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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. Here, we focused on the two main subtypes of circulating monocytes, classical (CM) and non-classical (NCM).SSc patients were recruited from the Prospective Registry for Early SSc registry. Clinical data were collected as well as peripheral blood for isolation of CM and NCM. Age-, sex-, and race-matched healthy volunteers were recruited as controls. Bulk macrophages were isolated from skin in a separate cohort. All samples were assayed by RNA-seq.We used an unbiased approach to cluster patients into three groups (A-C) based on their transcriptional signatures of CM relative to controls. Further, each group maintained their characteristic transcriptional signature in NCM. Genes upregulated in Group C demonstrated the highest signature compared to the other groups in skin macrophages. Patients from Group B and C exhibited worse lung function than Group A, although there was no difference in skin disease at baseline. We validated our approach by applying our group classifications to published bulk monocyte RNA-seq data on SSc patients: we found that patients with no skin disease were most likely to be classified as Group A.We are the first to show that transcriptional signature of CM and NCM can be used to unbiasedly stratify SSc patients and correlate with disease activity outcome measures.

Background/PurposePatients with systemic sclerosis (SSc) display a complex clinical phenotype. There are numerous studies that relate transcriptional signatures from PBMC or whole skin of SSc patients to disease activity. However, analyses of whole tissue RNA-sequencing studies are subjected to changes in cellular composition that can drive gene expression signatures and a loss of the ability to detect biologically important transcriptional changes within minority cell populations. Here, we focused on circulating monocytes, which have been shown to exist as two central populations classical (CM) and non-classical (NCM).MethodsSSc patients were recruited from four different sites that form PRESS: Northwestern University, University of Texas, University of Michigan and University of Utah. Comprehensive clinical data was collected for all patients. We isolated CM and NCM from these patients and age, sex, and race-matched healthy volunteers were used as controls. RNA-seq was performed on CM and NCM populations as well as on isolated bulk macrophages from skin.ResultsWe first performed RNA-seq on CM, which are the predominant population in circulation. In order to capture the variability across the SSc cohort, we defined 1790 differentially expressed genes in each patient. We then used these genes to cluster patients into 3 subgroups: Groups A-C. Group A exhibited the strongest interferon signature and innate immune pathways. Group B patients expressed genes in the same pathways but was also enriched for response to cAMP and corticosteroids. Both Group B and Group C exhibited upregulation of genes associated with vasculature development and blood vessel formation. Group C uniquely upregulated TGFB pathways. Next, we performed RNA-seq on NCM isolated from the same patients. When NCM were clustered based on the same 1790 genes as CM, we found that Groups A and C were recapitulated, while Group B was less cohesive. Our analysis stratified SSc patients based on their transcriptional profiles in monocytes but was agnostic to their clinical presentation. We found that Group B and C patients exhibited significantly worsened lung function at the time of monocyte isolation than Group A patients. However, there were no significant differences in skin disease. We then isolated macrophages from skin biopsies of SSc patients and showed that the transcriptional profile of Group A and C in SSc patients was conserved. We also used gene expression data from another study on monocytes which stratified patients based on disease presentation. We found that Group A accurately distinguished dcSSc and ncSSc patients from controls, but not lcSSc.ConclusionWe are the first to show that transcriptomic analysis of classical and non-classical circulating monocytes can unbiasedly stratify SSc patients and correlate with disease activity outcome measures.

Monocytes are one of the most abundant immune cells infiltrating the inflamed organs. However, the majority of studies on monocytes focus on circulating cells, rather than those in the tissue. Here, we identify and characterize an intravascular (i.v.) and extravascular (e.v.) synovial population (Syn Ly6C- cells) which lack cell surface markers of classical monocytes (Ly6C and CD62) or tissue macrophages (CD64 and Tim4), are transcriptionally distinct and conserved in RA patients. e.v. Syn Ly6C- cells are independent of NR4A1 and CCR2, long-lived and embryonically derived while the i.v. Syn Ly6C- cells are dependent on NR4A1, short lived and derived from circulating NCM. e.v. Syn Ly6C- cells undergo increased proliferation and reverse diapedesis dependent on LFA1 in response to arthrogenic stimuli and are required for the development of RA-like disease. These findings uncover a new facet of mononuclear cell biology and are imperative to understanding tissue-resident myeloid cell function in RA.

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.

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.

Tsai et al. demonstrate that loss of Bim (BCL2L11) in myeloid cells in mice (LysMCreBimfl/fl) is sufficient to induce systemic autoimmunity. Kidney macrophages in LysMCreBimfl/fl mice possess a proinflammatory transcriptional signature and signal through TRIF to cause end-stage glomerulonephritis., 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-β) 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.

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.

Misharin et al. elucidate the fate and function of monocyte-derived alveolar macrophages during the course of pulmonary fibrosis. These cells persisted throughout the life span, were enriched for the expression of profibrotic genes, and their genetic ablation ameliorated development of pulmonary fibrosis., 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.

Background Currently we are in the golden age of therapy for patients with rheumatoid arthritis (RA). However, currently there exists no available assay to predict the response to a particular therapy for an individual patient. Today, rheumatologists do not have information at han for therapeutic decisions. It is clear that the target organ in RA patients, i.e. the synovium, has the potential to unlock the secret for determining therapeutic response. Ideally, a sufficient synovial sample would be obtained from each patient to perform histology, sorting of individual cell populations and transcriptional analyses. Objectives Our goal is to establish a minimally invasive ultrasound guided synovial biopsy program in the United States to obtain synovial tissue for determining therapeutic response. Methods Rheumatologists from six Universities in the United States were trained in ultrasound guided minimally invasive synovial tissue biopsy procedures. Only patients with a grey scale synovitis score of 2 or greater were selected. A disposable semi-automatic-guillotine type biopsy needle (Quick-Core) was utilized for all patients and 25/26 patients had the biopsy performed on the wrist. Histology was performed on whole tissue. RNA was extracted from whole tissue and from FACS sorted macrophages in order for RNA sequencing (RNA-seq) analysis to be performed. Results Our group has already performed over 26 minimally invasive ultrasound guided synovial tissue biopsies on RA patients with active disease. We had minimal adverse effects and patients tolerated the procedure very well. At least 6–12 needle biopsies of synovial tissue were obtained via biopsy per patient. A minimum of 4 needle biopsies were placed in formalin and synovial lining was confirmed via histologic analyses. The remaining pieces were used to prepare libraries for RNA-seq. We observed comparable RNA integrity numbers, a measure of RNA quality, between the whole synovial tissue from RA (biopsy obtained) and OA (surgically-obtained) patients. OA patients segregated together transcriptionally, while RA patients are more heterogeneous as demonstrated via RNAseq analysis. We also optimized a protocol for digestion of synovial tissue biopsies for isolation of macrophages. We identified genes differentially associated with macrophage activity in RA versus OA synovial macrophages that were not evident in the whole tissue transcriptional profile. Conclusions Ultrasound guided synovial tissue biopsies are feasible in the United States. Based on our recent success using minimally invasive ultrasound guided synovial biopsies, we believe that this procedure coupled with cutting-edge technologies will provide the critical information to rheumatologists to establish precision based medicine as a reality for RA patients. Disclosure of Interest None declared

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. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0912-y) contains supplementary material, which is available to authorized users.

We used computer-generated stimuli to assess the stimulus parameters eliciting prey-directed behaviors in four mantis species: Rhombodera basalis (60 mm body length), Deroplatys lobata (70 mm), Hierodula membranacea (80 mm), Miomantis sp (38 mm). All responded appetitively to erratically moving disks subtending 10° or more. R. basalis responded more consistently to checkered (versus solid black) disks; only D. lobata displayed a size preference (14–23 deg disks); Miomantis sp. struck at disks as large as did the largest species; only Miomantis sp. displayed approaching behavior. H. membranacea and R. basalis preferred black disks on a white background (versus the reverse), and red disks (versus blue or green) on a brightness-matched grey background. All preferred elongated stimuli moving parallel (versus perpendicular) to their long axes.

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.

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.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.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.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.

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.

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 (