The Role of TGFβ1 and Macrophage Differentiation in MSU Crystal-Induced Inflammation

Gout is a painful form of inflammatory arthritis that is caused by the deposition of monosodium urate (MSU) crystals in the joints. MSU crystals trigger a local inflammatory response initiated by resident macrophages followed by a large infiltration of leukocytes. The spontaneous resolution of acute gout is associated with the production of transforming growth factor β1 (TGFβ1). The overall objectives of this thesis were to investigate mechanisms that lead to TGFβ1 production and contribute to the resolution of acute gout, the effect of TGFβ1 on the functional phenotype of differentiated macrophages, and possible changes in surface marker expression by macrophages in response to MSU crystals. To determine macrophage-independent sources of TGFβ1 during the resolution of acute gout and how TGFβ1 production altered MSU crystal-recruited neutrophil functions, neutrophils were purified from MSU crystal-treated mice when levels of TGFβ1 were high. MSU crystal-recruited neutrophils and circulating blood neutrophils were identified as TGFβ1⁺ cells. The mechanism for TGFβ1 production by neutrophils was associated with their ability to phagocytose apoptotic neutrophils. TGFβ1 produced by canibalising neutrophils inhibited both respiratory burst and interleukin-1β (IL-1β) production by MSU crystal-activated neutrophils ex vivo. Importantly, neutrophils from MSU crystal-challenged mice treated with TGFβ1 neutralising antibody in vivo produced elevated levels of superoxide but neutrophil IL-1β production was unaffected. These results show that TGFβ1 produced by canibalising neutrophils can actively suppress neutrophil inflammatory functions and therefore make a significant contribution towards the resolution of gouty inflammation. To investigate the effect of TGFβ1 on macrophage differentiation in vitro, granulocyte macrophage colony-stimulating factor (GM-CSF) bone marrow macrophages (GM-BMMs) and macrophage colony-stimulating factor (M-CSF) bone marrow macrophages (M-BMMs) were generated in the presence of TGFβ1. TGFβ1 was found to drive a hyper-inflammatory GM-BMM phenotype, while contributing to the differentiation of a hypo-inflammatory M-BMM phenotype specifically in response to MSU crystals. Increased IL-1β production by TGFβ1-differentiated GM-BMMs was associated with enhanced NOD like receptor family, pyrin domain-containing 3 (NLRP3) in ammasome activation and caspase 1/caspase 8 interaction, and a down-regulation of receptor-interacting serine/threonine-protein kinase 3 (RIP3) triggered by MSU crystals. At the same, TGFβ1 inhibited antigen-specific T cell proliferation by GM-BMMs. In contrast, TGFβ1-treated M-BMMs down-regulated the expression of active IL-1β that correlated with decreased IL-1β production, and upregulated RIP3 expression in response to MSU crystals. These data indicate that TGFβ1-treated GM-BMMs exhibited a hyper-inflammatory response to MSU crystal stimulation, whereas M-BMMs were found to be hypo-responsive. Macrophages were found to upregulate the surface marker NK1.1, which is primarily expressed on natural killer (NK) cells, and occured as a consequence of phagocytosis. Following phagocytosis of MSU crystals, activated macrophages produced IL-1β and tumour necrosis factor ⍺ (TNF⍺), which triggered the upregulation of NK1.1 expression. Macrophage NK1.1 expression is an activation-driven event specifc to MSU crystals. However, phagocytosis of apoptotic neutrophils also triggered the upregulation of NK1.1 by macrophages, a non-inflammatory event that is characteristic for the resolution of acute inflammation. These findings suggest that macrophages may develop NK cell-like properties initiated by an activation-driven or apoptotic cell clearance mechanism. Taken together, the results of this thesis indicate that canibalising neutrophils self-regulate their inflammatory functions via TGFβ1 and that TGFβ1 drives a hyper-inflammatory GM-BMM phenotype, while shutting down inflammatory functions of M-BMMs. These data highlight a regulatory role for TGFβ1 during acute gouty inflammation

Biological drugs for systemic lupus erythematosus or active lupus nephritis and rates of infectious complications. Evidence from large clinical trials

Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease that frequently affects the kidneys, known as lupus nephritis (LN). Such patients are treated with antimalarials, corticosteroids or immunosuppressive drugs, and more recently, target-specific biological drugs. Although efficacy of these therapies improved SLE-related outcomes, SLE remains associated with higher rates of infections. Here, we performed a comprehensive systemic review of infectious complications in clinical trials covering drug interventions for SLE or specifically for active LN. Our search in 15 online registries yielded a total of 1477 studies of which 14 matched our prespecified criteria. These covered the biological drugs anifrolumab, belimumab, and rituximab that were tested in patients with non-renal SLE and active LN.The available safety data from the SLE trials indicated that infectious complications such as herpes zoster, upper respiratory tract infection, nasopharyngitis, bronchitis, and urinary tract infection in patients receiving placebo were quite prevalent especially in the EXPLORER (rituximab) trial. Infections occurred mostly during the first year of LN therapy. Serious adverse events and infectious complications occurred more frequently in placebo-treated patients with active LN, especially in the BLISS-LN (belimumab) and LUNAR (rituximab) trials. Anifrolumab and rituximab increased the number of clinically relevant episodes of herpes zoster compared to belimumab in patients with active LN. Anifrolumab displayed a similar trend for influenza infections, which is consistent with the specific mechanisms-of-action of anifrolumab; highlighting drug-specific effects on infectious complications. In addition, standard-of-care therapy, e.g., MMF and immunosuppressants, as well as a longer SLE duration may also affect the incidence of serious adverse events and certain infectious complications in SLE patients with active LN.Infectious complications are common in SLE but even more common in patients with active LN, especially herpes zoster is strongly associated with active LN and anifrolumab therapy (OR 2.8, 95% CI 1.18 to 6.66, p = 0.018). Immunotherapy seems to impose unspecific and specific risks for infections. The latter may imply specific precautions such as preemptive vaccination and individual risk-benefit assessments

A guide to crystal‐related and nano‐ or microparticle‐related tissue responses

Crystals and nano‐ and microparticles form inside the human body from intrinsic proteins, minerals, or metabolites or enter the body as particulate matter from occupational and environmental sources. Associated tissue injuries and diseases mostly develop from cellular responses to such crystal deposits and include inflammation, cell necrosis, granuloma formation, tissue fibrosis, and stone‐related obstruction of excretory organs. But how do crystals and nano‐ and microparticles trigger these biological processes? Which pathomechanisms are identical across different particle types, sizes, and shapes? In addition, which mechanisms are specific to the atomic or molecular structure of crystals or to specific sizes or shapes? Do specific cellular or molecular mechanisms qualify as target for therapeutic interventions? Here, we provide a guide to approach this diverse and multidisciplinary research domain. We give an overview about the clinical spectrum of crystallopathies, about shared and specific pathomechanisms as a conceptual overview before digging deeper into the specialty field of interest

Sodium glucose transporter-2 inhibition has no renoprotective effects on non-diabetic chronic kidney disease

Sodium glucose transporter (SGLT)-2 inhibition has renoprotective effects in diabetic kidney disease. Whether similar effects can be achieved also in non-diabetic kidney disease is speculative. Chronic kidney disease was induced in C57BL/6N mice by feeding an oxalate-rich diet for 14days, known to induce nephrocalcinosis-related tubular atrophy and interstitial fibrosis without directly affecting the glomerular compartment. Empagliflozin treatment started from day 0 of oxalate feeding had no effect on the decline of glomerular filtration rate, crystal deposition, blood urea nitrogen or serum creatinine levels on day 7 and 14. Tissue morphometry of tubular injury and kidney mRNA levels of kidney injury molecule-1 or tissue inhibitor of metalloproteinase-2 were comparable between empagliflozin- and vehicle-treated mice with oxalate nephropathy on day 7 and 14. Similarly, empagliflozin did not affect markers of interstitial fibrosis, including silver, alpha smooth muscle actin (alpha SMA) and collagen 1 staining, and mRNA levels of fibronectin-1, collagen 1 alpha 1, fibroblast-specific protein-1, and transforming growth factor (TGF)-beta 2 on day 7 and 14. Thus, the specific renoprotective mechanisms-of-action of SGLT2 inhibition in diabetic kidney disease do not apply to chronic oxalosis, a non-diabetic form of chronic kidney disease

IRF8-Dependent Type I Conventional Dendritic Cells (cDC1s) Control Post-Ischemic Inflammation and Mildly Protect Against Post-Ischemic Acute Kidney Injury and Disease

Post-ischemic acute kidney injury and disease (AKI/AKD) involve acute tubular necrosis and irreversible nephron loss. Mononuclear phagocytes including conventional dendritic cells (cDCs) are present during different phases of injury and repair, but the functional contribution of this subset remains controversial. Transcription factor interferon regulatory factor 8 (IRF8) is required for the development of type I conventional dendritic cells (cDC1s) lineage and helps to define distinct cDC1 subsets. We identified one distinct subset among mononuclear phagocyte subsets according to the expression patterns of CD11b and CD11c in healthy kidney and lymphoid organs, of which IRF8 was significantly expressed in the CD11blowCD11chigh subset that mainly comprised cDC1s. Next, we applied a Irf8-deficient mouse line (Irf8fl/flClec9acre mice) to specifically target Clec9a-expressing cDC1s in vivo. During post-ischemic AKI/AKD, these mice lacked cDC1s in the kidney without affecting cDC2s. The absence of cDC1s mildly aggravated the loss of living primary tubule and decline of kidney function, which was associated with decreased anti-inflammatory Tregs-related immune responses, but increased T helper type 1 (TH1)-related and pro-inflammatory cytokines, infiltrating neutrophils and acute tubular cell death, while we also observed a reduced number of cytotoxic CD8+ T cells in the kidney when cDC1s were absent. Together, our data show that IRF8 is indispensable for kidney cDC1s. Kidney cDC1s mildly protect against post-ischemic AKI/AKD, probably via suppressing tissue inflammation and damage, which implies an immunoregulatory role for cDC1s

Soluble Uric Acid Is an Intrinsic Negative Regulator of Monocyte Activation in Monosodium Urate Crystal–Induced Tissue Inflammation

Abstract Although monosodium urate (MSU) crystals are known to trigger inflammation, published data on soluble uric acid (sUA) in this context are discrepant. We hypothesized that diverse sUA preparation methods account for this discrepancy and that an animal model with clinically relevant levels of asymptomatic hyperuricemia and gouty arthritis can ultimately clarify this issue. To test this, we cultured human monocytes with different sUA preparation solutions and found that solubilizing uric acid (UA) by prewarming created erroneous results because of UA microcrystal contaminants triggering IL-1β release. Solubilizing UA with NaOH avoided this artifact, and this microcrystal-free preparation suppressed LPS- or MSU crystal–induced monocyte activation, a process depending on the intracellular uptake of sUA via the urate transporter SLC2A9/GLUT9. CD14+ monocytes isolated from hyperuricemic patients were less responsive to inflammatory stimuli compared with monocytes from healthy individuals. Treatment with plasma from hyperuricemic patients impaired the inflammatory function of CD14+ monocytes, an effect fully reversible by removing sUA from hyperuricemic plasma. Moreover, Alb-creERT2;Glut9lox/lox mice with hyperuricemia (serum UA of 9–11 mg/dl) showed a suppressed inflammatory response to MSU crystals compared with Glut9lox/lox controls without hyperuricemia. Taken together, we unravel a technical explanation for discrepancies in the published literature on immune effects of sUA and identify hyperuricemia as an intrinsic suppressor of innate immunity, in which sUA modulates the capacity of monocytes to respond to danger signals. Thus, sUA is not only a substrate for the formation of MSU crystals but also an intrinsic inhibitor of MSU crystal–induced tissue inflammation

The Long Pentraxin PTX3 Is an Endogenous Inhibitor of Hyperoxaluria-Related Nephrocalcinosis and Chronic Kidney Disease

The long pentraxin 3 (PTX3) exerts a variety of regulatory functions in acute and chronic tissue inflammation. In particular, PTX3 acts as an opsonin for a variety of pathogens and endogenous particles. We hypothesized that PTX3 would exhibit opsonin-like functions toward calcium oxalate crystals, too, and inhibit crystal growth. This process is fundamental in kidney stone disease as well as in hyperoxaluria-related nephrocalcinosis, the paradigmatic cause of chronic kidney disease (CKD) in children with primary hyperoxaluria type I due to genetic defects in oxalate metabolism. Direct effects of PTX3 on calcium oxalate crystals were investigated in chemico by adding recombinant PTX3 to supersaturated calcium and oxalate solutions. PTX3, but not isomolar concentrations of albumin, dose-dependently inhibited crystal growth. In vivo, the PTX3 protein was undetectable in tubular epithelial cells and urine of wild-type mice under physiological conditions. However, its levels increased within 3 weeks of feeding an oxalate-rich diet, an exposure inducing hyperoxaluria-related nephrocalcinosis and CKD in selected mouse strains (male and female C57BL/6N and male Balb/c mice) but not in others (male and female 129SV and CD-1, male and female Balb/c mice). Genetic ablation of ptx3 in nephrocalcinosis un-susceptible B6;129 mice was sufficient to raise the oxalate nephropathy phenotype observed in susceptible strains. We conclude that PTX3 is an endogenous inhibitor of calcium oxalate crystal growth. This mechanism limits hyperoxaluria-related nephrocalcinosis, e.g., in primary or secondary hyperoxaluria, and potentially also in the more prevalent kidney stone disease

IFN regulatory factor 4 controls post-ischemic inflammation and prevents chronic kidney disease

Ischemia reperfusion injury (IRI) of the kidney results in interferon regulatory factor 4 (IRF4)–mediated counter-regulation of the acute inflammatory response. Beyond that, IRF4 exerts important functions in controlling the cytokine milieu, T-cell differentiation, and macrophage polarization. The latter has been implicated in tissue remodeling. It therefore remains elusive what the role of IRF4 is in terms of long-term outcome following IRI. We hypothesized that an inability to resolve chronic inflammation in Irf4−/− mice would promote chronic kidney disease (CKD) progression. To evaluate the effects of IRF4 in chronic upon acute injury in vivo, a mouse model of chronic injury following acute IRI was employed. The expression of Irf4 increased within 10 days after IRI in renal tissue. Both mRNA and protein levels remained high up to 5 weeks upon IRI, suggesting a regulatory function in the chronic phase. Mice deficient in IRF4 display increased tubular cell loss and defective clearance of infiltrating macrophages. These phenomena were associated with increased expression of pro-inflammatory macrophage markers together with reduced expression of alternatively activated macrophage markers. In addition, IRF4-deficient mice showed defective development of alternatively activated macrophages. Hints of a residual M1 macrophage signature were further observed in human biopsy specimens of patients with hypertensive nephropathy vs. living donor specimens. Thus, IRF4 restricts CKD progression and kidney fibrosis following IRI, potentially by enabling M2 macrophage polarization and restricting a Th1 cytokine response. Deteriorated alternative macrophage subpopulations in Irf4−/− mice provoke chronic intrarenal inflammation, tubular epithelial cell loss, and renal fibrosis in the long course after IRI in mice. The clinical significance of these finding for human CKD remains uncertain at present and warrants further studies

Activated platelets induce MLKL-driven neutrophil necroptosis and release of neutrophil extracellular traps in venous thrombosis

Venous thromboembolic (VIE) disease, often manifesting as deep vein thrombosis or pulmonary embolism, involves clot formation consisting of blood cells and platelets locked in plasma protein and chromatin networks. The latter derives from neutrophil extracellular traps released by dying neutrophils;however, the molecular mechanisms of neutrophil death in VIE remains unknown. We speculated that mixed lineage kinase-like (MLKL)-driven neutrophil necroptosis contributes to VTE. Indeed, human inferior venous cava thrombus material stained positive for phosphorylated MLKL, the activated version of MLKL that executes necroptotic cell death. In mice, MLKL immunostaining showed co-localization of MLKL with citrullinated histone H3, a marker of neutrophil extracellular trap (NET) formation. These data provide indirect support for a role of MLKL-mediated necroptosis. As a functional proof, both the stabilizer of receptor-interacting protein kinase-1 (RIPK1) and necroptosis inhibitor necrostatin-1s as well as genetic deficiency of MLKL partially prevented clot formation upon inferior vena cava ligation in mice. In both experiments terminal deoxynucleotidyl transferase dUTP nick-end labeling, RIPK3, and citrullinated histone H3+ areas were markedly reduced within the remnant thrombus. In vitro, thrombin-activated platelets induced cell death and NET formation in human neutrophils, which was inhibited by necrostatin-1s treatment. Necrostatin-1s and necrosulfonamide also inhibited neutrophil-platelet aggregate formation induced by tumor necrosis factor-a but had no effect on platelet activation itself. We conclude that in VTE, activated platelets, and possibly other triggers, induce neutrophil necroptosis, a process contributing to clot formation by releasing chromatin in the extracellular space