A tagged parathyroid hormone derivative as a carrier of antibody cargoes transported by the G protein coupled PTH1 receptor

Based on the known fact that the parathyroid hormone (PTH) might be extended at its C-terminus with biotechnological protein cargoes, a vector directing the secretion of PTH1–84 C-terminally fused with the antigenic epitope myc (PTH-myc) was exploited. The functional properties and potential of this analog for imaging PTH1R-expressing cells were examined. The PTH-myc construct was recombinantly produced as a conditioned medium (CM) of transfected HEK 293a cells (typical concentrations of 187 nM estimated with ELISAs for PTH). PTH-myc CM induced cyclic AMP formations (10 min), with a minor loss of potency relative to authentic PTH1–84, and c-Fos expression (1–3 h). Treatment of recipient HEK 293a cells transiently expressing PTH1R with PTH-myc CM (supplemented with a fluorescent monoclonal anti-myc tag antibody, either 4A6 or 9E10) allowed the labeling of endosomal structures positive for Rab5 and/or for β-arrestin1 (microscopy, cytofluorometry). Authentic PTH was inactive in this respect, ruling out a non-specific form of endocytosis like pinocytosis. Using a horseradish peroxidase-conjugated secondary antibody, the endocytosis of the PTH-myc-based antibody complex by endogenous PTH1R was evidenced in MG-63 osteoblastoid cells. The secreted construct PTH-myc represents a bona fide agonist that supports the feasibility of transporting cargoes of considerable molecular weight inside cells using arrestin and Rab5-mediated PTH1R endocytosis. PTH-myc is also transported into cells that express PTH1R at a physiological level. Such tagged peptide hormones may be part of a cancer chemotherapy scheme exploiting a modular cytotoxic secondary antibody and the receptor repertoire expressed in a given tumor

Autoantibodies in Systemic Lupus Erythematosus Target Mitochondrial RNA

The mitochondrion supplies energy to the cell and regulates apoptosis. Unlike other mammalian organelles, mitochondria are formed by binary fission and cannot be directly produced by the cell. They contain numerous copies of a compact circular genome that encodes RNA molecules and proteins involved in mitochondrial oxidative phosphorylation. Whereas, mitochondrial DNA (mtDNA) activates the innate immune system if present in the cytosol or the extracellular milieu, it is also the target of circulating autoantibodies in systemic lupus erythematosus (SLE). However, it is not known whether mitochondrial RNA is also recognized by autoantibodies in SLE. In the present study, we evaluated the presence of autoantibodies targeting mitochondrial RNA (AmtRNA) in SLE. We quantified AmtRNA in an inducible model of murine SLE. The AmtRNA were also determined in SLE patients and healthy volunteers. AmtRNA titers were measured in both our induced model of murine SLE and in human SLE, and biostatistical analyses were performed to determine whether the presence and/or levels of AmtRNA were associated with clinical features expressed by SLE patients. Both IgG and IgM classes of AmtRNA were increased in SLE patients (n = 86) compared to healthy controls (n = 30) (p < 0.0001 and p = 0.0493, respectively). AmtRNA IgG levels correlated with anti-mtDNA-IgG titers (rs = 0.54, p < 0.0001) as well as with both IgG and IgM against β-2-glycoprotein I (anti-β2GPI; rs = 0.22, p = 0.05), and AmtRNA-IgG antibodies were present at higher levels when patients were positive for autoantibodies to double-stranded-genomic DNA (p < 0.0001). AmtRNA-IgG were able to specifically discriminate SLE patients from healthy controls, and were negatively associated with plaque formation (p = 0.04) and lupus nephritis (p = 0.03). Conversely, AmtRNA-IgM titers correlated with those of anti-β2GPI-IgM (rs = 0.48, p < 0.0001). AmtRNA-IgM were higher when patients were positive for anticardiolipin antibodies (aCL-IgG: p = 0.01; aCL-IgM: p = 0.002), but AmtRNA-IgM were not associated with any of the clinical manifestations assessed. These findings identify mtRNA as a novel mitochondrial antigen target in SLE, and support the concept that mitochondria may provide an important source of circulating autoantigens in SLE

Neutrophile et immunité

Le polynucléaire neutrophile, longtemps considéré comme une simple cellule phagocytaire à durée de vie très courte éliminant les intrus de l’organisme, est, en fait, une cellule dont les diverses facettes fonctionnelles connues en font un acteur indispensable des défenses de première ligne, certes, mais aussi des défenses acquises. À cet aspect multifonctionnel s’associent un retard de sa mort programmée, une plasticité éclairant sa capacité à se transdifférencier en fonction des besoins locaux du processus inflammatoire et infectieux, mais aussi une hétérogénéité de cette cellule constituant des sous-populations variables en fonction des individus. Le neutrophile est certainement la cellule hématopoïétique qui suscite le plus de controverses, dont bien des propriétés restent à découvrir, et dont les secrets fonctionnels cachent sans doute d’étonnantes capacities

SARS-CoV-2 Nsp2 Contributes to Inflammation by Activating NF-κB

COVID-19 is associated with robust inflammation and partially impaired antiviral responses. The modulation of inflammatory gene expression by SARS-CoV-2 is not completely understood. In this study, we characterized the inflammatory and antiviral responses mounted during SARS-CoV-2 infection. K18-hACE2 mice were infected with a Wuhan-like strain of SARS-CoV-2, and the transcriptional and translational expression interferons (IFNs), cytokines, and chemokines were analyzed in mouse lung homogenates. Our results show that the infection of mice with SARS-CoV-2 induces the expression of several pro-inflammatory CC and CXC chemokines activated through NF-κB but weakly IL1β and IL18 whose expression are more characteristic of inflammasome formation. We also observed the downregulation of several inflammasome effectors. The modulation of innate response, following expressions of non-structural protein 2 (Nsp2) and SARS-CoV-2 infection, was assessed by measuring IFNβ expression and NF-κB modulation in human pulmonary cells. A robust activation of the NF-κB p65 subunit was induced following the infection of human cells with the corresponding NF-κB-driven inflammatory signature. We identified that Nsp2 expression induced the activation of the IFNβ promoter through its NF-κB regulatory domain as well as activation of p65 subunit phosphorylation. The present studies suggest that SARS-CoV-2 skews the antiviral response in favor of an NF-κB-driven inflammatory response, a hallmark of acute COVID-19 and for which Nsp2 should be considered an important contributor

SARS-CoV-2 Nsp2 Contributes to Inflammation by Activating NF-κB

COVID-19 is associated with robust inflammation and partially impaired antiviral responses. The modulation of inflammatory gene expression by SARS-CoV-2 is not completely understood. In this study, we characterized the inflammatory and antiviral responses mounted during SARS-CoV-2 infection. K18-hACE2 mice were infected with a Wuhan-like strain of SARS-CoV-2, and the transcriptional and translational expression interferons (IFNs), cytokines, and chemokines were analyzed in mouse lung homogenates. Our results show that the infection of mice with SARS-CoV-2 induces the expression of several pro-inflammatory CC and CXC chemokines activated through NF-κB but weakly IL1β and IL18 whose expression are more characteristic of inflammasome formation. We also observed the downregulation of several inflammasome effectors. The modulation of innate response, following expressions of non-structural protein 2 (Nsp2) and SARS-CoV-2 infection, was assessed by measuring IFNβ expression and NF-κB modulation in human pulmonary cells. A robust activation of the NF-κB p65 subunit was induced following the infection of human cells with the corresponding NF-κB-driven inflammatory signature. We identified that Nsp2 expression induced the activation of the IFNβ promoter through its NF-κB regulatory domain as well as activation of p65 subunit phosphorylation. The present studies suggest that SARS-CoV-2 skews the antiviral response in favor of an NF-κB-driven inflammatory response, a hallmark of acute COVID-19 and for which Nsp2 should be considered an important contributor

Effects of pharmacological inhibitors on IL-32γ-induced sustained amounts of MCL-1 protein.

<p>Human neutrophils were pretreated for 30 min with 20 µM LY249002 (PI-3 kinase inhibitor), or 10 µM U0126 (MEK inhibitor), or 25 µM SB202190 (p38 MAP kinase inhibitor) or their vehicle (DMSO). Neutrophils were then incubated with IL-32γ (300 ng.ml⁻¹) for 20 hrs prior to cell lysis and immunoblot analysis. Immunoblot representative of 3 donors (<b>A</b>). Densitometric analyses of MCL-1 and actin expression were performed and ratios between MCL-1 and actin were calculated to normalize the data (<b>B</b>). Results are means ± s.e.m. of 3 different donors. Statistics: one-way ANOVA followed by Bonferroni multiple-comparison test, * p<0.05.</p

Effects of signaling inhibitors on IL-32γ delayed apoptosis.

<p>IL-32γ-delayed apoptosis is dependent on kinase activation. Neutrophils were pre-incubated 30 min with an inhibitor of PI-3 kinase (LY249002 at 20 µM), an inhibitor of MEK (U0126 at 10 µM), an inhibitor of p38 MAP kinase (SB202190 at 25 µM), and an inhibitor of JN Kinase (SP600125 at 10 µM) or their vehicle (DMSO). Neutrophils were then stimulated with IL-32γ (300 ng.ml⁻¹) for 20 h and further analyzed by FACS to evaluate their annexin V-FITC binding in conjunction with propidium iodide staining. The effects of signaling inhibitors were compared to the experimental condition in which neutrophils were incubated with IL-32γ + inhibitor vehicle (DMSO); this condition corresponds to 100% of delayed apoptosis. Results are expressed as percentage of IL-32γ inhibitory effect. Results are means ± s.e.m. of 5–9 different donors. Statistics: paired Student <i>t</i> test, p values are indicated in the table. NS  =  non-significant.</p><p>Effects of signaling inhibitors on IL-32γ delayed apoptosis.</p

IL-32γ delays neutrophil apoptosis.

<p>Neutrophils were incubated with graded concentrations of IL-32γ or its vehicle. Cells were analyzed by flow cytometry for annexin V-FITC binding in conjunction with propidium iodide (PI) staining. Representative FACS analysis of neutrophils incubated for 24 hrs with vehicle (Control) or 300 ng.ml⁻¹ of IL-32γ and stained with annexin V-FITC and PI (<b>A</b>). Neutrophils were incubated with vehicle (0) or graded concentrations of IL-32γ (10, 30, 100, 300 and 1000 ng.ml⁻¹) for 24 hrs (<b>B</b>) or 48 hrs (<b>C</b>). Results are illustrated in (A) as annexin V-FITC negative and PI negative cells (non-apoptotic cells; present in the lower left quadrant of FACS), annexin V-FITC positive and PI negative cells (early apoptotic cells; lower right quadrant), annexin V-FITC positive and PI positive cells (late apoptotic cells; upper right quadrant) or annexin V-FITC negative and PI positive cells (necrotic cells; upper left quadrant). Apoptosis was also evaluated by measurement of neutrophil pan-caspase activity after 48 hrs of incubation with IL-32γ or its vehicle. Activated caspases were labelled with a FITC-conjugated pan-caspase inhibitor before being analyzed by FACS (<b>D</b>). Results represent the means ± s.e.m. of 4 different donors. Statistical significance was assessed using one-way ANOVA followed by Bonferroni multiple comparison test: means without a common letter differ (P<0.05).</p

IL-32γ induces sustained steady state levels of MCL-1 protein.

<p>Human neutrophils were incubated for 8, 20 and 30 hrs with IL-32γ (300 ng.ml⁻¹) or its vehicle (absence of IL-32γ). Cell lysates were subjected to SDS-PAGE and transferred to PVDF membranes. Western blotting was then performed with anti-MCL-1 or anti-β-actin (control of protein loading) antibodies, and proteins were revealed with Western Lightning Plus ECL. Representative of 4 different donors (<b>A</b>). Densitometric analyses of MCL-1 and actin expression were performed and ratios between MCL-1 and actin were calculated to normalize the data. These normalized values were then used to calculate the percentages of MCL-1 variations in neutrophils incubated with vehicle at 20 and 30 hrs of incubation or with IL-32γ at 8, 20 and 30 hrs of incubation, considering that the normalized value of MCL-1 in neutrophils incubated with vehicle at 8 hrs represents 100% MCL-1 (neutrophils at 8 hrs of incubation with vehicle were 96% non-apoptotic) (<b>B</b>). Results are means ± s.e.m. of 4 different donors. Statistics: paired Student <i>t</i> test, * p<0.05.</p