Killer cell proteases can target viral immediate-early proteins to control human cytomegalovirus infection in a noncytotoxic manner.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and can trigger devastating disease in immune-suppressed patients. Cytotoxic lymphocytes (CD8+ T cells and NK cells) control HCMV infection by releasing interferon-γ and five granzymes (GrA, GrB, GrH, GrK, GrM), which are believed to kill infected host cells through cleavage of intracellular death substrates. However, it has recently been demonstrated that the in vivo killing capacity of cytotoxic T cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we demonstrate that (primary) cytotoxic lymphocytes can block HCMV dissemination independent of host cell death, and interferon-α/ß/γ. Prior to killing, cytotoxic lymphocytes induce the degradation of viral immediate-early (IE) proteins IE1 and IE2 in HCMV-infected cells. Intriguingly, both IE1 and/or IE2 are directly proteolyzed by all human granzymes, with GrB and GrM being most efficient. GrB and GrM cleave IE1 after Asp398 and Leu414, respectively, likely resulting in IE1 aberrant cellular localization, IE1 instability, and functional impairment of IE1 to interfere with the JAK-STAT signaling pathway. Furthermore, GrB and GrM cleave IE2 after Asp184 and Leu173, respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data indicate that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV infection, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic infection.

Activation of Human NK Cells by Bordetella pertussis Requires Inflammasome Activation in Macrophages.

Pertussis is a highly contagious respiratory infection caused by the bacterium Bordetella pertussis. Humans are the only known natural reservoir of B. pertussis. In mice, macrophages and NK cells have a key role in confining B. pertussis to the respiratory tract. However, the mechanisms underlying this process, particularly during human infections, remain unclear. Here we characterized the activation of human macrophages and NK cells in response to B. pertussis and unraveled the role of inflammasomes in this process. NLRP3 inflammasome activation by B. pertussis in human macrophage-like THP-1 cells and primary monocyte-derived macrophages (mo-MΦ) was shown by the visualization of ASC-speck formation, pyroptosis, and the secretion of caspase-mediated IL-1ß and IL-18. In contrast to macrophages, stimulation of human CD56+CD3- NK cells by B. pertussis alone did not result in activation of these cells. However, co-culture of B. pertussis-stimulated mo-MΦ and autologous NK cells resulted in high amounts of IFNγ secretion and an increased frequency of IL-2Rα+ and HLA-DR+ NK cells, indicating NK cell activation. This activation was significantly reduced upon inhibition of inflammasome activity or blocking of IL-18 in the mo-MΦ/NK cell co-culture. Furthermore, we observed increased secretion of proinflammatory cytokines in the B. pertussis-stimulated mo-MΦ/NK co-culture compared to the mo-MΦ single culture. Our results demonstrate that B. pertussis induces inflammasome activation in human macrophages and that the IL-18 produced by these cells is required for the activation of human NK cells, which in turn enhances the pro-inflammatory response to this pathogen. Our data provides a better understanding of the underlying mechanisms involved in the induction of innate immune responses against B. pertussis. These findings contribute to the knowledge required for the development of improved intervention strategies to control this highly contagious disease.