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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.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding. We performed combined single cell RNA transcriptomic and TCRα/β sequencing on rBx from patients with ACR under differing immunosuppression (IS): tacrolimus, iscalimab, and belatacept. TCR analysis revealed a highly restricted CD8+T cell clonal expansion (CD8EXP), independent of HLA mismatch or IS type. Subcloning of TCRα/β cDNAs from CD8EXPinto Jurkat76 cells (TCR-/-) conferred alloreactivity by mixed lymphocyte reaction. scRNAseq analysis of CD8EXPrevealed effector, memory, and exhausted phenotypes that were influenced by IS type. Successful anti-rejection treatment decreased, but did not eliminate, CD8EXP, while CD8EXPwere maintained during treatment-refractory rejection. Finally, most rBx-derived CD8EXPwere also observed in matching urine samples. Overall, our data define the clonal CD8+T cell response to ACR, providing novel insights to improve detection, assessment, and treatment of rejection.

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.

Neovascular age-related macular degeneration (nAMD) commonly causes vision loss from aberrant angiogenesis, termed choroidal neovascularization (CNV). Interleukin-6 (IL6) is a pro-inflammatory and pro-angiogenic cytokine that is correlated with AMD progression and nAMD activity. We hypothesize that anti-IL6 therapy is a potential nAMD therapeutic. We found that IL6 levels were increased after laser injury and expressed by macrophages. Il6-deficiency decreased laser-induced CNV area and exogenous IL6 addition increased choroidal sprouting angiogenesis. Il6-null mice demonstrated equally increased macrophage numbers as wildtype mice. At steady state, IL6R expression was detected on peripheral blood and ocular monocytes. After laser injury, the number of IL6R+Ly6C+ monocytes in blood and IL6R+ macrophages in the eye were increased. In human choroid, macrophages expressed IL6, IL6R, and IL6ST. Furthermore, IL6R+ macrophages displayed a transcriptional profile consistent with STAT3 (signal transducer and activator of transcription 3) activation and angiogenesis. Our data show that IL6 is both necessary and sufficient for choroidal angiogenesis. Macrophage-derived IL6 may stimulate choroidal angiogenesis via classical activation of IL6R+ macrophages, which then stimulate angiogenesis. Targeting IL6 or the IL6R could be an effective adjunctive therapy for treatment-resistant nAMD patients.

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.

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

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.

Macrophages fulfill critical functions in maintaining tissue homeostasis in steady-state, as well as in inflammation and immune response. Inflammation is not considered a major driver of osteoarthritis (OA), but macrophages have been implicated in its pathogenesis. Here, we use two mouse models of experiment OA – collagenase-induced osteoarthritis (CIOA) and destabilization of the medial meniscus (DMM) – to quantify the immune cell infiltration into the knee joint during the early stages of disease. We find that the peak of inflammation occurs at day 3 in CIOA and is characterized by a transitory increase in neutrophils and monocytes and a longer-lived expansion of synovial macrophages. Macrophage sub-populations are disproportionally expanded with CX3CR1+ cells accounting for a larger proportion of the macrophage compartment. Transcriptional profiling demonstrates that synovial macrophages up-regulate inflammatory genes coinciding with peak inflammation and down-regulate genes associated with homeostasis and tissue-residence. Female mice exhibit a similar expansion of macrophages post-CIOA indicating that the inflammatory phase is not sex-specific. Finally, we find day 7 post-DMM is also characterized by increases in neutrophil, monocyte, and macrophage sub-population numbers. These results support a role for macrophages in early stages of OA through driving the inflammatory phase. Further investigation may elucidate potential targets for the prevention or attenuation of OA-associated cartilage damage.

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.

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.

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms. We applied lineage tracing, spatial methods and single-cell RNA-seq to a spatially-restricted model of asbestos-induced pulmonary fibrosis. We demonstrate that while tissue-resident interstitial macrophages, tissue-resident alveolar macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche, only monocyte-derived alveolar macrophages are causally related to fibrosis. Monocyte-derived alveolar macrophages were specifically localized to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including PDGFA. Moreover, we identified autocrine M-CSF/M-CSFR signaling in monocyte-derived alveolar macrophages as a novel mechanism promoting their self-maintenance and persistence in the fibrotic niche. Pharmacological blockade of M-CSF signaling led to disappearance of the established population of monocyte-derived alveolar macrophages. Thus, our data indicate that monocyte-derived alveolar macrophages are specifically recruited to the fibrotic niche where they are maintained by autocrine signaling and drive fibrosis by stimulating fibroblast proliferation.

Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple organs, including the brain. Though 50% of patients may experience neuropsychiatric symptoms (NPSLE), disease mechanisms remain largely unknown. Microglia, the resident innate immune cell of the brain, have recently been implicated in NPSLE. Microglia are heterogenous and may be associated with a homeostatic microglia (CD11clo) or a disease-associated microglia (DAM; CD11chi) state common to neurodegenerative disease and aging. Yet, few studies have examined microglia subsets in NPSLE. We showed that expression of a shared NPSLE transcriptional signature and DAM-associated genes correlates with the severity of behavioral deficits in microglia isolated from two NPSLE models prior to overt systemic disease. Further, our single-cell RNA-seq data identify homeostatic microglia and DAM states in control and NPSLE-prone mice. DAM in NPSLE are enriched for genes associated with antigen presentation but depleted for genes associated with phagocytosis, which is in contrast to DAM in neurodegenerative disease that are critical for phagocytic functions. Moreover, NPSLE CD11clo microglia upregulate genes linked to synapse pruning and phagocytosis, consistent with exacerbated synaptic pruning by microglia in models of NPSLE. We also find that restricted expression of the DAM transcriptional program in NPSLE DAM corresponds to improved behavioral outcomes in NPSLE-prone mice following treatment with fingolimod, a sphingosine-1-phosphate receptor modulator that reduces microglia activation and improves blood brain barrier integrity. These discoveries mark the first to implicate DAM as a potentially pathogenic microglia subset in NPSLE.

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) TCRαβhi 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.

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.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease affecting multiple organs, including the brain. Though 50% of patients may experience neuropsychiatric symptoms (NPSLE), disease mechanisms remain largely unknown. Microglia are the resident innate immune cell of the brain and suggested to act as drivers of neurological conditions ranging from neurodevelopmental (autism) to neurodegenerative (Alzheimer’s disease) disorders. The majority of investigations into microglia, particularly the recently discovered disease-associated microglia (DAM) subset, have occurred in models of neurodegenerative disease. However, few studies have examined microglia, particularly DAM, in NPSLE. We performed RNA-seq on microglia isolated from two NPSLE-prone mouse strains to correlate gene signatures with behavioral deficits. Similar to patients, both NPSLE models exhibit cognitive impairment. Of the significantly upregulated genes found in NPSLE microglia compared to their respective controls, a common 18-gene ‘NPSLE signature’ is shared and enriched for genes associated with lipid metabolism, scavenger receptor activity and downregulating inflammatory responses and cell chemotaxis processes. NPSLE microglia are also enriched for DAM-associated genes and expression of ‘NPSLE’ and ‘DAM’ signatures significantly correlates with the severity of behavioral deficits. The discovery of our novel ‘NPSLE signature’ and enrichment of the ‘DAM signature’ represents the first to connect microglia-specific transcriptional signatures with clinical outcomes. In future studies, we will assess the penetrance of these signatures to further interrogate how defective microglial function may incite NPSLE.

The etiology and pathogenesis of systemic sclerosis (SSc) are poorly understood. Circulating monocytes likely play a critical role in SSc progression through secretion of proinflammatory molecules and as precursors of macrophages that can reorganize the extracellular matrix, thereby leading to development of end-organ fibrosis. Here, we evaluate the transcriptional similarities between circulating classical monocytes (CMo) and skin macrophages of SSc patients. Bulk RNA-seq was performed on sorted CMo from SSc blood obtained through the Prospective Registry of Early Systemic Sclerosis (PRESS) cohort, and transcriptional profiles were analyzed along with profiles from matched control samples. Additionally, sorted CD45+ cells from skin biopsies of one SSc patient and one control patient were prepared for single-cell RNA-seq. There was a numerical expansion of skin macrophages from the control to the SSc sample, but no significant difference in the quantity of circulating CMo between control and SSc patients. Of the 152 significantly upregulated genes (DESeq2, p2) observed in circulating CMo population and the 290 upregulated genes (fold change>2) in the skin macrophage cluster compared to their respective controls, we found 23 genes in common (p

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.

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 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. The MRI derived data 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.

Background Neuropsychiatric manifestations of systemic lupus erythematosus (NPSLE) affect approximately 40% of patients. Lipocalin-2 (LCN2), an acute-phase reactant protein, is an established urinary biomarker in lupus patients. Previous studies using LCN2-deficient mice have demonstrated its role in glial migration and chemokine regulation in the brain. We therefore hypothesized that LCN2 is involved in NPSLE pathogenesis. Methods We investigated the lupus prone B6.Sle1.Sle3 (Sle1,3) mouse and the effects of LCN2 deficiency on the development of the neuropsychiatric phenotype exhibited by this strain. Sle1,3, B6.LCN2KO, B6, and Sle1,3-LCN2KO mice (6–10 month old; n=5–10/group) underwent comprehensive neurobehavioral assessment, and brains were evaluated by flow cytometry and RNA sequencing. Results Sle1,3 mice exhibited significant impairment in spatial (p Conclusions Our findings establish the Sle1,3 mouse as an NPSLE model and demonstrate that LCN2 deficiency attenuates neurobehavioral deficits and regulates microglial expression of genes essential to NPSLE development.

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.

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.

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. 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. 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. 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.

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 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 phosph atase (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. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1375-0) contains supplementary material, which is available to authorized users.

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.

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 and aims There are numerous endogenous Bcl-2 antagonists that share similar homology, structure, topology and expression pattern, yet only the loss of Bim in mice is sufficient to lead to the development of a systemic autoimmunity. Methods We investigated the contribution of Bim in monocytes/macrophages and its effect on systemic autoimmunity by establishing conditionally Bim-deleted mice in the monocyte/macrophage compartment (Cre LysM Bim fl/fl mice) and examined the development of lupus-like disease over time. Results Patients with lupus display decreased expression of Bim in circulating monocytes and reduced Bim expression in kidney macrophages. Cre LysM Bim flox/flox mice develop a lupus-like disease that mirrors aged Bim -/- mice including loss of the marginal zone macrophages, splenomegaly, lymphadenopathy, autoantibodies including anti-DNA IgG, and a type I interferon signature as compared to control mice. Cre LysM Bim flox/flox mice also exhibit increased mortality attributed to immune complex deposition and increased numbers of kidney macrophages all of which contribute to glomerulonephritis. The loss of Bim in macrophages is sufficient to break tolerance as adoptive transfer of wild-type lymphocytes into Cre LysM Bim flox/flox Rag -/- mice leads to systemic autoimmunity. We also identified that the loss of TLR signalling adaptor protein TRIF but not MyD88 is essential for progression to GN phase but is dispensable for systemic autoimmunity. RNA seq analysis of sorted kidney macrophages revealed a novel Bim and lupus specific signatures. Conclusions These data add another facet to the conventional dogma that Bim’s central role in autoimmune disease is to prevent the escape of autoreactive lymphocytes from apoptosis. Thus, Bim may be a novel therapeutic target for treating SLE.

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.

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 in situ 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., Monocyte-derived alveolar macrophages orchestrate the development of the fibrotic niche, causally related to fibrosis and maintained via M-CSF/M-CSFR signalling http://bit.ly/2nDjS20

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. Elevated levels of signalling mediators of both the extrinsic and intrinsic apoptotic as well as the autophagy death pathways are observed in the joints of patients with rheumatoid arthritis. Furthermore, in rheumatoid arthritis patients, risk polymorphisms are present in signalling molecules of the extrinsic apoptotic and autophagy death pathways. Although research into the mechanisms underlying these death 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 therapeutic targets for rheumatoid arthritis and other rheumatic diseases.

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.

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.

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 CreLysMCasp8fl/fl mice were bred via a cross between Casp8fl/fl mice and CreLysM mice, and RIPK3−/−CreLysMCasp8fl/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. CreLysMCasp8fl/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 Ly6ChighCD11b+F4/80+ splenic cells, and oral antibiotic treatment to remove microbiota prevents splenomegaly and lymphadenopathy in CreLysMCasp8fl/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. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0794-z) contains supplementary material, which is available to authorized users.