[Dysnatremias - concepts and clinical work-up]. / Dysnatriämien ­ Konzepte und klinische Aufarbeitung.

Osmotic gradients over cell membranes lead to water movement into or out of cells. An intact osmoregulation prevents osmotic gradients, thereby protecting cells from swelling or shrinking. Na+ is the major cation in the extracellular fluid (ECF) and the major determinant of the osmolarity in the ECF, including plasma. Therefore, the plasma-Na+ concentration needs to be tightly regulated. An excess of electrolyte-free water decreases the concentration of osmolytes leading to hyponatremia. In contrast, a free water deficit increases the osmolyte concentration leading to hypernatremia. Pathophysiology-oriented approaches to dysnatremic patients help both clinicians and patients. Therapeutic interventions depend on the differentiation between acute and chronic, asymptomatic, and symptomatic dysnatremia, and on the patient's extracellular volume status. The therapeutic armamentarium for hyponatremia consists of water restriction, hypertonic infusions, urea, V2 receptor-blockers, and sodium-glucose cotransporter 2 (SGLT2) inhibitors. Patients with hypernatremia are treated with electrolyte-free water or hypotonic sodium-containing solutions depending on their volume status. Basic concepts in the management of dysnatremic patients are discussed.

CD19-targeting CAR T cells protect from ANCA-induced acute kidney injury.

OBJECTIVES: Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) are life-threatening systemic autoimmune diseases manifesting in the kidneys as necrotizing crescentic glomerulonephritis (NCGN). ANCA antigens are myeloperoxidase (MPO) or proteinase 3. Current treatments include steroids, cytotoxic drugs and B cell-depleting antibodies. The use of chimeric antigen receptor (CAR) T cells in autoimmune diseases is a promising new therapeutic approach. We tested the hypothesis that CAR T cells targeting CD19 deplete B cells, including MPO-ANCA-producing B cells, thereby protecting from ANCA-induced NCGN. METHODS: We tested this hypothesis in a preclinical MPO-AAV mouse model. NCGN was established by immunisation of MPO-/- mice with murine MPO, followed by irradiation and transplantation with haematopoietic cells from wild-type mice alone or together with either CD19-targeting CAR T cells or control CAR T cells. RESULTS: CD19 CAR T cells efficiently migrated to and persisted in bone marrow, spleen, peripheral blood and kidneys for up to 8 weeks. CD19 CAR T cells, but not control CAR T cells, depleted B cells and plasmablasts, enhanced the MPO-ANCA decline, and most importantly protected from NCGN. CONCLUSION: Our proof-of-principle study may encourage further exploration of CAR T cells as a treatment for ANCA-vasculitis patients with the goal of drug-free remission.

Potassium homeostasis - Physiology and pharmacology in a clinical context.

Membrane voltage controls the function of excitable cells and is mainly a consequence of the ratio between the extra- and intracellular potassium concentration. Potassium homeostasis is safeguarded by balancing the extra-/intracellular distribution and systemic elimination of potassium to the dietary potassium intake. These processes adjust the plasma potassium concentration between 3.5 and 4.5 mmol/L. Several genetic and acquired diseases but also pharmacological interventions cause dyskalemias that are associated with increased morbidity and mortality. The thresholds at which serum K+ not only associates but also causes increased mortality are hotly debated. We discuss physiologic, pathophysiologic, and pharmacologic aspects of potassium regulation and provide informative case vignettes. Our aim is to help clinicians, epidemiologists, and pharmacologists to understand the complexity of the potassium homeostasis in health and disease and to initiate appropriate treatment strategies in dyskalemic patients.

Urinary T Cells Identify Renal Antineutrophil Cytoplasmic Antibody-Associated Vasculitis and Predict Prognosis: A Proof of Concept Study.

Introduction: Necrotizing crescentic glomerulonephritis is a major contributor to morbidity and mortality in Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Because therapy relies on immunosuppressive agents with potentially severe adverse effects, a reliable noninvasive biomarker of disease activity is needed to guide treatment. Methods: We used flow cytometry to quantify T cell subsets in blood and urine samples from 95 patients with AAV and 8 controls to evaluate their biomarker characteristics. These were compared to soluble markers, monocyte chemoattractant protein-1 (MCP-1), soluble CD163 (sCD163), soluble CD25 (sCD25), and complement C5a (C5a), measured using multiplex analysis. Available kidney biopsies (n = 21) were classified according to Berden. Results: Patients with active renal AAV (rAAV) showed significantly higher urinary cell counts than those in remission, or those with extrarenal manifestation, or healthy controls. Urinary T cells showed robust discrimination of disease activity with superior performance compared to MCP-1 and sCD163. Patients whose kidney biopsies had been classified as "crescentic" according to Berden classification showed higher urinary T cell counts. Discordant regulatory T cells (Treg) proportions and CD4+/CD8+ ratio in blood and urine suggested that urinary cells reflect tissue migration rather than mere micro-bleeding. Furthermore, urinary Treg and T helper cells (TH17) patterns were associated with clinical response and risk of renal relapse. Conclusion: Urinary T cells reflect the renal inflammatory milieu in AAV and provide further insights into the pathogenesis of this chronic condition. Their promising potential as noninvasive diagnostic and prognostic biomarkers deserves further exploitation.

Dipeptidyl peptidase 1 inhibition as a potential therapeutic approach in neutrophil-mediated inflammatory disease.

Neutrophils have a critical role in the innate immune response to infection and the control of inflammation. A key component of this process is the release of neutrophil serine proteases (NSPs), primarily neutrophil elastase, proteinase 3, cathepsin G, and NSP4, which have essential functions in immune modulation and tissue repair following injury. Normally, NSP activity is controlled and modulated by endogenous antiproteases. However, disruption of this homeostatic relationship can cause diseases in which neutrophilic inflammation is central to the pathology, such as chronic obstructive pulmonary disease (COPD), alpha-1 antitrypsin deficiency, bronchiectasis, and cystic fibrosis, as well as many non-pulmonary pathologies. Although the pathobiology of these diseases varies, evidence indicates that excessive NSP activity is common and a principal mediator of tissue damage and clinical decline. NSPs are synthesized as inactive zymogens and activated primarily by the ubiquitous enzyme dipeptidyl peptidase 1, also known as cathepsin C. Preclinical data confirm that inactivation of this protease reduces activation of NSPs. Thus, pharmacological inhibition of dipeptidyl peptidase 1 potentially reduces the contribution of aberrant NSP activity to the severity and/or progression of multiple inflammatory diseases. Initial clinical data support this view. Ongoing research continues to explore the role of NSP activation by dipeptidyl peptidase 1 in different disease states and the potential clinical benefits of dipeptidyl peptidase 1 inhibition.

Protective α1-antitrypsin effects in autoimmune vasculitis are compromised by methionine oxidation.

BackgroundAntineutrophil cytoplasmic autoantibody-associated (ANCA-associated) vasculitidies (AAV) are life-threatening systemic autoimmune conditions. ANCAs directed against proteinase 3 (PR3) or myeloperoxidase (MPO) bind their cell surface-presented antigen, activate neutrophils, and cause vasculitis. An imbalance between PR3 and its major inhibitor α1-antitrypsin (AAT) was proposed to underlie PR3- but not MPO-AAV. We measured AAT and PR3 in healthy individuals and patients with AAV and studied protective AAT effects pertaining to PR3- and MPO-ANCA.MethodsPlasma and blood neutrophils were assessed for PR3 and AAT. WT, mutant, and oxidation-resistant AAT species were produced to characterize AAT-PR3 interactions by flow cytometry, immunoblotting, fluorescence resonance energy transfer assays, and surface plasmon resonance measurements. Neutrophil activation was measured using the ferricytochrome C assay and AAT methionine-oxidation by Parallel Reaction Monitoring.ResultsWe found significantly increased PR3 and AAT pools in patients with both PR3- and MPO-AAV; however, only in PR3-AAV did the PR3 pool correlate with the ANCA titer, inflammatory response, and disease severity. Mechanistically, AAT prevented PR3 from binding to CD177, thereby reducing neutrophil surface antigen for ligation by PR3-ANCA. Active patients with PR3-AAV showed critical methionine-oxidation in plasma AAT that was recapitulated by ANCA-activated neutrophils. The protective PR3-related AAT effects were compromised by methionine-oxidation in the AAT reactive center loop but preserved when 2 critical methionines were substituted with valine and leucine.ConclusionPathogenic differences between PR3- and MPO-AAV are related to AAT regulation of membrane-PR3, attenuating neutrophil activation by PR3-ANCA rather than MPO-ANCA. Oxidation-resistant AAT could serve as adjunctive therapy in PR3-AAV.FUNDINGThis work was supported by KE 576/10-1 from the Deutsche Forschungsgemeinschaft, SCHR 771/8-1 from the Deutsche Forschungsgemeinschaft, grant 394046635 - SFB 1365 from the Deutsche Forschungsgemeinschaft, and ECRC grants.

CSF2-dependent monocyte education in the pathogenesis of ANCA-induced glomerulonephritis.

OBJECTIVES: Myeloid cell activation by antineutrophil cytoplasmic antibody (ANCA) is pivotal for necrotising vasculitis, including necrotising crescentic glomerulonephritis (NCGN). In contrast to neutrophils, the contribution of classical monocyte (CM) and non-classical monocyte (NCM) remains poorly defined. We tested the hypothesis that CMs contribute to antineutrophil cytoplasmic antibody-associated vasculitis (AAV) and that colony-stimulating factor-2 (CSF2, granulocyte-macrophage colony-stimulating factor (GM-CSF)) is an important monocyte-directed disease modifier. METHODS: Myeloperoxidase (MPO)-immunised MPO-/- mice were transplanted with haematopoietic cells from wild-type (WT) mice, C-C chemokine receptor 2 (CCR2)-/- mice to abrogate CM, or transcription factor CCAAT-enhancer-binding protein beta (C/EBPß)-/- mice to reduce NCM, respectively. Monocytes were stimulated with CSF2, and CSF2 receptor subunit beta (CSF2rb)-deficient mice were used. Urinary monocytes and CSF2 were quantified and kidney Csf2 expression was analysed. CSF2-blocking antibody was used in the nephrotoxic nephritis (NTN) model. RESULTS: Compared with WT mice, CCR2-/- chimeric mice showed reduced circulating CM and were protected from NCGN. C/EBPß-/- chimeric mice lacked NCM but developed NCGN similar to WT chimeric mice. Kidney and urinary CSF2 were upregulated in AAV mice. CSF2 increased the ability of ANCA-stimulated monocytes to generate interleukin-1ß and to promote TH17 effector cell polarisation. CSF2rb-/- chimeric mice harboured reduced numbers of kidney TH17 cells and were protected from NCGN. CSF2 neutralisation reduced renal damage in the NTN model. Finally, patients with active AAV displayed increased urinary CM numbers, CSF2 levels and expression of GM-CSF in infiltrating renal cells. CONCLUSIONS: CMs but not NCMs are important for inducing kidney damage in AAV. CSF2 is a crucial pathological factor by modulating monocyte proinflammatory functions and thereby TH17 cell polarisation.

Targeting Cathepsin C in PR3-ANCA Vasculitis.

BACKGROUND: The ANCA autoantigens proteinase 3 (PR3) and myeloperoxidase (MPO) are exclusively expressed by neutrophils and monocytes. ANCA-mediated activation of these cells is the key driver of the vascular injury process in ANCA-associated vasculitis (AAV), and neutrophil serine proteases (NSPs) are disease mediators. Cathepsin C (CatC) from zymogens activates the proteolytic function of NSPs, including PR3. Lack of NSP zymogen activation results in neutrophils with strongly reduced NSP proteins. METHODS: To explore AAV-relevant consequences of blocking NSP zymogen activation by CatC, we used myeloid cells from patients with Papillon-Lefèvre syndrome, a genetic deficiency of CatC, to assess NSPs and NSP-mediated endothelial cell injury. We also examined pharmacologic CatC inhibition in neutrophil-differentiated human hematopoietic stem cells, primary human umbilical vein cells, and primary glomerular microvascular endothelial cells. RESULTS: Patients with Papillon-Lefèvre syndrome showed strongly reduced NSPs in neutrophils and monocytes. Neutrophils from these patients produced a negative PR3-ANCA test, presented less PR3 on the surface of viable and apoptotic cells, and caused significantly less damage in human umbilical vein cells. These findings were recapitulated in human stem cells, in which a highly specific CatC inhibitor, but not prednisolone, reduced NSPs without affecting neutrophil differentiation, reduced membrane PR3, and diminished neutrophil activation upon PR3-ANCA but not MPO-ANCA stimulation. Compared with healthy controls, neutrophils from patients with Papillon-Lefèvre syndrome transferred less proteolytically active NSPs to glomerular microvascular endothelial cells, the cell type targeted in ANCA-induced necrotizing crescentic glomerulonephritis. Finally, both genetic CatC deficiency and pharmacologic inhibition, but not prednisolone, reduced neutrophil-induced glomerular microvascular endothelial cell damage. CONCLUSIONS: These findings may offer encouragement for clinical studies of adjunctive CatC inhibitor in patients with PR3-AAV.

Competitively disrupting the neutrophil-specific receptor-autoantigen CD177:proteinase 3 membrane complex reduces anti-PR3 antibody-induced neutrophil activation.

CD177 is a neutrophil-specific receptor presenting the proteinase 3 (PR3) autoantigen on the neutrophil surface. CD177 expression is restricted to a neutrophil subset, resulting in CD177pos/mPR3high and CD177neg/mPR3low populations. The CD177pos/mPR3high subset has implications for antineutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis, wherein patients harbor PR3-specific ANCAs that activate neutrophils for degranulation. Here, we generated high-affinity anti-CD177 monoclonal antibodies, some of which interfered with PR3 binding to CD177 (PR3 "blockers") as determined by surface plasmon resonance spectroscopy and used them to test the effect of competing PR3 from the surface of CD177pos neutrophils. Because intact anti-CD177 antibodies also caused neutrophil activation, we prepared nonactivating Fab fragments of a PR3 blocker and nonblocker that bound specifically to CD177pos neutrophils. We observed that Fab blocker clone 40, but not nonblocker clone 80, dose-dependently reduced anti-PR3 antibody binding to CD177pos neutrophils. Importantly, preincubation with clone 40 significantly reduced respiratory burst in primed neutrophils challenged with either monoclonal antibodies to PR3 or PR3-ANCA immunoglobulin G from ANCA-associated autoimmune vasculitis patients. After separating the two CD177/mPR3 neutrophil subsets from individual donors by magnetic sorting, we found that PR3-ANCAs provoked significantly more superoxide production in CD177pos/mPR3high than in CD177neg/mPR3low neutrophils, and that anti-CD177 Fab clone 40 reduced the superoxide production of CD177pos cells to the level of the CD177neg cells. Our data demonstrate the importance of the CD177:PR3 membrane complex in maintaining a high ANCA epitope density and thereby underscore the contribution of CD177 to the severity of PR3-ANCA diseases.

Cathepsin C inhibition as a potential treatment strategy in cancer.

Epidemiological studies established an association between chronic inflammation and higher risk of cancer. Inhibition of proteolytic enzymes represents a potential treatment strategy for cancer and prevention of cancer metastasis. Cathepsin C (CatC) is a highly conserved lysosomal cysteine dipeptidyl aminopeptidase required for the activation of pro-inflammatory neutrophil serine proteases (NSPs, elastase, proteinase 3, cathepsin G and NSP-4). NSPs are locally released by activated neutrophils in response to pathogens and non-infectious danger signals. Activated neutrophils also release neutrophil extracellular traps (NETs) that are decorated with several neutrophil proteins, including NSPs. NSPs are not only NETs constituents but also play a role in NET formation and release. Although immune cells harbor large amounts of CatC, additional cell sources for this protease exists. Upregulation of CatC expression was observed in different tissues during carcinogenesis and correlated with metastasis and poor patient survival. Recent mechanistic studies indicated an important interaction of tumor-associated CatC, NSPs, and NETs in cancer development and metastasis and suggested CatC as a therapeutic target in a several cancer types. Cancer cell-derived CatC promotes neutrophil recruitment in the inflammatory tumor microenvironment. Because the clinical consequences of genetic CatC deficiency in humans resulting in the elimination of NSPs are mild, small molecule inhibitors of CatC are assumed as safe drugs to reduce the NSP burden. Brensocatib, a nitrile CatC inhibitor is currently tested in a phase 3 clinical trial as a novel anti-inflammatory therapy for patients with bronchiectasis. However, recently developed CatC inhibitors possibly have protective effects beyond inflammation. In this review, we describe the pathophysiological function of CatC and discuss molecular mechanisms substantiating pharmacological CatC inhibition as a potential strategy for cancer treatment.

Genetic Background but Not Intestinal Microbiota After Co-Housing Determines Hyperoxaluria-Related Nephrocalcinosis in Common Inbred Mouse Strains.

Calcium oxalate (CaOx) crystal formation, aggregation and growth is a common cause of kidney stone disease and nephrocalcinosis-related chronic kidney disease (CKD). Genetically modified mouse strains are frequently used as an experimental tool in this context but observed phenotypes may also relate to the genetic background or intestinal microbiota. We hypothesized that the genetic background or intestinal microbiota of mice determine CaOx crystal deposition and thus the outcome of nephrocalcinosis. Indeed, Casp1-/-, Cybb-/- or Casp1-/-/Cybb-/- knockout mice on a 129/C57BL/6J (B6J) background that were fed an oxalate-rich diet for 14 days did neither encounter intrarenal CaOx crystal deposits nor nephrocalcinosis-related CKD. To test our assumption, we fed C57BL/6N (B6N), 129, B6J and Balb/c mice an oxalate-rich diet for 14 days. Only B6N mice displayed CaOx crystal deposits and developed CKD associated with tubular injury, inflammation and interstitial fibrosis. Intrarenal mRNA expression profiling of 64 known nephrocalcinosis-related genes revealed that healthy B6N mice had lower mRNA levels of uromodulin (Umod) compared to the other three strains. Feeding an oxalate-rich diet caused an increase in uromodulin protein expression and CaOx crystal deposition in the kidney as well as in urinary uromodulin excretion in B6N mice but not 129, B6J and Balb/c mice. However, backcrossing 129 mice on a B6N background resulted in a gradual increase in CaOx crystal deposits from F2 to F7, of which all B6N/129 mice from the 7th generation developed CaOx-related nephropathy similar to B6N mice. Co-housing experiments tested for a putative role of the intestinal microbiota but B6N co-housed with 129 mice or B6N/129 (3rd and 6th generation) mice did not affect nephrocalcinosis. In summary, genetic background but not the intestinal microbiome account for strain-specific crystal formation and, the levels of uromodulin secretion may contribute to this phenomenon. Our results imply that only littermate controls of the identical genetic background strain are appropriate when performing knockout mouse studies in this context, while co-housing is optional.

[Volume Management in Chronic kidney disease]. / Volumenmanagement bei chronischen Nierenerkrankungen.

Understanding the (patho-)physiology of volume regulation and osmoregulation is fundamental to guide patient advice and therapy in chronic kidney disease (CKD). Volume regulation primarily impacts the amount of sodium in the body, and it mainly affects the extracellular space, while osmoregulation primarily impacts the amount of free water, and it affects both the intra- and extracellular space. The kidneys control water and sodium homeostasis both through their sensor (e. g. tubuloglomerular feedback) and regulator systems (e. g. sodium reabsorption). Many CKD patients are advised by non-nephrologists to a high fluid intake, although they often do not require a daily intake of more than 1.5 litres. Many CKD patients are hypervolemic, and sodium restriction is of key importance in patients' effort to utilize lifestyle changes as therapeutic means. Pharmacologically, (particularly loop) diuretics are the basis of therapy, increasing sodium excretion. Recent developments shift the focus towards classes of drugs ameliorating prognosis in CKD: sodium-glucose linked transporter 2 (SGLT2) inhibitors have proven beneficial in heart and renal failure - by sodium and fluid excretion, among others; additionally, a novel mineralocorticoid receptor antagonist (MRA), finerenone, was recently shown to improve prognosis in CKD.

Fluorine (19F) MRI for Assessing Inflammatory Cells in the Kidney: Experimental Protocol.

Inflammation is one underlying contributing factor in the pathology of acute and chronic kidney disorders. Phagocytes such as monocytes, neutrophils and dendritic cells are considered to play a deleterious role in the progression of kidney disease but may also contribute to organ homeostasis. The kidney is a target of life-threatening autoimmune disorders such as the antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV). Neutrophils and monocytes express ANCA antigens and play an important role in the pathogenesis of AAV. Noninvasive in vivo methods that can quantify the distribution of inflammatory cells in the kidney as well as other organs in vivo would be vital to identify the causality and significance of inflammation during disease progression. Here we describe an noninvasive technique to study renal inflammation in rodents in vivo using fluorine (19F) MRI. In this protocol we chose a murine ANCA-AAV model of renal inflammation and made use of nanoparticles prepared from perfluoro-5-crown-15-ether (PFCE) for renal 19F MRI.This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This experimental protocol chapter is complemented by two separate chapters describing the basic concept and data analysis.

[Hyponatremia - causes, diagnostic evaluation and treatment]. / Hyponatriämie ­ Ursachen, Diagnostik und Therapie.

Intact osmoregulation prevents osmotic gradients thereby limiting cell swelling and shrinking. Hyponatremia is a state of an osmole-free water excess compared to the amounts of solutes and clinical management of hyponatremic patients requires pathophysiology-oriented approaches to select appropriate treatments. Clinicians need to assess the patient's volume status to differentiate hyponatremia with volume depletion, expansion or normovolemia, respectively. In addition, work-up includes differentiation between acute and chronic and asymptomatic and symptomatic hyponatremia. Estimation of free water-clearance helps predicting Serum-Na+ changes and is important to prevent overcorrection of hyponatremia. Water restriction, hypertonic salt, urea, V2-receptor-blockers and recently sodium glucose cotransporter 2 (SGLT2) inhibitors were employed to treat patients with hyponatremia.

Hypoxia-inducible factors not only regulate but also are myeloid-cell treatment targets.

Hypoxia describes limited oxygen availability at the cellular level. Myeloid cells are exposed to hypoxia at various bodily sites and even contribute to hypoxia by consuming large amounts of oxygen during respiratory burst. Hypoxia-inducible factors (HIFs) are ubiquitously expressed heterodimeric transcription factors, composed of an oxygen-dependent α and a constitutive ß subunit. The stability of HIF-1α and HIF-2α is regulated by oxygen-sensing prolyl-hydroxylases (PHD). HIF-1α and HIF-2α modify the innate immune response and are context dependent. We provide a historic perspective of HIF discovery, discuss the molecular components of the HIF pathway, and how HIF-dependent mechanisms modify myeloid cell functions. HIFs enable myeloid-cell adaptation to hypoxia by up-regulating anaerobic glycolysis. In addition to effects on metabolism, HIFs control chemotaxis, phagocytosis, degranulation, oxidative burst, and apoptosis. HIF-1α enables efficient infection defense by myeloid cells. HIF-2α delays inflammation resolution and decreases antitumor effects by promoting tumor-associated myeloid-cell hibernation. PHDs not only control HIF degradation, but also regulate the crosstalk between innate and adaptive immune cells thereby suppressing autoimmunity. HIF-modifying pharmacologic compounds are entering clinical practice. Current indications include renal anemia and certain cancers. Beneficial and adverse effects on myeloid cells should be considered and could possibly lead to drug repurposing for inflammatory disorders.

Neutrophil Gelatinase-Associated Lipocalin Protects from ANCA-Induced GN by Inhibiting TH17 Immunity.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a diagnostic marker of intrinsic kidney injury produced by damaged renal cells and by neutrophils. ANCA-associated vasculitis features necrotizing crescentic GN (NCGN), and ANCA-activated neutrophils contribute to NCGN. Whether NGAL plays a mechanistic role in ANCA-associated vasculitis is unknown. METHODS: We measured NGAL in patients with ANCA-associated vasculitis and mice with anti-myeloperoxidase (anti-MPO) antibody-induced NCGN. We compared kidney histology, neutrophil functions, T cell proliferation and polarization, renal infiltrating cells, and cytokines in wild-type and NGAL-deficient chimeric mice with anti-MPO antibody-induced NCGN. To assess the role of TH17 immunity, we transplanted irradiated MPO-immunized MPO-deficient mice with bone marrow from either wild-type or NGAL-deficient mice; we also transplanted irradiated MPO-immunized MPO/IL-17A double-deficient mice with bone marrow from either IL-17A-deficient or NGAL/IL-17A double-deficient mice. RESULTS: Mice and patients with active ANCA-associated vasculitis demonstrated strongly increased serum and urinary NGAL levels. ANCA-stimulated neutrophils released NGAL. Mice with NGAL-deficient bone marrow developed worsened MPO-ANCA-induced NCGN. Intrinsic neutrophil functions were similar in NGAL-deficient and wild-type neutrophils, whereas T cell immunity was increased in chimeric mice with NGAL-deficient neutrophils with more renal infiltrating TH17 cells. NGAL-expressing neutrophils and CD3+ T cells were in close proximity in kidney and spleen. CD4+ T cells showed no intrinsic difference in proliferation and polarization in vitro, whereas iron siderophore-loaded NGAL suppressed TH17 polarization. We found significantly attenuated NCGN in IL-17A-deficient chimeras compared with MPO-deficient mice receiving wild-type bone marrow, as well as in NGAL/IL-17A-deficient chimeras compared with NGAL-deficient chimeras. CONCLUSIONS: Our findings support that bone marrow-derived, presumably neutrophil, NGAL protects from ANCA-induced NCGN by downregulating TH17 immunity.

Control of neutrophil influx during peritonitis by transcriptional cross-regulation of chemokine CXCL1 by IL-17 and IFN-γ.

Neutrophil infiltration is a hallmark of peritoneal inflammation, but mechanisms regulating neutrophil recruitment in patients with peritoneal dialysis (PD)-related peritonitis are not fully defined. We examined 104 samples of PD effluent collected during acute peritonitis for correspondence between a broad range of soluble parameters and neutrophil counts. We observed an association between peritoneal IL-17 and neutrophil levels. This relationship was evident in effluent samples with low but not high IFN-γ levels, suggesting a differential effect of IFN-γ concentration on neutrophil infiltration. Surprisingly, there was no association of neutrophil numbers with the level of CXCL1, a key IL-17-induced neutrophil chemoattractant. We investigated therefore the production of CXCL1 by human peritoneal mesothelial cells (HPMCs) under in vitro conditions mimicking clinical peritonitis. Stimulation of HPMCs with IL-17 increased CXCL1 production through induction of transcription factor SP1 and activation of the SP1-binding region of the CXCL1 promoter. These effects were amplified by TNFα. In contrast, IFN-γ dose-dependently suppressed IL-17-induced SP1 activation and CXCL1 production through a transcriptional mechanism involving STAT1. The SP1-mediated induction of CXCL1 was also observed in HPMCs exposed to PD effluent collected during peritonitis and containing IL-17 and TNFα, but not IFN-γ. Supplementation of the effluent with IFN-γ led to a dose-dependent activation of STAT1 and a resultant inhibition of SP1-induced CXCL1 expression. Transmesothelial migration of neutrophils in vitro increased upon stimulation of HPMCs with IL-17 and was reduced by IFN-γ. In addition, HPMCs were capable of binding CXCL1 at their apical cell surface. These observations indicate that changes in relative peritoneal concentrations of IL-17 and IFN-γ can differently engage SP1-STAT1, impacting on mesothelial cell transcription of CXCL1, whose release and binding to HPMC surface may determine optimal neutrophil recruitment and retention during peritonitis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.