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

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.

Visual stimulus characteristics that elicit tracking and striking in the praying mantises Parasphendale affinis, Popa spurca and Sphodromantis lineola.

We tested three species of praying mantis, Parasphendale affinis, Popa spurca and Sphodromantis lineola, with computer-generated stimuli that differed in size, contrast, configuration and movement pattern to determine the effects of these parameters on visual tracking and striking behavior. Overall, black disks moving erratically against a white background were strong releasers of both behaviors. When stimulus presentation order was randomized by size, P. affinis and P. spurca struck at progressively higher rates as the stimuli enlarged up to 44 deg; S. lineola struck most at intermediate sized (10-20 deg) disks. When disks were size-ordered from small to large, P. affinis and S. lineola struck at the smaller disks at higher rates; however, when the order was reversed, the early appearance of large disks suppressed subsequent responses to the smaller disks. Stimulus order did not differentially affect the responses of P. spurca. All species responded at higher rates to black disks moving against a white background versus the reverse. However, only P. spurca and S. lineola responded at higher rates to relatively darker grey disks, only P. affinis responded to mottled grey disks moving against an identically patterned background, and only P. spurca struck more frequently in response to rectangular stimuli oriented parallel (versus orthogonal) to their direction of movement. In conjunction with data on other species, these results support the hypothesis that praying mantises recognize prey based on assessment of several category-specific, spatiotemporal features, e.g. size, contrast, speed, movement pattern and leading edge length.

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.

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.

Visual stimuli that elicit appetitive behaviors in three morphologically distinct species of praying mantis.

We assessed the differences in appetitive responses to visual stimuli by three species of praying mantis (Insecta: Mantodea), Tenodera aridifolia sinensis, Mantis religiosa, and Cilnia humeralis. Tethered, adult females watched computer generated stimuli (erratically moving disks or linearly moving rectangles) that varied along predetermined parameters. Three responses were scored: tracking, approaching, and striking. Threshold stimulus size (diameter) for tracking and striking at disks ranged from 3.5 deg (C. humeralis) to 7.8 deg (M. religiosa), and from 3.3 deg (C. humeralis) to 11.7 deg (M. religiosa), respectively. Unlike the other species which struck at disks as large as 44 deg, T. a. sinensis displayed a preference for 14 deg disks. Disks moving at 143 deg/s were preferred by all species. M. religiosa exhibited the most approaching behavior, and with T. a. sinensis distinguished between rectangular stimuli moving parallel versus perpendicular to their long axes. C. humeralis did not make this distinction. Stimulus sizes that elicited the target behaviors were not related to mantis size. However, differences in compound eye morphology may be related to species differences: C. humeralis' eyes are farthest apart, and it has an apparently narrower binocular visual field which may affect retinal inputs to movement-sensitive visual interneurons.

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

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.

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.

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

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.

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

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

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

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

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

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.

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

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-1ß 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.

Visual stimuli that elicit visual tracking, approaching and striking behavior from an unusual praying mantis, Euchomenella macrops (Insecta: Mantodea).

In comparison to other similarly sized mantis species examined in previous studies, Euchomenella macrops has a significantly smaller head, shorter foreleg tibia, but longer prothorax which have been interpreted as specializations for the capture of smaller, slower prey. We tested this conjecture by assessing the rates at which computer generated stimuli elicit visual tracking, approaching, and striking behaviors by adult females. When presented with black disks moving erratically against a white background, strike rate rose progressively as disks enlarged up to 44 deg (visual angle) if the disks moved rapidly (e.g., 143 deg/s); at slower speeds (113, 127 deg/s), smaller disks (<27 deg) were preferred. When black moved linearly from the visual periphery to visual field center (at 73 or 143 deg/s) and then stopped, E. macrops struck consistently at disks as small as 5 deg after movement ceased. E. macrops also struck at higher rates in response to 23 deg erratically moving (subjective) red (versus subjective blue or green) disks that were luminance matched to a grey background although they tracked all colors at equally high rates. Unlike some other species, E. macrops did not strike at higher rates in response to elongated rectangular stimuli moving parallel (versus perpendicular) to their long axis, although the former elicited higher rates of approaching. An analysis of tracking behavior revealed that virtually all tracking movements were a result of head (versus) prothorax rotation.