Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Immune and metabolic markers for identifying and investigating severe Coronavirus disease and Sepsis in children and young people (pSeP/COVID ChYP study): protocol for a prospective cohort study.

INTRODUCTION: Early recognition and appropriate management of paediatric sepsis are known to improve outcomes. A previous system's biology investigation of the systemic immune response in neonates to sepsis identified immune and metabolic markers that showed high accuracy for detecting bacterial infection. Further gene expression markers have also been reported previously in the paediatric age group for discriminating sepsis from control cases. More recently, specific gene signatures were identified to discriminate between COVID-19 and its associated inflammatory sequelae. Through the current prospective cohort study, we aim to evaluate immune and metabolic blood markers which discriminate between sepses (including COVID-19) from other acute illnesses in critically unwell children and young persons, up to 18 years of age. METHODS AND ANALYSIS: We describe a prospective cohort study for comparing the immune and metabolic whole-blood markers in patients with sepsis, COVID-19 and other illnesses. Clinical phenotyping and blood culture test results will provide a reference standard to evaluate the performance of blood markers from the research sample analysis. Serial sampling of whole blood (50 µL each) will be collected from children admitted to intensive care and with an acute illness to follow time dependent changes in biomarkers. An integrated lipidomics and RNASeq transcriptomics analyses will be conducted to evaluate immune-metabolic networks that discriminate sepsis and COVID-19 from other acute illnesses. This study received approval for deferred consent. ETHICS AND DISSEMINATION: The study has received research ethics committee approval from the Yorkshire and Humber Leeds West Research Ethics Committee 2 (reference 20/YH/0214; IRAS reference 250612). Submission of study results for publication will involve making available all anonymised primary and processed data on public repository sites. TRIAL REGISTRATION NUMBER: NCT04904523.

Immune-metabolic adaptations in pregnancy: A potential stepping-stone to sepsis.

Physiological shifts during pregnancy predispose women to a higher risk of developing sepsis resulting from a maladapted host-response to infection. Insightful studies have delineated subtle point-changes to the immune system during pregnancy. Here, we present an overlay of these point-changes, asking what changes and when, at a physiological, cellular, and molecular systems-level in the context of sepsis. We identify distinct immune phases in pregnancy delineated by placental hormone-driven changes in homeostasis setpoints of the immune and metabolic systems that subtly mirrors changes observed in sepsis. We propose that pregnancy immune-metabolic setpoint changes impact feedback thresholds that increase risk for a maladapted host-response to infection and thus act as a stepping-stone to sepsis. Defining maternal immune-metabolic setpoint changes is not only vital for tailoring the right diagnostic tools for early management of maternal sepsis but will facilitate an unravelling of the pathophysiological pathways that predispose an individual to sepsis.

mSep: investigating physiological and immune-metabolic biomarkers in septic and healthy pregnant women to predict feto-maternal immune health - a prospective observational cohort study protocol.

INTRODUCTION: Maternal sepsis remains a leading cause of death in pregnancy. Physiological adaptations to pregnancy obscure early signs of sepsis and can result in delays in recognition and treatment. Identifying biomarkers that can reliably diagnose sepsis will reduce morbidity and mortality and antibiotic overuse. We have previously identified an immune-metabolic biomarker network comprising three pathways with a >99% accuracy for detecting bacterial neonatal sepsis. In this prospective study, we will describe physiological parameters and novel biomarkers in two cohorts-healthy pregnant women and pregnant women with suspected sepsis-with the aim of mapping pathophysiological drivers and evaluating predictive biomarkers for diagnosing maternal sepsis. METHODS AND ANALYSIS: Women aged over 18 with an ultrasound-confirmed pregnancy will be recruited to a pilot and two main study cohorts. The pilot will involve blood sample collection from 30 pregnant women undergoing an elective caesarean section. Cohort A will follow 100 healthy pregnant women throughout their pregnancy journey, with collection of blood samples from participants at routine time points in their pregnancy: week 12 'booking', week 28 and during labour. Cohort B will follow 100 pregnant women who present with suspected sepsis in pregnancy or labour and will have at least two blood samples taken during their care pathway. Study blood samples will be collected during routine clinical blood sampling. Detailed medical history and physiological parameters at the time of blood sampling will be recorded, along with the results of routine biochemical tests, including C reactive protein, lactate and white blood cell count. In addition, study blood samples will be processed and analysed for transcriptomic, lipidomic and metabolomic analyses and both qualitative and functional immunophenotyping. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the Wales Research Ethics Committee 2 (SPON1752-19, 30 October 2019). TRIAL REGISTRATION NUMBER: NCT05023954.

Oxylipin metabolism is controlled by mitochondrial ß-oxidation during bacterial inflammation.

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial ß-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin ß-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by ß-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial ß-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

nSeP: immune and metabolic biomarkers for early detection of neonatal sepsis-protocol for a prospective multicohort study.

INTRODUCTION: Diagnosing neonatal sepsis is heavily dependent on clinical phenotyping as culture-positive body fluid has poor sensitivity, and existing blood biomarkers have poor specificity.A combination of machine learning, statistical and deep pathway biology analyses led to the identification of a tripartite panel of biologically connected immune and metabolic markers that showed greater than 99% accuracy for detecting bacterial infection with 100% sensitivity. The cohort study described here is designed as a large-scale clinical validation of this previous work. METHODS AND ANALYSIS: This multicentre observational study will prospectively recruit a total of 1445 newborn infants (all gestations)-1084 with suspected early-or late-onset sepsis, and 361 controls-over 4 years. A small volume of whole blood will be collected from infants with suspected sepsis at the time of presentation. This sample will be used for integrated transcriptomic, lipidomic and targeted proteomics profiling. In addition, a subset of samples will be subjected to cellular phenotype and proteomic analyses. A second sample from the same patient will be collected at 24 hours, with an opportunistic sampling for stool culture. For control infants, only one set of blood and stool sample will be collected to coincide with clinical blood sampling. Along with detailed clinical information, blood and stool samples will be analysed and the information will be used to identify and validate the efficacy of immune-metabolic networks in the diagnosis of bacterial neonatal sepsis and to identify new host biomarkers for viral sepsis. ETHICS AND DISSEMINATION: The study has received research ethics committee approval from the Wales Research Ethics Committee 2 (reference 19/WA/0008) and operational approval from Health and Care Research Wales. Submission of study results for publication will involve making available all anonymised primary and processed data on public repository sites. TRIAL REGISTRATION NUMBER: NCT03777670.

Erratum: Effective In Vivo Gene Modification in Mouse Tissue-Resident Peritoneal Macrophages by Intraperitoneal Delivery of Lentiviral Vectors.

[This corrects the article DOI: 10.1016/j.omtm.2019.10.004.].

Tissue-resident macrophages actively suppress IL-1beta release via a reactive prostanoid/IL-10 pathway.

The alarm cytokine interleukin-1ß (IL-1ß) is a potent activator of the inflammatory cascade following pathogen recognition. IL-1ß production typically requires two signals: first, priming by recognition of pathogen-associated molecular patterns leads to the production of immature pro-IL-1ß; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of IL-1ß from its pro-form. However, despite the important role of IL-1ß in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissue-resident macrophages use an active inhibitory pathway, to suppress IL-1ß processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of IL-10. In the absence of secondary signal, IL-10 potently inhibits IL-1ß processing, providing a previously unrecognized control of IL-1ß in tissue-resident macrophages.

Nitric oxide orchestrates metabolic rewiring in M1 macrophages by targeting aconitase 2 and pyruvate dehydrogenase.

Profound metabolic changes are characteristic of macrophages during classical activation and have been implicated in this phenotype. Here we demonstrate that nitric oxide (NO) produced by murine macrophages is responsible for TCA cycle alterations and citrate accumulation associated with polarization. 13C tracing and mitochondrial respiration experiments map NO-mediated suppression of metabolism to mitochondrial aconitase (ACO2). Moreover, we find that inflammatory macrophages reroute pyruvate away from pyruvate dehydrogenase (PDH) in an NO-dependent and hypoxia-inducible factor 1α (Hif1α)-independent manner, thereby promoting glutamine-based anaplerosis. Ultimately, NO accumulation leads to suppression and loss of mitochondrial electron transport chain (ETC) complexes. Our data reveal that macrophages metabolic rewiring, in vitro and in vivo, is dependent on NO targeting specific pathways, resulting in reduced production of inflammatory mediators. Our findings require modification to current models of macrophage biology and demonstrate that reprogramming of metabolism should be considered a result rather than a mediator of inflammatory polarization.

Effective In Vivo Gene Modification in Mouse Tissue-Resident Peritoneal Macrophages by Intraperitoneal Delivery of Lentiviral Vectors.

Tissue-resident macrophages exhibit specialized phenotypes dependent on their in vivo physiological niche. Investigation of their function often relies upon complex whole mouse transgenic studies. While some appropriate lineage-associated promoters exist, there are no options for tissue-specific targeting of macrophages. We have developed full protocols for in vivo productive infection (defined by stable transgene expression) of tissue-resident macrophages with lentiviral vectors, enabling RNA and protein overexpression, including expression of small RNA species such as shRNA, to knock down and modulate gene expression. These approaches allow robust infection of peritoneal tissue-resident macrophages without significant infection of other cell populations. They permit rapid functional study of macrophages in homeostatic and inflammatory settings, such as thioglycolate-induced peritonitis, while maintaining the cells in their physiological context. Here we provide detailed protocols for the whole workflow: viral production, purification, and quality control; safety considerations for administration of the virus to mice; and assessment of in vivo transduction efficiency and the low background levels of inflammation induced by the virus. In summary, we present a quick and accessible protocol for the rapid assessment of gene function in peritoneal tissue-resident macrophages in vivo.

Innate immunology in COVID-19-a living review. Part I: viral entry, sensing and evasion.

The coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a world health concern and can cause severe disease and high mortality in susceptible groups. While vaccines offer a chance to treat disease, prophylactic and anti-viral treatments are still of vital importance, especially in context of the mutative ability of this group of viruses. Therefore, it is essential to elucidate the molecular mechanisms of viral entry, innate sensing and immune evasion of SARS-CoV-2, which control the triggers of the subsequent excessive inflammatory response. Viral evasion strategies directly target anti-viral immunity, counteracting host restriction factors and hijacking signalling pathways to interfere with interferon production. In Part I of this review, we examine SARS-CoV-2 viral entry and the described immune evasion mechanisms to provide a perspective on how the failure in initial viral sensing by infected cells can lead to immune dysregulation causing fatal COVID-19, discussed in Part II.

Innate immunology in COVID-19-a living review. Part II: dysregulated inflammation drives immunopathology.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a global health crisis and will likely continue to impact public health for years. As the effectiveness of the innate immune response is crucial to patient outcome, huge efforts have been made to understand how dysregulated immune responses may contribute to disease progression. Here we have reviewed current knowledge of cellular innate immune responses to SARS-CoV-2 infection, highlighting areas for further investigation and suggesting potential strategies for intervention. We conclude that in severe COVID-19 initial innate responses, primarily type I interferon, are suppressed or sabotaged which results in an early interleukin (IL)-6, IL-10 and IL-1ß-enhanced hyperinflammation. This inflammatory environment is driven by aberrant function of innate immune cells: monocytes, macrophages and natural killer cells dispersing viral pathogen-associated molecular patterns and damage-associated molecular patterns into tissues. This results in primarily neutrophil-driven pathology including fibrosis that causes acute respiratory distress syndrome. Activated leukocytes and neutrophil extracellular traps also promote immunothrombotic clots that embed into the lungs and kidneys of severe COVID-19 patients, are worsened by immobility in the intensive care unit and are perhaps responsible for the high mortality. Therefore, treatments that target inflammation and coagulation are promising strategies for reducing mortality in COVID-19.

The protective effect of inflammatory monocytes during systemic C. albicans infection is dependent on collaboration between C-type lectin-like receptors.

Invasive candidiasis, mainly caused by Candida albicans, is a serious healthcare problem with high mortality rates, particularly in immunocompromised patients. Innate immune cells express pathogen recognition receptors (PRRs) including C-type lectin-like receptors (CLRs) that bind C. albicans to initiate an immune response. Multiple CLRs including Dectin-1, Dectin-2 and Mincle have been proposed individually to contribute to the immune response to C. albicans. However how these receptors collaborate to clear a fungal infection is unknown. Herein, we used novel multi-CLR knockout (KO) mice to decipher the individual, collaborative and collective roles of Dectin-1, Dectin-2 and Mincle during systemic C. albicans infection. These studies revealed an unappreciated and profound role for CLR co-operation in anti-fungal immunity. The protective effect of multiple CLRs was markedly greater than any single receptor, and was mediated through inflammatory monocytes via recognition and phagocytosis of C. albicans, and production of C. albicans-induced cytokines and chemokines. These CLRs were dispensable for mediating similar responses from neutrophils, likely due to lower expression of these CLRs on neutrophils compared to inflammatory monocytes. Concurrent deletion of Dectin-1 and Dectin-2, or all three CLRs, resulted in dramatically increased susceptibility to systemic C. albicans infection compared to mice lacking a single CLR. Multi-CLR KO mice were unable to control fungal growth due to an inadequate early inflammatory monocyte-mediated response. In response to excessive fungal growth, the multi-CLR KO mice mounted a hyper-inflammatory response, likely leading to multiple organ failure. Thus, these data reveal a critical role for CLR co-operation in the effective control of C. albicans and maintenance of organ function during infection.

Diversity and environmental adaptation of phagocytic cell metabolism.

Phagocytes are cells of the immune system that play important roles in phagocytosis, respiratory burst and degranulation-key components of innate immunity and response to infection. This diverse group of cells includes monocytes, macrophages, dendritic cells, neutrophils, eosinophils, and basophils-heterogeneous cell populations possessing cell and tissue-specific functions of which cellular metabolism comprises a critical underpinning. Core functions of phagocytic cells are diverse and sensitive to alterations in environmental- and tissue-specific nutrients and growth factors. As phagocytic cells adapt to these extracellular cues, cellular processes are altered and may contribute to pathogenesis. The considerable degree of functional heterogeneity among monocyte, neutrophil, and other phagocytic cell populations necessitates diverse metabolism. As we review our current understanding of metabolism in phagocytic cells, gaps are focused on to highlight the need for additional studies that hopefully enable improved cell-based strategies for counteracting cancer and other diseases.

Tumour-elicited neutrophils engage mitochondrial metabolism to circumvent nutrient limitations and maintain immune suppression.

Neutrophils are a vital component of immune protection, yet in cancer they may promote tumour progression, partly by generating reactive oxygen species (ROS) that disrupts lymphocyte functions. Metabolically, neutrophils are often discounted as purely glycolytic. Here we show that immature, c-Kit+ neutrophils subsets can engage in oxidative mitochondrial metabolism. With limited glucose supply, oxidative neutrophils use mitochondrial fatty acid oxidation to support NADPH oxidase-dependent ROS production. In 4T1 tumour-bearing mice, mitochondrial fitness is enhanced in splenic neutrophils and is driven by c-Kit signalling. Concordantly, tumour-elicited oxidative neutrophils are able to maintain ROS production and T cell suppression when glucose utilisation is restricted. Consistent with these findings, peripheral blood neutrophils from patients with cancer also display increased immaturity, mitochondrial content and oxidative phosphorylation. Together, our data suggest that the glucose-restricted tumour microenvironment induces metabolically adapted, oxidative neutrophils to maintain local immune suppression.

Itaconic acid mediates crosstalk between macrophage metabolism and peritoneal tumors.

Control of cellular metabolism is critical for efficient cell function, although little is known about the interplay between cell subset-specific metabolites in situ, especially in the tumor setting. Here, we determined how a macrophage-specific (Mϕ-specific) metabolite, itaconic acid, can regulate tumor progression in the peritoneum. We show that peritoneal tumors (B16 melanoma or ID8 ovarian carcinoma) elicited a fatty acid oxidation-mediated increase in oxidative phosphorylation (OXPHOS) and glycolysis in peritoneal tissue-resident macrophages (pResMϕ). Unbiased metabolomics identified itaconic acid, the product of immune-responsive gene 1-mediated (Irg1-mediated) catabolism of mitochondrial cis-aconitate, among the most highly upregulated metabolites in pResMϕ of tumor-bearing mice. Administration of lentivirally encoded Irg1 shRNA significantly reduced peritoneal tumors. This resulted in reductions in OXPHOS and OXPHOS-driven production of ROS in pResMϕ and ROS-mediated MAPK activation in tumor cells. Our findings demonstrate that tumors profoundly alter pResMϕ metabolism, leading to the production of itaconic acid, which potentiates tumor growth. Monocytes isolated from ovarian carcinoma patients' ascites fluid expressed significantly elevated levels of IRG1. Therefore, IRG1 in pResMϕ represents a potential therapeutic target for peritoneal tumors.

Peritoneal tissue-resident macrophages are metabolically poised to engage microbes using tissue-niche fuels.

The importance of metabolism in macrophage function has been reported, but the in vivo relevance of the in vitro observations is still unclear. Here we show that macrophage metabolites are defined in a specific tissue context, and these metabolites are crucially linked to tissue-resident macrophage functions. We find the peritoneum to be rich in glutamate, a glutaminolysis-fuel that is exploited by peritoneal-resident macrophages to maintain respiratory burst during phagocytosis via enhancing mitochondrial complex-II metabolism. This niche-supported, inducible mitochondrial function is dependent on protein kinase C activity, and is required to fine-tune the cytokine responses that control inflammation. In addition, we find that peritoneal-resident macrophage mitochondria are recruited to phagosomes and produce mitochondrially derived reactive oxygen species, which are necessary for microbial killing. We propose that tissue-resident macrophages are metabolically poised in situ to protect and exploit their tissue-niche by utilising locally available fuels to implement specific metabolic programmes upon microbial sensing.

Interleukin-27 Is a Potential Rescue Therapy for Acute Severe Colitis Through Interleukin-10-Dependent, T-Cell-Independent Attenuation of Colonic Mucosal Innate Immune Responses.

BACKGROUND: If treatment with intravenous steroids fail, inflammatory bowel disease patients with acute severe colitis face systemic anti-tumor necrosis factor biologic rescue therapy or colectomy. Interleukin (IL)-27 is a cytokine with an immunosuppressive role in adaptive immune responses. However, the IL-27 receptor complex is also expressed on innate immune cells, and there is evidence that IL-27 can impact the function of innate cell subsets, although this particular functionality in vivo is not understood. Our aim was to define the efficacy of IL-27 in acute severe colitis and characterize novel IL-27-driven mechanisms of immunosuppression in the colonic mucosa. METHODS: We assessed oral delivery of Lactococcus lactis expressing an IL-27 hyperkine on the innate immune response in vivo in a genetically intact, noninfective, acute murine colitis model induced by intrarectal instillation of 2,4,6-trinitrobenzenesulfonic acid in SJL/J mice. RESULTS: IL-27 attenuates acute severe colitis through the reduction of colonic mucosal neutrophil infiltrate associated with a decreased CXC chemokine gradient. This suppression was T cell independent and IL-10 dependent, initially featuring enhanced mucosal IL-10. IL-27 was associated with a reduction in colonic proinflammatory cytokines and induced a multifocal, strong, positive nuclear expression of phosphorylated STAT-1 in mucosal epithelial cells. CONCLUSION: We have defined novel mechanisms of IL-27 immunosuppression toward colonic innate immune responses in vivo. Mucosal delivery of IL-27 has translational potential as a novel therapeutic for inflammatory bowel disease, and it is a future mucosal directed rescue therapy in acute severe inflammatory bowel disease.

Autocrine IL-10 functions as a rheostat for M1 macrophage glycolytic commitment by tuning nitric oxide production.

Inflammatory maturation of M1 macrophages by proinflammatory stimuli such as toll like receptor ligands results in profound metabolic reprogramming resulting in commitment to aerobic glycolysis as evidenced by repression of mitochondrial oxidative phosphorylation (OXPHOS) and enhanced glucose utilization. In contrast, "alternatively activated" macrophages adopt a metabolic program dominated by fatty acid-fueled OXPHOS. Despite the known importance of these developmental stages on the qualitative aspects of an inflammatory response, relatively little is know regarding the regulation of these metabolic adjustments. Here we provide evidence that the immunosuppressive cytokine IL-10 defines a metabolic regulatory loop. Our data show for the first time that lipopolysaccharide (LPS)-induced glycolytic flux controls IL-10-production via regulation of mammalian target of rapamycin (mTOR) and that autocrine IL-10 in turn regulates macrophage nitric oxide (NO) production. Genetic and pharmacological manipulation of IL-10 and nitric oxide (NO) establish that metabolically regulated autocrine IL-10 controls glycolytic commitment by limiting NO-mediated suppression of OXPHOS. Together these data support a model where autocine IL-10 production is controlled by glycolytic flux in turn regulating glycolytic commitment by preserving OXPHOS via suppression of NO. We propose that this IL-10-driven metabolic rheostat maintains metabolic equilibrium during M1 macrophage differentiation and that perturbation of this regulatory loop, either directly by exogenous cellular sources of IL-10 or indirectly via limitations in glucose availability, skews the cellular metabolic program altering the balance between inflammatory and immunosuppressive phenotypes.

IL-10 differentially controls the infiltration of inflammatory macrophages and antigen-presenting cells during inflammation.

The inflammatory activation and recruitment of defined myeloid populations is essential for controlling the bridge between innate and adaptive immunity and shaping the immune response to microbial challenge. However, these cells exhibit significant functional heterogeneity and the inflammatory signals that differentially influence their effector characteristics are poorly characterized. In this study, we defined the phenotype of discrete subsets of effective antigen-presenting cells (APCs) in the peritoneal cavity during peritonitis. When the functional properties of these cells were compared to inflammatory monocyte-derived macrophages we noted differential responses to the immune-modulatory cytokine IL-10. In contrast to the suppressive actions of IL-10 on inflammatory macrophages, the recruitment of APCs was relatively refractory and we found no evidence for selective inhibition of APC differentiation. This differential response of myeloid cell subsets to IL-10 may thus have limited impact on development of potentially tissue-damaging adaptive immune responses, while restricting the magnitude of the inflammatory response. These findings may have clinical relevance in the context of peritoneal dialysis patients, where recurrent infections are associated with immune-mediated membrane dysfunction, treatment failure, and increased morbidity.