Deep Phenotyping of Urinary Leukocytes by Mass Cytometry Reveals a Leukocyte Signature for Early and Non-Invasive Prediction of Response to Treatment in Active Lupus Nephritis.

Martina Bertolo,Anette Peddinghaus,Pawel Durek,Henrik Mei,Thomas Rose,Philipp Enghard,Andreas Grützkau,Sabine Baumgart

doi:10.3389/fimmu.2020.00256

Martina Bertolo, Anette Peddinghaus + Show 6 more

Open Access

https://doi.org/10.3389/fimmu.2020.00256

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Abstract

Non-invasive biomarkers are necessary for diagnosis and monitoring disease activity in lupus nephritis (LN) to circumvent risks and limitations of renal biopsies. To identify new non-invasive cellular biomarkers in the urine sediment of LN patients, which may reflect kidney inflammation and can be used to predict treatment outcome, we performed in-depth urinary immune cell profiling by mass cytometry. We established a mass cytometric workflow to comparatively analyze the cellular composition of urine and peripheral blood (PB) in 13 patients with systemic lupus erythematosus (SLE) with active, biopsy-proven proliferative LN. Clinical and laboratory data were collected at the time of sampling and 6 months after induction of therapy in order to evaluate the clinical response of each patient. Six patients with different acute inflammatory renal diseases were included as comparison group. Leukocyte phenotypes and composition differed significantly between urine and paired PB samples. In urine, neutrophils and monocytes/macrophages were identified as the most prominent cell populations comprising together about 30%–83% of nucleated cells, while T and B lymphocytes, eosinophils, and natural killer (NK) cells were detectable at frequencies of <10% each. The majority of urinary T cells showed phenotypical characteristics of activated effector memory T cells (EM) as indicated by the co-expression of CD38 and CD69 – a phenotype that was not detectable in PB. Kidney inflammation was also reflected by tissue-imprinted macrophages, which phenotypically differed from PB monocytes by an increased expression of HLA-DR and CD11c. The presence of activated urinary T cells and macrophages could be used for differential diagnosis of proliferative LN forms and other renal pathologies. Most interestingly, the amount of EM in the urine sediment could be used as a biomarker to stratify LN patients in terms of response to induction therapy. Deep immunophenotypic profiling of urinary cells in LN allowed us to identify a signature of activated T cells and macrophages, which appear to reflect leukocytic infiltrates in the kidney. This explorative study has not only confirmed but also extended the knowledge about urinary cells as a future non-invasive biomarker platform for diagnosis and precision medicine in inflammatory renal diseases.

Highlights

Systemic lupus erythematosus (SLE) is a rare potentially lifethreatening autoimmune disease with a complex and not fully understood immune pathophysiology [1].Almost half of SLE patients develop lupus nephritis (LN) [2], which is responsible for a significant increase in mortality (18% over 5 years) and morbidity [3]
While stable amounts of live single CD45+ cells were recovered from peripheral blood (PB) samples, corresponding urine samples of LN patients showed large variations with respect to cell density in the urine and in cell viability ranging from 5% to 55%
The application of a comprehensive mass cytometry antibody panel allowed an in-depth analysis of the urinary leukocyte composition in comparison to autologous blood samples

Summary

INTRODUCTION

Systemic lupus erythematosus (SLE) is a rare potentially lifethreatening autoimmune disease with a complex and not fully understood immune pathophysiology [1]. Several clinical situations might occur in which kidney biopsy is contraindicated, due to severe hypertension, bleeding diathesis, or solitary kidney, or is not performed, such as in mild proteinuria and silent LN [7] In these cases, the diagnosis is uncertain and initiation of induction therapy might be delayed, potentially resulting in ongoing inflammation and subsequent chronic organ damage, while early diagnosed and treated LN patients have a better outcome [8, 9]. To overcome the limitations of kidney biopsy and to improve LN prognosis, reliable non-invasive biomarkers for early diagnosis and outcome prediction are required For this purpose, the urine sediment of LN patients was already investigated by conventional flow cytometry before, and the presence of mononuclear cells in urine of patients with active renal disease was described [13,14,15].

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