186/s13075-017-1375-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518154PMC
July 2017

Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span.

J Exp Med 2017 Aug 10;214(8):2387-2404. Epub 2017 Jul 10.

Division of Thoracic Surgery, Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL.

Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.
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http://dx.doi.org/10.1084/jem.20162152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551573PMC
August 2017

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

Shock 2017 09;48(3):276-283

*Division of Trauma and Critical Care, Department of Surgery, Northwestern University, Chicago, Illinois †Division of Rheumatology, Department of Medicine, Northwestern University, Chicago, Illinois.

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.
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http://dx.doi.org/10.1097/SHK.0000000000000859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555778PMC
September 2017

Temporal Expression of Bim Limits the Development of Agonist-Selected Thymocytes and Skews Their TCRβ Repertoire.

J Immunol 2017 01 16;198(1):257-269. Epub 2016 Nov 16.

Division of Immunobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229;

CD8αα TCRαβ intestinal intraepithelial lymphocytes play a critical role in promoting intestinal homeostasis, although mechanisms controlling their development and peripheral homeostasis remain unclear. In this study, we examined the spatiotemporal role of Bim in the thymic selection of CD8αα precursors and the fate of these cells in the periphery. We found that T cell-specific expression of Bim during early/cortical, but not late/medullary, thymic development controls the agonist selection of CD8αα precursors and limits their private TCRβ repertoire. During this process, agonist-selected double-positive cells lose CD4/8 coreceptor expression and masquerade as double-negative (DN) TCRαβ thymocytes. Although these DN thymocytes fail to re-express coreceptors after OP9-DL1 culture, they eventually mature and accumulate in the spleen where TCR and IL-15/STAT5 signaling promotes their conversion to CD8αα cells and their expression of gut-homing receptors. Adoptive transfer of splenic DN cells gives rise to CD8αα cells in the gut, establishing their precursor relationship in vivo. Interestingly, Bim does not restrict the IL-15-driven maturation of CD8αα cells that is critical for intestinal homeostasis. Thus, we found a temporal and tissue-specific role for Bim in limiting thymic agonist selection of CD8αα precursors and their TCRβ repertoire, but not in the maintenance of CD8αα intraepithelial lymphocytes in the intestine.
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http://dx.doi.org/10.4049/jimmunol.1601200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568849PMC
January 2017

The contribution of the programmed cell death machinery in innate immune cells to lupus nephritis.

Clin Immunol 2017 12 22;185:74-85. Epub 2016 Oct 22.

Northwestern University, Feinberg School of Medicine, Department of Medicine, Division of Rheumatology, 240 East Huron Street, Room M300, Chicago, IL 60611, USA. Electronic address:

Systemic lupus erythematosus (SLE) is a chronic multi-factorial autoimmune disease initiated by genetic and environmental factors, which in combination trigger disease onset in susceptible individuals. Damage to the kidney as a consequence of lupus nephritis (LN) is one of the most prevalent and severe outcomes, as LN affects up to 60% of SLE patients and accounts for much of SLE-associated morbidity and mortality. As remarkable strides have been made in unlocking new inflammatory mechanisms associated with signaling molecules of programmed cell death pathways, this review explores the available evidence implicating the action of these pathways specifically within dendritic cells and macrophages in the control of kidney disease. Although advancements into the underlying mechanisms responsible for inducing cell death inflammatory pathways have been made, there still exist areas of unmet need. By understanding the molecular mechanisms by which dendritic cells and macrophages contribute to LN pathogenesis, we can improve their viability as potential therapeutic targets to promote remission.
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http://dx.doi.org/10.1016/j.clim.2016.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5401656PMC
December 2017

The inflammatory role of phagocyte apoptotic pathways in rheumatic diseases.

Nat Rev Rheumatol 2016 08;12(9):543-58

Northwestern University, Feinberg School of Medicine, Department of Medicine, Division of Rheumatology, 240 East Huron Street, Chicago, Illinois 60611, USA.

Rheumatoid arthritis affects nearly 1% of the world's population and is a debilitating autoimmune condition that can result in joint destruction. During the past decade, inflammatory functions have been described for signalling molecules classically involved in apoptotic and non-apoptotic death pathways, including, but not limited to, Toll-like receptor signalling, inflammasome activation, cytokine production, macrophage polarization and antigen citrullination. In light of these remarkable advances in the understanding of inflammatory mechanisms of the death machinery, this Review provides a snapshot of the available evidence implicating death pathways, especially within the phagocyte populations of the innate immune system, in the perpetuation of rheumatoid arthritis and other rheumatic diseases. Elevated levels of signalling mediators of both extrinsic and intrinsic apoptosis, as well as the autophagy, are observed in the joints of patients with rheumatoid arthritis. Furthermore, risk polymorphisms are present in signalling molecules of the extrinsic apoptotic and autophagy death pathways. Although research into the mechanisms underlying these pathways has made considerable progress, this Review highlights areas where further investigation is particularly needed. This exploration is critical, as new discoveries in this field could lead to the development of novel therapies for rheumatoid arthritis and other rheumatic diseases.
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http://dx.doi.org/10.1038/nrrheum.2016.132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297631PMC
August 2016

ApoE deficiency exacerbates the development and sustainment of a semi-chronic K/BxN serum transfer-induced arthritis model.

J Transl Med 2016 06 10;14(1):170. Epub 2016 Jun 10.

Division of Rheumatology, Department of Medicine, Northwestern University Feinberg School of Medicine, 240 East Huron Street, McGaw M338, Chicago, IL, 60611, USA.

Background: The risk for developing cardiovascular disease is greater in patients with rheumatoid arthritis (RA) than in the general population. While patients with RA also have dyslipidemia, the impact of dyslipidemia on the severity of inflammatory arthritis and associated cardiovascular disease is unclear. Currently, there are conflicting results regarding arthritis incidence in apolipoprotein E (ApoE) deficient mice, which spontaneously exhibit both hyperlipidemia and atherosclerosis. Here, we utilize a distinct approach to investigate the contribution of a hyperlipidemic environment on the development of arthritis and atherosclerosis in mice lacking ApoE.

Methods: K/BxN serum transfer-induced arthritis (STIA) was assessed in C57BL/6 (control) and ApoE(-/-) mice using clinical indices and immunohistochemical staining. Ankle synoviums were processed for flow cytometry. Aortic atherosclerosis was quantitated using Sudan IV staining. Serum cholesterol and cytokine levels were determined via enzymatic and luminex bead-based assays, respectively.

Results: ApoE(-/-) mice developed a sustained and enhanced semi-chronic inflammatory arthritis as compared to control mice. ApoE(-/-) mice had increased numbers of foamy macrophages, enhanced joint inflammation and amplified collagen deposition versus controls. The presence of arthritis did not exacerbate serum cholesterol levels or significantly augment the level of atherosclerosis in ApoE(-/-) mice. However, arthritic ApoE(-/-) mice exhibited a marked elevation of IL-6 as compared to non-arthritic ApoE(-/-) mice and arthritic C57BL/6 mice.

Conclusions: Loss of ApoE potentiates a semi-chronic inflammatory arthritis. This heightened inflammatory response was associated with an increase in circulating IL-6 and in the number of foamy macrophages within the joint. Moreover, the foamy macrophages within the arthritic joint are reminiscent of those within unstable atherosclerotic lesions and suggest a pathologic role for foamy macrophages in propagating arthritis.
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http://dx.doi.org/10.1186/s12967-016-0912-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901400PMC
June 2016

Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner.

Arthritis Res Ther 2015 Oct 16;17:291. Epub 2015 Oct 16.

Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 East Huron Street, Room M300, Chicago, IL, 60611, USA.

Introduction: Although caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. As cells of the innate immune system, and in particular macrophages, are now at the forefront of autoimmune disease pathogenesis, we examined the potential involvement of caspase-8 within this population.

Methods: Cre (LysM) Casp8 (fl/fl) mice were bred via a cross between Casp8 (fl/fl) mice and Cre (LysM) mice, and RIPK3 (-/-) Cre (LysM) Casp8 (fl/fl) mice were generated to assess the contribution of receptor-interacting serine-threonine kinase (RIPK)3. Immunohistochemical and immunofluorescence analyses were used to examine renal damage. Flow cytometric analysis was employed to characterize splenocyte distribution and activation. Cre (LysM) Casp8 (fl/fl) mice were treated with either Toll-like receptor (TLR) agonists or oral antibiotics to assess their response to TLR activation or TLR agonist removal. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure cytokine/chemokine and immunoglobulin levels in serum and cytokine levels in cell culture studies. In vitro cell culture was used to assess macrophage response to cell death stimuli, TLR activation, and M1/M2 polarization. Data were compared using the Mann-Whitney U test.

Results: Loss of caspase-8 expression in macrophages promotes onset of a mild systemic inflammatory disease, which is preventable by the deletion of RIPK3. In vitro cell culture studies reveal that caspase-8-deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8-deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations. Loss of caspase-8 in macrophages dictates the response to TLR activation, as injection of TLR ligands upregulates expression of costimulatory CD86 on the Ly6C(high)CD11b(+)F4/80(+) splenic cells, and oral antibiotic treatment to remove microbiota prevents splenomegaly and lymphadenopathy in Cre (LysM) Casp8 (fl/fl) mice. Further, caspase-8-deficient macrophages are hyperresponsive to TLR activation and exhibit aberrant M1 macrophage polarization due to RIPK activity.

Conclusions: These data demonstrate that caspase-8 functions uniquely in macrophages by controlling the response to TLR activation and macrophage polarization in an RIPK-dependent manner.
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http://dx.doi.org/10.1186/s13075-015-0794-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608154PMC
October 2015

Promotion of Inflammatory Arthritis by Interferon Regulatory Factor 5 in a Mouse Model.

Arthritis Rheumatol 2015 Dec;67(12):3146-57

Boston University School of Medicine, Boston, Massachusetts.

Objective: Polymorphisms in the transcription factor interferon regulatory factor 5 (IRF5) are associated with an increased risk of developing rheumatoid arthritis (RA). This study was undertaken to determine the role of IRF5 in a mouse model of arthritis development.

Methods: K/BxN serum-transfer arthritis was induced in mice deficient in IRF5, or lacking IRF5 only in myeloid cells, and arthritis severity was evaluated. K/BxN arthritis was also induced in mice deficient in TRIF, Toll-like receptor 2 (TLR2), TLR3, TLR4, and TLR7 to determine the pathways through which IRF5 might promote arthritis. In vitro studies were performed to determine the role of IRF5 in interleukin-1 (IL-1) receptor and TLR signaling.

Results: Arthritis severity was reduced in IRF5-deficient, TRIF-deficient, TLR3-deficient, and TLR7-deficient mice. The expression of multiple genes regulating neutrophil recruitment or function and bioactive IL-1β formation was reduced in the joints during active arthritis in IRF5-deficient mice. In vitro studies showed that TLR7 and the TRIF-dependent TLR3 pathway induce proinflammatory cytokine production in disease-relevant cell types in an IRF5-dependent manner.

Conclusion: Our findings indicate that IRF5 contributes to disease pathogenesis in inflammatory arthritis. This is likely due at least in part to the role of IRF5 in mediating proinflammatory cytokine production downstream of TLR7 and TLR3. Since TLR7 and TLR3 are both RNA-sensing TLRs, this suggests that endogenous RNA ligands present in the inflamed joint promote arthritis development. These findings may be relevant to human RA, since RNA capable of activating TLR7 and TLR3 is present in synovial fluid and TLR7 and TLR3 are up-regulated in the joints of RA patients.
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http://dx.doi.org/10.1002/art.39321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661118PMC
December 2015

Differential Activation of Infiltrating Monocyte-Derived Cells After Mild and Severe Traumatic Brain Injury.

Shock 2015 Mar;43(3):255-60

*Department of Surgery, Division of Trauma and Critical Care, and †Department of Medicine, Division of Rheumatology, Northwestern University, Chicago, Illinois.

Microglia are the resident innate immune cells of the brain. Although embryologically and functionally distinct, they are morphologically similar to peripheral monocyte-derived cells, resulting in a poor ability to discriminate between the two cell types. The purpose of this study was to develop a rapid and reliable method to simultaneously characterize, quantify, and discriminate between whole populations of myeloid cells from the brain in a murine model of traumatic brain injury. Male C57BL/6 mice underwent traumatic brain injury (n = 16) or sham injury (n = 14). Brains were harvested at 24 h after injury. Multiparameter flow cytometry and sequential gating analysis were performed, allowing for discrimination between microglia and infiltrating leukocytes as well as for the characterization and quantification of individual subtypes within the infiltrating population. The proportion of infiltrating leukocytes within the brain increased with the severity of injury, and the predominant cell types within the infiltrating population were monocyte derived (P = 0.01). In addition, the severity of injury altered the overall makeup of the infiltrating monocyte-derived cells. In conclusion, we describe a flow cytometry-based technique for gross discrimination between infiltrating leukocytes and microglia as well as the ability to simultaneously characterize and quantify individual myeloid subtypes and their maturation states within these populations.
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http://dx.doi.org/10.1097/SHK.0000000000000291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476068PMC
March 2015

Nonclassical Ly6C(-) monocytes drive the development of inflammatory arthritis in mice.

Cell Rep 2014 Oct 16;9(2):591-604. Epub 2014 Oct 16.

Division of Rheumatology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. Electronic address:

Different subsets and/or polarized phenotypes of monocytes and macrophages may play distinct roles during the development and resolution of inflammation. Here, we demonstrate in a murine model of rheumatoid arthritis that nonclassical Ly6C(-) monocytes are required for the initiation and progression of sterile joint inflammation. Moreover, nonclassical Ly6C(-) monocytes differentiate into inflammatory macrophages (M1), which drive disease pathogenesis and display plasticity during the resolution phase. During the development of arthritis, these cells polarize toward an alternatively activated phenotype (M2), promoting the resolution of joint inflammation. The influx of Ly6C(-) monocytes and their subsequent classical and then alternative activation occurs without changes in synovial tissue-resident macrophages, which express markers of M2 polarization throughout the course of the arthritis and attenuate joint inflammation during the initiation phase. These data suggest that circulating Ly6C(-) monocytes recruited to the joint upon injury orchestrate the development and resolution of autoimmune joint inflammation.
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http://dx.doi.org/10.1016/j.celrep.2014.09.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4223808PMC
October 2014

Caspase-8 acts as a molecular rheostat to limit RIPK1- and MyD88-mediated dendritic cell activation.

J Immunol 2014 Jun 7;192(12):5548-60. Epub 2014 May 7.

Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611;

Caspase-8, an executioner enzyme in the death receptor pathway, was shown to initiate apoptosis and suppress necroptosis. In this study, we identify a novel, cell death-independent role for caspase-8 in dendritic cells (DCs): DC-specific expression of caspase-8 prevents the onset of systemic autoimmunity. Failure to express caspase-8 has no effect on the lifespan of DCs but instead leads to an enhanced intrinsic activation and, subsequently, more mature and autoreactive lymphocytes. Uncontrolled TLR activation in a RIPK1-dependent manner is responsible for the enhanced functionality of caspase-8-deficient DCs, because deletion of the TLR-signaling mediator, MyD88, ameliorates systemic autoimmunity induced by caspase-8 deficiency. Taken together, these data demonstrate that caspase-8 functions in a cell type-specific manner and acts uniquely in DCs to maintain tolerance.
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http://dx.doi.org/10.4049/jimmunol.1400122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4074511PMC
June 2014

The PYRIN domain-only protein POP3 inhibits ALR inflammasomes and regulates responses to infection with DNA viruses.

Nat Immunol 2014 Apr 16;15(4):343-53. Epub 2014 Feb 16.

1] Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA. [2] Robert H. Lurie Comprehensive Cancer Center, Interdepartmental Immunobiology Center and Skin Disease Research Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages.
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http://dx.doi.org/10.1038/ni.2829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4123781PMC
April 2014

Fas signaling in macrophages promotes chronicity in K/BxN serum-induced arthritis.

Arthritis Rheumatol 2014 Jan;66(1):68-77

Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Objective: A nonapoptotic role of Fas signaling has been implicated in the regulation of inflammation and innate immunity. This study was undertaken to elucidate the contribution of Fas signaling in macrophages to the development of arthritis.

Methods: K/BxN serum-transfer arthritis was induced in a mouse line in which Fas was conditionally deleted in the myeloid lineage (Cre(LysM) Fas(flox/flox) mice). The arthritis was assessed clinically and histologically. Expression of interleukin-1β (IL-1β), CXCL5, IL-10, IL-6, and gp96 was determined by enzyme-linked immunosorbent assay. Bone marrow-derived macrophages were activated with IL-1β and gp96. Cell phenotype and apoptosis were analyzed by flow cytometry.

Results: Arthritis onset in Cre(LysM) Fas(flox/flox) mice was comparable with that observed in control mice; however, resolution was accelerated during the chronic phase. The attenuated arthritis was associated with reduced articular expression of the endogenous Toll-like receptor 2 (TLR-2) ligand gp96 and the neutrophil chemotactic chemokine CXCL5, and enhanced expression of IL-10. Activation with IL-1β or gp96 induced increased IL-10 expression in Fas-deficient murine macrophages compared with control macrophages. IL-10 suppressed IL-6 and CXCL5 expression induced by IL-1β plus gp96. IL-1β-mediated activation of ERK, which regulates IL-10 expression, was increased in Fas-deficient mouse macrophages.

Conclusion: Taken together, our findings indicate that impaired Fas signaling results in enhanced expression of antiinflammatory IL-10 and reduced expression of gp96, and these effects are associated with accelerated resolution of inflammation during the chronic phase of arthritis. These observations suggest that strategies to reduce endogenous TLR ligands and increase IL-10 may be beneficial in the treatment of rheumatoid arthritis.
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http://dx.doi.org/10.1002/art.38198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5407456PMC
January 2014

Pre-B cell leukemia homeobox 1 is associated with lupus susceptibility in mice and humans.

J Immunol 2012 Jan 16;188(2):604-14. Epub 2011 Dec 16.

Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA.

Sle1a.1 is part of the Sle1 susceptibility locus, which has the strongest association with lupus nephritis in the NZM2410 mouse model. In this study, we show that Sle1a.1 results in the production of activated and autoreactive CD4(+) T cells. Additionally, Sle1a.1 expression reduces the peripheral regulatory T cell pool, as well as induces a defective response of CD4(+) T cells to the retinoic acid expansion of TGF-β-induced regulatory T cells. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d overexpression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells and to decrease their apoptotic response to retinoic acid. PBX1-d is expressed more frequently in the CD4(+) T cells from lupus patients than from healthy controls, and its presence correlates with an increased central memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance.
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http://dx.doi.org/10.4049/jimmunol.1002362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253202PMC
January 2012

Requirement of myeloid cell-specific Fas expression for prevention of systemic autoimmunity in mice.

Arthritis Rheum 2012 Mar;64(3):808-20

Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

Objective: The death receptor Fas is a critical mediator of the extrinsic apoptotic pathway, and its role in mediating lymphoproliferation has been extensively examined. The present study was undertaken to investigate the impact of myeloid cell-specific loss of Fas.

Methods: Mice with Fas flanked by loxP sites (Fas(flox/flox) ) were crossed with mice expressing Cre under control of the murine lysozyme M gene promoter (Cre(LysM) ), which functions in mature lysozyme-expressing cells of the myelomonocytic lineage. The genotype for Cre(LysM) Fas(flox/flox) mice was verified by polymerase chain reaction and flow cytometric analysis. Flow cytometric analysis was also used to characterize myeloid, dendritic, and lymphoid cell distribution and activation in bone marrow, blood, and spleen. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure serum cytokine/chemokine and immunoglobulin levels. Renal damage or dysfunction was examined by immunohistochemical and immunofluorescence analysis.

Results: Cre(LysM) Fas(flox/flox) mice exhibited a systemic lupus erythematosus (SLE)-like disease that included leukocytosis, splenomegaly, hypergammaglobulinemia, antinuclear autoantibody and proinflammatory cytokine production, and glomerulonephritis. Loss of Fas in myeloid cells increased levels of both Gr-1(low) and Gr-1(intermediate) blood monocytes and splenic macrophages and, in a paracrine manner, incited activation of conventional dendritic cells and lymphocytes in Cre(LysM) Fas(flox/flox) mice.

Conclusion: Taken together, these results suggest that loss of Fas in myeloid cells is sufficient to induce inflammatory phenotypes in mice, reminiscent of an SLE-like disease. Thus, Fas in myeloid cells may be considered a suppressor of systemic autoimmunity.
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http://dx.doi.org/10.1002/art.34317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3290732PMC
March 2012