Identification of an osteoclastogenic CD4+ T cell population producing IL-17 and TNFα

Poster presentationInternational audienc

Functional characterization of Na+/H+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na(+)/H(+) exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na(+)/H(+) exchangers used for the selection

Epitope Spreading of Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy

International audienceThe phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy. However, the value of anti-PLA2R1 antibody titers in predicting patient outcomes is unknown. Here, we screened serum samples from 50 patients positive for PLA2R1 for immunoreactivity against a series of PLA2R1 deletion mutants covering the extracellular domains. We identified reactive epitopes in the cysteine-rich (CysR), C-type lectin domain 1 (CTLD1), and C-type lectin domain 7 (CTLD7) domains and confirmed the reactivity with soluble forms of each domain. We then used ELISAs to stratify 69 patients positive for PLA2R1 by serum reactivity to one or more of these domains: CysR (n=23), CysRC1 (n=14), and CysRC1C7 (n=32). Median ELISA titers measured using the full-length PLA2R1 antigens were not statistically different between subgroups. Patients with anti-CysR-restricted activity were younger (P=0.008), had less nephrotic range proteinuria (P=0.02), and exhibited a higher rate of spontaneous remission (P=0.03) and lower rates of renal failure progression (P=0.002) and ESRD (P=0.01) during follow-up. Overall, 31 of 69 patients had poor renal prognosis (urinary protein/creatinine ratio >4 g/g or eGFR<45 ml/min per 1.73 m(2) at end of follow-up). High anti-PLA2R1 activity and epitope spreading beyond the CysR epitope were independent risk factors of poor renal prognosis in multivariable Cox regression analysis. Epitope spreading during follow-up associated with disease worsening (n=3), whereas reverse spreading from a CysRC1C7 profile back to a CysR profile associated with favorable outcome (n=1). We conclude that analysis of the PLA2R1 epitope profile and spreading is a powerful tool for monitoring disease severity and stratifying patients by renal prognosis