Ilka Schulze

Myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity

The granule enzyme myeloperoxidase (MPO) plays an important role in neutrophil antimicrobial responses. However, the severity of immunodeficiency in patients carrying mutations in MPO is variable. Serious microbial infections, especially with Candida species, have been observed in a subset of completely MPO-deficient patients. Here we show that neutrophils from donors who are completely deficient in MPO fail to form neutrophil extracellular traps (NETs), indicating that MPO is required for NET formation. In contrast, neutrophils from partially MPO-deficient donors make NETs, and pharmacological inhibition of MPO only delays and reduces NET formation. Extracellular products of MPO do not rescue NET formation, suggesting that MPO acts cell-autonomously. Finally, NET-dependent inhibition of Candida albicans growth is compromised in MPO-deficient neutrophils. The inability to form NETs may contribute in part to the host defense defects observed in completely MPO-deficient individuals.

ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis

Lymphocytes mediate cytotoxicity by polarized release of the contents of cytotoxic granules toward their target cells. Here, we have studied the role of the calcium release-activated calcium channel ORAI1 in human lymphocyte cytotoxicity. Natural killer (NK) cells obtained from an ORAI1-deficient patient displayed defective store-operated Ca2+ entry (SOCE) and severely defective cytotoxic granule exocytosis leading to impaired target cell lysis. Similar findings were obtained using NK cells from a stromal interaction molecule 1-deficient patient. The defect occurred at a late stage of the signaling process, because activation of leukocyte functional antigen (LFA)-1 and cytotoxic granule polarization were not impaired. Moreover, pharmacological inhibition of SOCE interfered with degranulation and target cell lysis by freshly isolated NK cells and CD8+ effector T cells from healthy donors. In addition to effects on lymphocyte cytotoxicity, synthesis of the chemokine macrophage inflammatory protein-1β and the cytokines TNF-α and IFN-γ on target cell recognition was impaired in ORAI1-deficient NK cells, as previously described for T cells. By contrast, NK cell cytokine production induced by combinations of IL-12, IL-15, and IL-18 was not impaired by ORAI1 deficiency. Taken together, these results identify a critical role for ORAI1-mediated Ca2+ influx in granule exocytosis for lymphocyte cytotoxicity as well as for cytokine production induced by target cell recognition.

Btk is a positive regulator in the TREM-1/DAP12 signaling pathway

The triggering receptor expressed on myeloid cells 1 (TREM-1) has been implicated in the production of proinflammatory cytokines and chemokines during bacterial infection and sepsis. For downstream signal transduction, TREM-1 is coupled to the ITAM-containing adaptor DAP12. Here, we demonstrate that Bruton tyrosine kinase (Btk), a member of the Tec kinases, becomes phosphorylated upon TREM-1 triggering. In U937-derived cell lines, in which expression of Btk was diminished by shRNA-mediated knockdown, phosphorylation of Erk1/2 and PLCγ1 and Ca²⁺ mobilization were reduced after TREM-1 stimulation. Importantly, TREM-1-induced production of the pro-inflammatory cytokines, TNF-α and IL-8, and up-regulation of activation/differentiation cell surface markers were impaired in Btk knockdown cells. Similar results were obtained upon TREM-1 stimulation of BMDCs of Btk(-/-) mice. The analysis of cells containing Btk mutants revealed that intact membrane localization and a functional kinase domain were required for TREM-1-mediated signaling. Finally, after TREM-1 engagement, TNF-α production by PBMCs was reduced in the majority of patients suffering from X-linked agammaglobulinemia (XLA), a rare hereditary disease caused by mutations in the BTK gene. In conclusion, our data identify Btk as a positive regulator in the ITAM-mediated TREM-1/DAP12 pathway and suggest its implication in inflammatory processes.