Publications
137 results found
Gudd C, Mitchell E, Atkinson S, et al., 2024, Therapeutic inhibition of monocyte recruitment prevents checkpoint inhibitor-induced hepatitis, Journal for ImmunoTherapy of Cancer, ISSN: 2051-1426
Asowata EO, Romoli S, Sargeant R, et al., 2024, Multi-omics and imaging mass cytometry characterization of human kidneys to identify pathways and phenotypes associated with impaired kidney function., Kidney Int
Despite the recent advances in our understanding of the role of lipids, metabolites, and related enzymes in mediating kidney injury, there is limited integrated multi-omics data identifying potential metabolic pathways driving impaired kidney function. The limited availability of kidney biopsies from living donors with acute kidney injury has remained a major constraint. Here, we validated the use of deceased transplant donor kidneys as a good model to study acute kidney injury in humans and characterized these kidneys using imaging and multi-omics approaches. We noted consistent changes in kidney injury and inflammatory markers in donors with reduced kidney function. Neighborhood and correlation analyses of imaging mass cytometry data showed that subsets of kidney cells (proximal tubular cells and fibroblasts) are associated with the expression profile of kidney immune cells, potentially linking these cells to kidney inflammation. Integrated transcriptomic and metabolomic analysis of human kidneys showed that kidney arachidonic acid metabolism and seven other metabolic pathways were upregulated following diminished kidney function. To validate the arachidonic acid pathway in impaired kidney function we demonstrated increased levels of cytosolic phospholipase A2 protein and related lipid mediators (prostaglandin E2) in the injured kidneys. Further, inhibition of cytosolic phospholipase A2 reduced injury and inflammation in human kidney proximal tubular epithelial cells in vitro. Thus, our study identified cell types and metabolic pathways that may be critical for controlling inflammation associated with impaired kidney function in humans.
Tsujimoto H, Hoshina A, Mae S-I, et al., 2024, Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells, Cell Reports, Vol: 43, ISSN: 2211-1247
Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures in vitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages in vitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.
Jackson WD, Giacomassi C, Ward S, et al., 2023, TLR7 activation at epithelial barriers promotes emergency myelopoiesis and lung antiviral immunity, eLife, Vol: 12, ISSN: 2050-084X
Monocytes are heterogeneous innate effector leukocytes generated in the bone marrow and released into circulation in a CCR2-dependent manner. During infection or inflammation, myelopoiesis is modulated to rapidly meet the demand for more effector cells. Danger signals from peripheral tissues can influence this process. Herein we demonstrate that repetitive TLR7 stimulation via the epithelial barriers drove a potent emergency bone marrow monocyte response in mice. This process was unique to TLR7 activation and occurred independently of the canonical CCR2 and CX3CR1 axes or prototypical cytokines. The monocytes egressing the bone marrow had an immature Ly6C-high profile and differentiated into vascular Ly6C-low monocytes and tissue macrophages in multiple organs. They displayed a blunted cytokine response to further TLR7 stimulation and reduced lung viral load after RSV and influenza virus infection. These data provide insights into the emergency myelopoiesis likely to occur in response to the encounter of single-stranded RNA viruses at barrier sites.
Marin EL, Musial B, Seth A, et al., 2023, FRONTIER-1: A PHASE 2B STUDY OF IL-33 BLOCKADE IN DIABETIC KIDNEY DISEASE, 60th ERA Congress, Publisher: OXFORD UNIV PRESS, Pages: I644-I644, ISSN: 0931-0509
Zeller J, Shing KSCT, Nero TL, et al., 2023, A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change in C-reactive protein, EMBO MOLECULAR MEDICINE, Vol: 15, ISSN: 1757-4676
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- Citations: 9
Yousif DE, Ye X, Stuard S, et al., 2023, Biphasic dynamics of inflammatory markers following hemodialysis initiation: results from the international MONitoring Dialysis Outcome initiative, Kidney International Reports, Vol: 8, Pages: 75-80, ISSN: 2468-0249
IntroductionInflammation is highly prevalent among patients with end-stage kidney disease and is associated with adverse outcomes. We aimed to investigate longitudinal changes in inflammatory markers in a diverse international incident hemodialysis patient population.MethodsThe MONitoring Dialysis Outcomes (MONDO) Consortium encompasses hemodialysis databases from 31 countries in Europe, North America, South America, and Asia. The MONDO database was queried for inflammatory markers (total white blood cell count [WBC], neutrophil count, lymphocyte count, serum albumin, and C-reactive protein [CRP]) and hemoglobin levels in incident hemodialysis patients. Laboratory parameters were measured every month. Patients were stratified by survival time (≤6 months, >6 to 12 months, >12 to 18 months, >18 to 24 months, >24 to 30 months, >30 to 36 months, and >36 months) following dialysis initiation. We used cubic B-spline basis function to evaluate temporal changes in inflammatory parameters in relationship with patient survival.ResultsWe studied 18,726 incident hemodialysis patients. Their age at dialysis initiation was 71.3 ± 11.9 years; 10,802 (58%) were males. Within the first 6 months, 2068 (11%) patients died, and 12,295 patients (67%) survived >36 months (survivor cohort). Hemodialysis patients who died showed a distinct biphasic pattern of change in inflammatory markers where an initial decline of inflammation was followed by a rapid rise that was consistently evident approximately 6 months before death. This pattern was similar in all patients who died and was consistent across the survival time intervals. In contrast, in the survivor cohort, we observed initial decline of inflammation followed by sustained low levels of inflammatory biomarkers.ConclusionOur international study of incident hemodialysis patients highlights a temporal relationship between serial measurements of inflammatory markers and patient survival. This finding may inf
Buvall L, Menzies RI, Williams J, et al., 2022, Selecting the right therapeutic target for kidney disease, Frontiers in Pharmacology, Vol: 13, ISSN: 1663-9812
Kidney disease is a complex disease with several different etiologies and underlying associated pathophysiology. This is reflected by the lack of effective treatment therapies in chronic kidney disease (CKD) that stop disease progression. However, novel strategies, recent scientific breakthroughs, and technological advances have revealed new possibilities for finding novel disease drivers in CKD. This review describes some of the latest advances in the field and brings them together in a more holistic framework as applied to identification and validation of disease drivers in CKD. It uses high-resolution ‘patient-centric’ omics data sets, advanced in silico tools (systems biology, connectivity mapping, and machine learning) and ‘state-of-the-art‘ experimental systems (complex 3D systems in vitro, CRISPR gene editing, and various model biological systems in vivo). Application of such a framework is expected to increase the likelihood of successful identification of novel drug candidates based on strong human target validation and a better scientific understanding of underlying mechanisms.
Hofherr A, Williams J, Gan L-M, et al., 2022, Targeting inflammation for the treatment of Diabetic Kidney Disease: a five-compartment mechanistic model, BMC Nephrology, Vol: 23, ISSN: 1471-2369
Diabetic kidney disease (DKD) is the leading cause of kidney failure worldwide. Mortality and morbidity associated with DKD are increasing with the global prevalence of type 2 diabetes. Chronic, sub-clinical, non-resolving inflammation contributes to the pathophysiology of renal and cardiovascular disease associated with diabetes. Inflammatory biomarkers correlate with poor renal outcomes and mortality in patients with DKD. Targeting chronic inflammation may therefore offer a route to novel therapeutics for DKD. However, the DKD patient population is highly heterogeneous, with varying etiology, presentation and disease progression. This heterogeneity is a challenge for clinical trials of novel anti-inflammatory therapies. Here, we present a conceptual model of how chronic inflammation affects kidney function in five compartments: immune cell recruitment and activation; filtration; resorption and secretion; extracellular matrix regulation; and perfusion. We believe that the rigorous alignment of pathophysiological insights, appropriate animal models and pathology-specific biomarkers may facilitate a mechanism-based shift from recruiting 'all comers' with DKD to stratification of patients based on the principal compartments of inflammatory disease activity.
Constantinescu-Bercu A, Wang YA, Woollard KJ, et al., 2022, The GPIbα intracellular tail – role in transducing VWF- and collagen/GPVI-mediated signaling, Haematologica, Vol: 107, Pages: 933-946, ISSN: 0390-6078
<jats:p>The GPIbT-VWF A1 domain interaction is essential for platelet tethering under high shear. Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GpIbαΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail that harbors the 14-3-3 and phosphoinositide-3 kinase binding sites. GPIbαΔsig/Δsig platelets bound VWF normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a oIIbI3 blocker, demonstrating that despite normal ligand binding, VWF-dependent signaling is diminished. Activation of GpIbαΔsig/Δsig platelets with ADP and thrombin was normal, but GpIbαΔsig/Δsig platelets stimulated with collagen-related-peptide (CRP) exhibited markedly decreased P-selectin exposure and eIIbI3 activation, suggesting a role for the GpIbaaintracellular tail in GPVI-mediated signaling. Consistent with this, while haemostasis was normal in GPIbαΔsig/Δsig mice, diminished tyrosine-phosphorylation, (particularly pSYK) was detected in CRP-stimulated GpIbαΔsig/Δsig platelets as well as reduced platelet spreading on CRP. Platelet responses to rhodocytin were also affected in GpIbαΔsig/Δsig platelets but to a lesser extent than those with CRP. GpIbαΔsig/Δsig platelets formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shear. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of bIIbI3- or GPVI-blockers suggested reduced bIIbI3 activation contributes to the phenotype of the GpIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbiiin transducing
Normahani P, Boyle JJ, Cave L, et al., 2022, Peripheral blood mononuclear cell gene expression and cytokine profiling in patients with intermittent claudication who exhibit exercise induced acute renal injury., PLoS One, Vol: 17, Pages: 1-15, ISSN: 1932-6203
BACKGROUND: Intermittent claudication (IC) is a common manifestation of peripheral arterial disease. Some patients with IC experience a rise in Urinary N-acetyl-β-D-Glucosaminidase (NAG)/ Creatinine (Cr) ratio, a marker of renal injury, following exercise. In this study, we aim to investigate whether peripheral blood mononuclear cells (PBMC) from patients with IC who exhibit a rise in urinary NAG/ Cr ratio following exercise exhibit differential IL-10/ IL-12 ratio and gene expression compared to those who do not have a rise in NAG/ Cr ratio. METHODS: We conducted a single center observational cohort study of patients diagnosed with IC. Blood and urine samples were collected at rest and following a standardised treadmill exercise protocol. For comparative analysis patients were separated into those with any rise in NAG/Cr ratio (Group 1) and those with no rise in NAG/Cr ratio (Group 2) post exercise. Isolated PBMC from pre- and post-exercise blood samples were analysed using flow cytometry. PBMC were also cultured for 20 hours to perform further analysis of IL-10 and IL-12 cytokine levels. RNA-sequencing analysis was performed to identify differentially expressed genes between the groups. RESULTS: 20 patients were recruited (Group 1, n = 8; Group 2, n = 12). We observed a significantly higher IL-10/IL-12 ratio in cell supernatant from participants in Group 1, as compared to Group 2, on exercise at 20 hours incubation; 47.24 (IQR 9.70-65.83) vs 6.13 (4.88-12.24), p = 0.04. 328 genes were significantly differentially expressed between Group 1 and 2. The modulated genes had signatures encompassing hypoxia, metabolic adaptation to starvation, inflammatory activation, renal protection, and oxidative stress. DISCUSSION: Our results suggest that some patients with IC have an altered immune status making them 'vulnerable' to systemic inflammation and renal injury following exercise. We have identified a panel of genes which are differentially expressed in this group of
Prendecki M, McAdoo SP, Turner-Stokes T, et al., 2022, Glomerulonephritis and autoimmune vasculitis are independent of P2RX7 but may depend on alternative inflammasome pathways., Journal of Pathology, Vol: 257, ISSN: 0022-3417
P2RX7, an ionotropic receptor for extracellular ATP, is expressed on immune cells, including macrophages, monocytes and dendritic cells and is up-regulated on non-immune cells following injury. P2RX7 plays a role in many biological processes, including production of pro-inflammatory cytokines such as IL-1β via the canonical inflammasome pathway. P2RX7 has been shown to be important in inflammation and fibrosis and may also play a role in autoimmunity. We have developed and phenotyped a novel P2RX7 knock-out (KO) inbred rat strain and taking advantage of the human-resembling unique histopathological features of rat models of glomerulonephritis, we induced three models of disease: nephrotoxic nephritis, experimental autoimmune glomerulonephritis, and experimental autoimmune vasculitis. We found that deletion of P2RX7 does not protect rats from models of experimental glomerulonephritis or the development of autoimmunity. Notably, treatment with A-438079, a P2RX7 antagonist, was equally protective in WKY WT and P2RX7 KO rats, revealing its 'off-target' properties. We identify a novel ATP/P2RX7/K+ efflux-independent and caspase-1/8-dependent pathway for production of IL-1β in rat dendritic cells, which was absent in macrophages. Taken together, these results comprehensively establish that inflammation and autoimmunity in glomerulonephritis is independent of P2RX7 and reveals the off-target properties of drugs previously known as selective P2RX7 antagonists. Rat mononuclear phagocytes may be able to utilise an 'alternative inflammasome' pathway to produce IL-1β independently of P2RX7, which may account for the susceptibility of P2RX7 KO rats to inflammation and autoimmunity in glomerulonephritis. This article is protected by copyright. All rights reserved.
Woollard K, 2022, Chronic kidney disease mediates cardiac dysfunction associated with increased resident cardiac macrophages, BMC Nephrology, Vol: 23, Pages: 1-15, ISSN: 1471-2369
Background – The leading cause of death in end-stage kidney disease is related tocardiovascular disease. Macrophages are known to be involved in both chronic kidney disease(CKD) and heart failure, however their role in the development of cardiorenal syndrome is lessclear. We thus sought to investigate the role of macrophages in uremic cardiac disease.Methods – We assessed cardiac response in two experimental models of CKD and testedmacrophage and chemokine implication in monocytopenic CCR2-/- and anti-CXCL10 treatedmice. We quantified CXCL10 in human CKD plasma and tested the response of human iPSCderived cardiomyocytes and primary cardiac fibroblasts to serum from CKD donors. Results– We found that reduced kidney function resulted in the expansion of cardiac macrophages,in particular through local proliferation of resident populations. Influx of circulating monocytescontributed to this increase. We identified CXCL10 as a crucial factor for cardiac macrophageexpansion in uremic disease. In humans, we found increased plasma CXCL10 concentrationsin advanced CKD, and identified the production of CXCL10 in cardiomyocytes and cardiacfibroblasts. Conclusions – This study provides new insight into the role of the innate immunesystem in uremic cardiomyopathy.
Prendecki M, Gulati K, Turner-Stokes T, et al., 2021, Characterisation of an enhanced preclinical model of experimental MPO-ANCA autoimmune vasculitis, Journal of Pathology, Vol: 255, Pages: 107-119, ISSN: 0022-3417
Experimental autoimmune vasculitis (EAV) is a model of antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) induced by immunisation of susceptible rat strains with myeloperoxidase (MPO). Animals develop circulating MPO-ANCA, pulmonary haemorrhage and glomerulonephritis, although renal injury is mild and recovers spontaneously without treatment. In this study we aimed to augment the severity of glomerulonephritis. Following induction of EAV on day 0, a sub-nephritogenic dose of nephrotoxic serum (NTS) containing heterologous antibodies to glomerular basement membrane was administered on day 14. This resulted in a significant increase in disease severity at day 28 compared to MPO immunisation alone - with more urinary abnormalities, infiltrating glomerular leucocytes, and crescent formation that progressed to glomerular and tubulointerstitial scarring by day 56, recapitulating important features of human disease. Importantly, the glomerulonephritis remained pauci-immune, and was strictly dependent on the presence of autoimmunity to MPO, as there was no evidence of renal disease following administration of sub-nephritogenic NTS alone or after immunisation with a control protein in place of MPO. Detailed phenotyping of glomerular leucocytes identified an early infiltrate of non-classical monocytes following NTS administration that, in the presence of autoimmunity to MPO, may initiate the subsequent influx of classical monocytes which augment glomerular injury. We also showed that this model can be used to test novel therapeutics by using a small molecule kinase inhibitor (fostamatinib) that rapidly attenuated both glomerular and pulmonary injury over a four-day treatment period. We believe that this enhanced model of MPO-AAV will prove useful for the study of glomerular leucocyte behaviour and novel therapeutics in AAV in the future. This article is protected by copyright. All rights reserved.
Shamshirgaran Y, Jonebring A, Svensson A, et al., 2021, Rapid target validation in a Cas9-inducible hiPSC derived kidney model, Scientific Reports, Vol: 11, Pages: 1-9, ISSN: 2045-2322
Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.
Possamai L, Gudd C, Au L, et al., 2021, Activation and transcriptional profile of monocytes and CD8+ T cells are altered in checkpoint inhibitor-related hepatitis, Journal of Hepatology, Vol: 75, Pages: 177-189, ISSN: 0168-8278
Background & Aims: Checkpoint inhibitor-related hepatitis (CPI-Hep) is an emerging clinical challenge. We aimed to gain insights into the immunopathology of CPI-Hep by comprehensively characterising myeloid and lymphoid subsets.Methods: CPI-treated patients with or without related hepatitis (CPI-Hep; n = 22 and CPI-noHep; n = 7) were recruited. Phenotypic and transcriptional profiling of peripheral immune subsets was performed and compared with 19 healthy controls (HCs). In vitro monocyte-derived macrophages (MoMFs) were assessed for activation and cytokine production. CD163, CCR2, CD68, CD3, CD8 and granzyme B expression was assessed using immunohistochemistry/immunofluorescence (n = 4).Results: A significant total monocyte depletion was observed in CPI-Hep compared with HCs (p = 0.04), along with a proportionate increase in the classical monocyte population (p = 0.0002) and significant upregulation of CCR2, CD163 and downregulation of CCR7. Soluble CD163 levels were significantly elevated in CPI-Hep compared with HCs (p <0.0001). In vitro MoMFs from CPI-Hep showed enhanced production of pro-inflammatory cytokines. CD8+ T cells demonstrated increased perforin, granzyme B, ICOS and HLA-DR expression in CPI-Hep. Transcriptional profiling indicated the presence of activated monocyte and enhanced effector CD8+ T cell populations in CPI-Hep. Immunohistochemistry demonstrated co-localisation of CD8+/granzyme B+ T cells with CD68+CCR2+/CD68+CD163+ macrophages in CPI-Hep liver tissue.Conclusions: CPI-Hep is associated with activation of peripheral monocytes and an enhanced cytotoxic, effector CD8+ T cell phenotype. These changes were reflected by liver inflammation composed of CD163+/CCR2+ macrophages and CD8+ T cells.Lay summary: Some patients who receive immunotherapy for cancer develop liver inflammation, which requires cessation of cancer treatment. Herein, we describe ways in which the white blood cells of patients who develop liver inflammation differ from tho
Gudd CLC, Antoniades CG, Turajlic S, et al., 2021, CCR2+monocytes induce hepatic accumulation and activation of tissue destructive CD8+T cells in a murine model of checkpoint inhibitor-induced hepatitis, Publisher: ELSEVIER, Pages: S224-S225, ISSN: 0168-8278
George MJ, Jasmin NH, Cummings VT, et al., 2021, Selective interleukin-6 trans-signaling blockade is more effective than panantagonism in reperfused myocardial infarction, JACC: Basic to Translational Science, Vol: 6, Pages: 431-443, ISSN: 2452-302X
Interleukin (IL)-6 is an emerging therapeutic target in myocardial infarction (MI). IL-6 has 2 distinct signaling pathways: trans-signaling, which mediates inflammation, and classic signaling, which also has anti-inflammatory effects. The novel recombinant fusion protein sgp130Fc achieves exclusive trans-signaling blockade, whereas anti-IL-6 antibodies (Abs) result in panantagonism. In a rat model of reperfused MI, sgp130Fc, but not anti-IL-6-Ab, attenuated neutrophil and macrophage infiltration into the myocardium, reduced infarct size, and preserved cardiac function 28 days after MI. These data demonstrate the efficacy of exclusive IL-6 trans-signaling blockade and support further investigation of sgp130Fc as a potential novel therapy in MI.
Kiefer J, Zeller J, Bogner B, et al., 2021, An unbiased flow cytometry-based approach to assess subset-specific circulating monocyte activation and cytokine profile in whole blood., Frontiers in Immunology, Vol: 12, ISSN: 1664-3224
Monocytes are the third most frequent type of leukocytes in humans, linking innate and adaptive immunity and are critical drivers in many inflammatory diseases. Based on the differential expression of surface antigens, three monocytic subpopulations have been suggested in humans and two in rats with varying inflammatory and phenotype characteristics. Potential intervention strategies that aim to manipulate these cells require an in-depth understanding of monocyte behavior under different conditions. However, monocytes are highly sensitive to their specific activation state and expression of surface markers, which can change during cell isolation and purification. Thus, there is an urgent need for an unbiased functional analysis of activation in monocyte subtypes, which is not affected by the isolation procedure. Here, we present a flow cytometry-based protocol for evaluating subset-specific activation and cytokine expression of circulating blood monocytes both in humans and rats using small whole blood samples (50 - 100 μL). In contrast to previously described monocyte isolation and flow cytometry visualization methods, the presented approach virtually leaves monocyte subsets in a resting state or fixes them in their current state and allows for an unbiased functional endpoint analysis without prior cell isolation. This protocol is a comprehensive tool for studying differential monocyte regulation in the inflammatory and allogeneic immune response in vitro and vivo.
Michell DL, Shihata WA, Andrews KL, et al., 2021, High intraluminal pressure promotes vascular inflammation via caveolin-1, Scientific Reports, Vol: 11, Pages: 1-15, ISSN: 2045-2322
The aetiology and progression of hypertension involves various endogenous systems, such as the renin angiotensin system, the sympathetic nervous system, and endothelial dysfunction. Recent data suggest that vascular inflammation may also play a key role in the pathogenesis of hypertension. This study sought to determine whether high intraluminal pressure results in vascular inflammation. Leukocyte adhesion was assessed in rat carotid arteries exposed to 1 h of high intraluminal pressure. The effect of intraluminal pressure on signaling mechanisms including reactive oxygen species production (ROS), arginase expression, and NFĸB translocation was monitored. 1 h exposure to high intraluminal pressure (120 mmHg) resulted in increased leukocyte adhesion and inflammatory gene expression in rat carotid arteries. High intraluminal pressure also resulted in a downstream signaling cascade of ROS production, arginase expression, and NFĸB translocation. This process was found to be angiotensin II-independent and mediated by the mechanosensor caveolae, as caveolin-1 (Cav1)-deficient endothelial cells and mice were protected from pressure-induced vascular inflammatory signaling and leukocyte adhesion. Cav1 deficiency also resulted in a reduction in pressure-induced glomerular macrophage infiltration in vivo. These findings demonstrate Cav1 is an important mechanosensor in pressure-induced vascular and renal inflammation.
Constantinescu-Bercu A, Wang YA, Woollard K, et al., 2020, The GPIbα intracellular tail - role in transducing VWF- and Collagen/GPVI-mediated signaling, Publisher: Cold Spring Harbor Laboratory
Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GPIbαΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail important for VWF-mediated signaling. GPIbαΔsig/Δsig platelets bound VWF normally under flow but formed fewer filopodia on VWF/botrocetin, demonstrating that the deleted region does not affect ligand binding but appreciably impairs VWF-dependent signaling. Notably, while haemostasis was normal in GPIbαΔsig/Δsig mice, GPIbαΔsig/Δsig platelets exhibited defective responses after collagen-related-peptide stimulation and formed smaller aggregates on collagen-coated microchannels at low and high shears. Flow assays performed with plasma-free blood or in the presence of αIIbβ3-or GPVI-blockers suggested reduced αIIbβ3 activation contributes to the phenotype of the GPIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbα in transducing both VWF-GPIbα and collagen-GPVI signaling events in platelets.
Triantafyllou E, Gudd C, Mawhin M-A, et al., 2020, PD-1 blockade improves Kupffer cell bacterial clearance in acute liver injury, Journal of Clinical Investigation, Vol: 131, Pages: 1-16, ISSN: 0021-9738
Acute liver failure (ALF) patients display systemic innate immune suppression and increased susceptibility to infections. PD-1 expression by macrophages has been associated with immune suppression during sepsis and cancer. We therefore examined the role of PD-1/PD-L1 pathway in regulating Kupffer cell inflammatory and antimicrobial responses in acetaminophen (APAP) induced acute liver injury. Using intravital imaging and flow cytometry we found impaired Kupffer cell bacterial clearance and systemic bacterial dissemination in mice with liver injury. Increased PD-1 and PD-L1 expression was detected in Kupffer cells and lymphocyte subsets, respectively, during resolution of injury. Gene expression profiling of PD-1+ Kupffer cells revealed an immune-suppressive profile and reduced pathogen responses. Compared to wild-type, PD-1 deficient or anti-PD-1 treated mice with liver injury showed improved Kupffer cell bacterial clearance, reduced tissue bacterial load and protection from sepsis. Blood sample analyses of ALF patients revealed enhanced PD-1 and PD-L1 expression of monocytes and lymphocytes, respectively, and that plasma soluble PD-L1 levels predict patient outcome and sepsis. PD-1 in vitro blockade restored monocyte functionality. Our study describes a role for PD-1/PD-L1 axis in suppressing Kupffer cell and monocyte antimicrobial responses after liver injury and suggests anti-PD-1 immunotherapy as a strategy to reduce infection susceptibility in ALF.
Pinheiro D, Mawhin M-A, Prendecki M, et al., 2020, In-silico analysis of myeloid cells across the animal kingdom reveals neutrophil evolution by colony-stimulating factors, ELIFE, Vol: 9, ISSN: 2050-084X
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Rahman MS, Haskard DO, Frost G, et al., 2020, Acute dietary saturated fat intake suppresses human monocyte subset inflammatory and chemokine responses, European-Society-of-Cardiology (ESC) Congress, Publisher: OXFORD UNIV PRESS, Pages: 3622-3622, ISSN: 0195-668X
Siggins MK, Lynskey NN, Lamb L, et al., 2020, Extracellular bacterial lymphatic metastasis drives Streptococcus pyogenes systemic infection, Nature Communications, Vol: 11, ISSN: 2041-1723
Unassisted metastasis through the lymphatic system is a mechanism of dissemination thus far ascribed only to cancer cells. Here, we report that Streptococcus pyogenes also hijack lymphatic vessels to escape a local infection site, transiting through sequential lymph nodes and efferent lymphatic vessels to enter the bloodstream. Contrasting with previously reported mechanisms of intracellular pathogen carriage by phagocytes, we show S. pyogenes remain extracellular during transit, first in afferent and then efferent lymphatics that carry the bacteria through successive draining lymph nodes. We identify streptococcal virulence mechanisms important for bacterial lymphatic dissemination and show that metastatic streptococci within infected lymph nodes resist and subvert clearance by phagocytes, enabling replication that can seed intense bloodstream infection. The findings establish the lymphatic system as both a survival niche and conduit to the bloodstream for S. pyogenes, explaining the phenomenon of occult bacteraemia. This work provides new perspectives in streptococcal pathogenesis with implications for immunity.
Turner-Stokes T, Garcia Diaz A, Pinheiro D, et al., 2020, Live imaging of monocyte subsets in immune complex-mediated glomerulonephritis reveals distinct phenotypes and effector functions., Journal of the American Society of Nephrology, Vol: 31, Pages: 1-1, ISSN: 1046-6673
BACKGROUND: Immune complexes within glomerular capillary walls cause crescentic GN (CrGN). Monocytes and macrophages are important in mediating CrGN, but little work has been done to phenotype the subpopulations involved and determine their respective contributions to glomerular inflammation. METHODS: Live glomerular imaging using confocal microscopy monitored intravascular monocyte subset behavior during nephrotoxic nephritis (NTN) in a novel WKY-hCD68-GFP monocyte/macrophage reporter rat strain. Flow cytometry and qPCR further analyzed ex vivo the glomerular leukocyte infiltrate during NTN. RESULTS: Non-classical monocytes surveyed the glomerular endothelium via lymphocyte function-associated antigen 1 (LFA-1) in the steady state. During NTN, non-classical monocytes were recruited first, but subsequent recruitment and retention of classical monocytes was associated with glomerular damage. Monocytes recruited to the glomerular vasculature did not undergo transendothelial migration. This finding suggests that inflammation in immune complex-mediated CrGN is predominantly intravascular, driven by dynamic interactions between intravascular blood monocytes and the endothelium. Glomerular endothelium and non-classical monocytes overexpressed a distinct chemokine axis, which may orchestrate inflammatory myeloid cell recruitment and expression of damage mediators. Reduced classical monocyte recruitment in Lewis rats during NTN confirmed a role for CD16 in mediating glomerular damage. CONCLUSIONS: Monocyte subsets with distinct phenotypes and effector functions may be important in driving inflammation in experimental CrGN resulting from immune complexes formed within the glomerular capillary wall. LFA-1-dependent endothelial surveillance by non-classical monocytes may detect immune complexes through CD16, orchestrating the inflammatory response through intravascular retention of classical monocytes, which results in glomerular damage and proteinuria.
Pinheiro D, Mahwin M-A, Prendecki M, et al., 2020, Analysis of receptor-ligand pairings and distribution of myeloid subpopulations across the animal kingdom reveals neutrophil evolution was facilitated by colony-stimulating factors
<jats:title>Abstract</jats:title><jats:p>Neutrophils or heterophils constitute the largest population of phagocytic granulocytes in the blood of mammals and birds. The development and function of neutrophils and monocytes is primarily governed by the granulocyte colony-stimulating factor receptor family (CSF3R/CSF3) and macrophage colony-stimulating factor receptor family (CSF1R/IL34/CSF1) respectively. Using various techniques this study considered how the emergence of receptor:ligand pairings shaped the distribution of blood myeloid cell populations. Comparative gene analysis supported the ancestral pairings of CSF1R/IL34 and CSF3R/CSF3, and the emergence of CSF1 later in tetrapod lineages after the advent of Jawed/Jawless fish. Further analysis suggested that the emergence of CSF3 lead to reorganisation of granulocyte distribution between amphibian and early reptiles. However, the advent of endothermy likely contributed to the dominance of the neutrophil/heterophil in modern-day mammals and birds. In summary, we show that the emergence of CSF3R/CSF3 was a key factor in the subsequent evolution of the modern-day mammalian neutrophil.</jats:p><jats:sec><jats:title>Impact statement</jats:title><jats:p>Colony-stimulating factors (CSFs) are important for myeloid phagocyte development. The emergence of CSF3/CSF3R in tetrapod lineages has uniquely contributed to physical, functional and structural adaptions observed in mammalian neutrophils.</jats:p></jats:sec>
Srivatsan A, Woollard K, 2020, Immunohistologic comparison and thrombus NET analysis in stroke and myocardial infarction, Neurology, Vol: 94, Pages: 955-956, ISSN: 0028-3878
Bright R, Ye X, Woollard K, et al., 2020, MONOCYTE-TO-LYMPHOCYTE RATIO, AN INDEPENDENT RISK FACTOR OF SURVIVAL IN HEMODIALYSIS PATIENTS: RESULTS FROM THE INTERNATIONAL MONDO CONSORTIUM, 57th ERA-EDTA Congress, Publisher: OXFORD UNIV PRESS, Pages: 66-66, ISSN: 0931-0509
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