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The immune system protects the host from a plethora of microorganisms and toxins through its unique ability to distinguish self from non-self. To perform this delicate but essential task, the immune system relies on two lines of defense. The innate immune system, which is by nature fast acting, represents the first line of defense. It involves anatomical barriers, physiological factors as well as a subset of haematopoietically-derived cells generically call leukocytes. Activation of the innate immune response leads to a state of inflammation that serves to both warn about and combat the ongoing infection and delivers the antigenic information of the invading pathogens to initiate the slower but highly potent and specific second line of defense, the adaptive immune system. The adaptive immune response calls on T lymphocytes as well as the B lymphocytes essential for the elimination of pathogens and the establishment of the immunological memory. Reactive oxygen species (ROS) have been implicated in many aspects of the immune responses to pathogens, mostly in innate immune functions, such as the respiratory burst and inflammasome activation. Here in this mini review, we focus on the role of ROS in adaptive immunity. We examine how ROS contribute to T-cell biology and discuss whether this activity can be extrapolated to B cells.

Malaria remains one of the greatest public health challenges worldwide, particularly in sub-Saharan Africa. The clinical outcome of individuals infected with Plasmodium falciparum parasites depends on many factors including host systemic inflammatory responses, parasite sequestration in tissues and vascular dysfunction. Production of pro-inflammatory cytokines and chemokines promotes endothelial activation as well as recruitment and infiltration of inflammatory cells, which in turn triggers further endothelial cell activation and parasite sequestration. Inflammatory responses are triggered in part by bioactive parasite products such as hemozoin and infected red blood cell-derived extracellular vesicles (iRBC-derived EVs). Here we demonstrate that such EVs contain functional miRNA-Argonaute 2 complexes that are derived from the host RBC. Moreover, we show that EVs are efficiently internalized by endothelial cells, where the miRNA-Argonaute 2 complexes modulate target gene expression and barrier properties. Altogether, these findings provide a mechanistic link between EVs and vascular dysfunction during malaria infection. Inflammatory response to malaria pathogen Plasmodium falciparum can be triggered by infected red blood cell-derived extracellular vesicles (EVs). This study shows these EVs contain functional microRNA-Argonaute 2 complex that modulates gene expression and alter vascular barrier properties.

Malaria infection caused by the Plasmodium species is a complex disease in which a fine balance between host and parasite factors determine the disease severity. While in some individuals, the infection will trigger only a mild and uncomplicated disease, other individuals will develop severe complications which lead to death. Extracellular vesicles (EVs) secreted by infected red blood cells (iRBCs), as well as other host cells, are important regulators of the balance that determines the disease outcome. In addition, EVs constitute a robust mode of cell-to-cell communication by transferring signaling cargoes between parasites, and between parasites and host, without requiring cellular contact. The transfer of membrane and cytosolic proteins, lipids, DNA, and RNA through EVs not only modulate the immune response, it also mediates cellular communication between parasites to synchronize the transmission stage. Here, we review the recent progress in understanding EV roles during malaria.

The parasite Plasmodium falciparum causes the most severe form of malaria. Cell communication between parasites is an important mechanism to control population density and differentiation. The infected red blood cells (iRBCs) release small extracellular vesicles (EVs) that transfer cargoes between cells. The EVs synchronize the differentiation of the asexual parasites into gametocytes to initiate the transmission to the mosquito. Beside their role in parasite communication, EVs regulate vascular function. So far, the exact cargoes responsible for cellular communication remain unknown. We isolated EVs from cultured iRBCs to determine their small RNA content. We identified several types of human and plasmodial regulatory RNAs. While the miRNAs and tRNA-derived fragments were the most abundant human RNAs, we also found Y-RNAs, vault RNAs, snoRNAs and piRNAs. Interestingly, we found about 120 plasmodial RNAs, including mRNAs coding for exported proteins and proteins involved in drug resistance, as well as non-coding RNAs, such as rRNAs, small nuclear (snRNAs) and tRNAs. These data show, that iRBC-EVs carry small regulatory RNAs. A role in cellular communication is possible since the RNAs were transferred to endothelial cells. Furthermore, the presence of Plasmodium RNAs, in EVs suggests that they may be used as biomarker to track and detect disease.

Cell death mediated by executioner caspases is essential during organ development and for organismal homeostasis. The mechanistic role of activated executioner caspases in antibacterial defense during infections with intracellular bacteria, such as Listeria monocytogenes , remains elusive. Cell death upon intracellular bacterial infections is considered altruistic to deprive the pathogens of their protective niche. To establish infections in a human host, Listeria monocytogenes deploy virulence mediators, including membranolytic listeriolysin O (LLO) and the invasion associated protein p60 (Iap), allowing phagosomal escape, intracellular replication and cell-to-cell spread. Here, by means of chemical and genetical modifications, we show that the executioner caspases-3 and -7 efficiently inhibit growth of intracellular Listeria monocytogenes in host cells. Comprehensive proteomics revealed multiple caspase-3 substrates in the Listeria secretome, including LLO, Iap and various other proteins crucially involved in pathogen-host interactions. Listeria secreting caspase-uncleavable LLO or Iap gained significant growth advantage in epithelial cells. With that, we uncovered an underappreciated defense barrier and a non-canonical role of executioner caspases to degrade virulence mediators, thus impairing intracellular Listeria growth.

Cell-mediated cytotoxicity is an essential immune defense mechanism to fight against viral, bacterial or parasitic infections. Upon recognition of an infected target cell, killer lymphocytes form an immunological synapse to release the content of their cytotoxic granules. Cytotoxic granules of humans contain two membrane-disrupting proteins, perforin and granulysin, as well as a homologous family of five death-inducing serine proteases, the granzymes. The granzymes, after delivery into infected host cells by the membrane disrupting proteins, may contribute to the clearance of microbial pathogens through different mechanisms. The granzymes can induce host cell apoptosis, which deprives intracellular pathogens of their protective niche, therefore limiting their replication. However, many obligate intracellular pathogens have evolved mechanisms to inhibit programed cells death. To overcome these limitations, the granzymes can exert non-cytolytic antimicrobial activities by directly degrading microbial substrates or hijacked host proteins crucial for the replication or survival of the pathogens. The granzymes may also attack factors that mediate microbial virulence, therefore directly affecting their pathogenicity. Many mechanisms applied by the granzymes to eliminate infected cells and microbial pathogens rely on the induction of reactive oxygen species. These reactive oxygen species may be directly cytotoxic or enhance death programs triggered by the granzymes. Here, in the light of the latest advances, we review the antimicrobial activities of the granzymes in regards to their cytolytic and non-cytolytic activities to inhibit pathogen replication and invasion. We also discuss how reactive oxygen species contribute to the various antimicrobial mechanisms exerted by the granzymes.

Mycobacterium tuberculosis (Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world’s population currently infected. Data indicate that γ 9 δ 2 T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of GZMA from γ 9 δ 2 T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA’s action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic.

Microglia are the chief immune cells of the brain and have been reported to be activated in severe malaria. Their activation may drive towards neuroinflammation in cerebral malaria. Malaria-infected red blood cell derived-extracellular vesicles (MiREVs) are produced during the blood stage of malaria infection. They mediate intercellular communication and immune regulation, among other functions. During cerebral malaria, the breakdown of the blood–brain barrier can promote the migration of substances such as MiREVs from the periphery into the brain, targeting cells such as microglia. Microglia and extracellular vesicle interactions in different pathological conditions have been reported to induce neuroinflammation. Unlike in astrocytes, microglia–extracellular vesicle interaction has not yet been described in malaria infection. Therefore, in this study, we aimed to investigate the uptake of MiREVs by human microglia cells and their cytokine response. Human blood monocyte-derived microglia (MoMi) were generated from buffy coats of anonymous healthy donors using Ficoll-Paque density gradient centrifugation. The MiREVs were isolated from the Plasmodium falciparum cultures. They were purified by ultracentrifugation and labeled with PKH67 green fluorescent dye. The internalization of MiREVs by MoMi was observed after 4 h of co-incubation on coverslips placed in a 24-well plate at 37 °C using confocal microscopy. Cytokine-gene expression was investigated using rt-qPCR, following the stimulation of the MoMi cells with supernatants from the parasite cultures at 2, 4, and 24 h, respectively. MiREVs were internalized by the microglia and accumulated in the perinuclear region. MiREVs-treated cells increased gene expression of the inflammatory cytokine TNFα and reduced gene expression of the immune suppressive IL-10. Overall, the results indicate that MiREVs may act on microglia, which would contribute to enhanced inflammation in cerebral malaria.

Malaria remains one of the most serious health problems in developing countries. The causative agent of malaria, Plasmodium spp., have a complex life cycle involving multiple developmental stages as well as different morphological, biochemical and metabolic requirements. We recently found that γδ T cells control parasite growth using pore-forming proteins to deliver their cytotoxic proteases, the granzymes, into blood residing parasites. Here, we follow up on the molecular mechanisms of parasite growth inhibition by human pore-forming proteins. We confirm that Plasmodium falciparum infection efficiently depletes the red blood cells of cholesterol, which renders the parasite surrounding membranes susceptible to lysis by prokaryotic membrane disrupting proteins, such as lymphocytic granulysin or the human cathelicidin LL-37. Interestingly, not the cholesterol depletion but rather the simultaneous exposure of phosphatidylserine, a negatively charged phospholipid, triggers resistance of late stage parasitized red blood cells towards the eukaryotic pore forming protein perforin. Overall, by revealing the molecular events we establish here a pathogen-host interaction that involves host cell membrane remodeling that defines the susceptibility towards cytolytic molecules.