Interleukin 6 receptor inhibition in primary Sjögren syndrome: a multicentre double-blind randomised placebo-controlled trial.

OBJECTIVES: No immunomodulatory drug has been approved for primary Sjögren's syndrome, a systemic autoimmune disease affecting 0.1% of the population. To demonstrate the efficacy of targeting interleukin 6 receptor in patients with Sjögren's syndrome-related systemic complications. METHODS: Multicentre double-blind randomised placebo-controlled trial between 24 July 2013 and 16 July 2018, with a follow-up of 44 weeks, involving 17 referral centres. Inclusion criteria were primary Sjögren's syndrome according to American European Consensus Group criteria and score ≥5 for the EULAR Sjögren's Syndrome Disease activity Index (ESSDAI, score of systemic complications). Patients were randomised to receive either 6 monthly infusions of tocilizumab or placebo. The primary endpoint was response to treatment at week 24. Response to treatment was defined by the combination of (1) a decrease of at least 3 points in the ESSDAI, (2) no occurrence of moderate or severe activity in any new domain of the ESSDAI and (3) lack of worsening in physician's global assessment on a Visual Numeric Scale ≥1/10, all as compared with enrolment. RESULTS: 110 patients were randomised, 55 patients to tocilizumab (mean (SD) age: 50.9 (12.4) years; women: 98.2%) and 55 patients to placebo (54.8 (10.7) years; 90.9%). At 24 weeks, the proportion of patients meeting the primary endpoint was 52.7% (29/55) in the tocilizumab group and 63.6% (35/55) in the placebo group, for a difference of -11.4% (95% credible interval -30.6 to 9.0) (Pr[Toc >Pla]=0.14). CONCLUSION: Among patients with primary Sjögren's syndrome, the use of tocilizumab did not improve systemic involvement and symptoms over 24 weeks of treatment compared with placebo. TRIAL REGISTRATION NUMBER: NCT01782235.

Internalization of staphylococcal leukotoxins that bind and divert the C5a receptor is required for intracellular Ca(2+) mobilization by human neutrophils.

A growing number of receptors, often associated with the innate immune response, are being identified as targets for bacterial toxins of the beta-stranded pore-forming family. These findings raise the new question of whether the receptors are activated or merely used as docking points facilitating the formation of a pore. To elucidate whether the Staphylococcus aureus Panton-Valentine leukocidin and the leukotoxin HlgC/HlgB act through the C5a receptor (C5aR) as agonists, antagonists or differ from the C5a complement-derived peptide, their activity is explored on C5aR-expressing cells. Both leukotoxins equally bound C5aR in neutrophils and in stable transfected U937 cells and initiated mobilization of intracellular Ca(2+) . HlgC/HlgB requires the presence of robust intracellular acidic Ca(2+) stores in order to evoke a rise in free [Ca(2+) ]i , while the LukS-PV/LukF-PV directly altered reticular Ca(2+) stores. Intracellular target specificity is conferred by the F-subunit associated to the S-subunit binding the receptor. Furthermore, internalization of the two leukotoxin components (S- and F-subunits) associated to C5aR is required for the initiation of [Ca(2+) ]i mobilization. Electrophysiological recordings on living cells demonstrated that LukS-PV/LukF-PV does not alter the membrane resistance of C5aR-expressing cells. The present observations suggest that part of the pore-forming process occurs in distinct intracellular compartments rather than at the plasma membrane.

Residues essential for Panton-Valentine leukocidin S component binding to its cell receptor suggest both plasticity and adaptability in its interaction surface.

Panton-Valentine leukocidin (PVL), a bicomponent staphylococcal leukotoxin, is involved in the poor prognosis of necrotizing pneumonia. The present study aimed to elucidate the binding mechanism of PVL and in particular its cell-binding domain. The class S component of PVL, LukS-PV, is known to ensure cell targeting and exhibits the highest affinity for the neutrophil membrane (Kd∼10(-10) M) compared to the class F component of PVL, LukF-PV (Kd∼10(-9) M). Alanine scanning mutagenesis was used to identify the residues involved in LukS-PV binding to the neutrophil surface. Nineteen single alanine mutations were performed in the rim domain previously described as implicated in cell membrane interactions. Positions were chosen in order to replace polar or exposed charged residues and according to conservation between leukotoxin class S components. Characterization studies enabled to identify a cluster of residues essential for LukS-PV binding, localized on two loops of the rim domain. The mutations R73A, Y184A, T244A, H245A and Y250A led to dramatically reduced binding affinities for both human leukocytes and undifferentiated U937 cells expressing the C5a receptor. The three-dimensional structure of five of the mutants was determined using X-ray crystallography. Structure analysis identified residues Y184 and Y250 as crucial in providing structural flexibility in the receptor-binding domain of LukS-PV.

Staphylococcal leukotoxins trigger free intracellular Ca(2+) rise in neurones, signalling through acidic stores and activation of store-operated channels.

Headache, muscle aches and chest pain of mild to medium intensity are among the most common clinical symptoms in moderate Staphylococcus aureus infections, with severe infections usually associated with worsening pain symptoms. These nociceptive responses of the body raise the question of how bacterial infection impinges on the nervous system. Does S. aureus, or its released virulence factors, act directly on neurones? To address this issue, we evaluated the potential effects on neurones of certain bi-component leukotoxins, which are virulent factors released by the bacterium. The activity of four different leukotoxins was verified by measuring the release of glutamate from rat cerebellar granular neurones. The bi-component γ-haemolysin HlgC/HlgB was the most potent leukotoxin, initiating transient rises in intracellular Ca(2+) concentration in cerebellar neurones and in primary sensory neurones from dorsal root ganglia, as probed with the Fura-2 Ca(2+) indicator dye. Using pharmacological antagonists of receptors and Ca(2+) channels, the variations in intracellular Ca(2+) concentration were found independent of the activation of voltage-operated Ca(2+) channels or glutamate receptors. Drugs targeting Sarco-Endoplasmic Reticulum Ca(2+)-ATPase (SERCA) or H(+)-ATPase and antagonists of the store-operated Ca(2+) entry complex blunted, or significantly reduced, the leukotoxin-induced elevation in intracellular Ca(2+). Moreover, activation of the ADP-ribosyl cyclase CD38 was also required to initiate the release of Ca(2+) from acidic stores. These findings suggest that, prior to forming a pore at the plasma membrane, leukotoxin HlgC/HlgB triggers a multistep process which initiates the release of Ca(2+) from lysosomes, modifies the steady-state level of reticular Ca(2+) stores and finally activates the Store-Operated Calcium Entry complex.