Extracellular vesicles from Mycobacterium tuberculosis-infected neutrophils induce maturation of monocyte-derived dendritic cells and activation of antigen-specific Th1 cells.

Tuberculosis remains one of the leading public health problems in the world. The mechanisms that lead to the activation of the immune response against Mycobacterium tuberculosis have been extensively studied, with a focus on the role of cytokines as the main signals for immune cell communication. However, less is known about the role of other signals, such as extracellular vesicles, in the communication between immune cells, particularly during the activation of the adaptive immune response. In this study, we determined that extracellular vesicles released by human neutrophils infected with M. tuberculosis contained several host proteins that are ectosome markers. In addition, we demonstrated that extracellular vesicles released by human neutrophils infected with M. tuberculosis released after only 30 min of infection carried mycobacterial antigens and pathogen-associated molecular patterns, and we identified 15 mycobacterial proteins that were consistently found in high concentrations in extracellular vesicles released by human neutrophils infected with M. tuberculosis; these proteins contain epitopes for CD4 T-cell activation. We found that extracellular vesicles released by human neutrophils infected with M. tuberculosis increased the expression of the costimulatory molecule CD80 and of the coinhibitory molecule PD-L1 on immature monocyte-derived dendritic cells. We also found that immature and mature dendritic cells treated with extracellular vesicles released by human neutrophils infected with M. tuberculosis were able to induce IFN-γ production by autologous M. tuberculosis antigen-specific CD4 T cells, indicating that these extracellular vesicles acted as antigen carriers and transferred mycobacterial proteins to the antigen-presenting cells. Our results provide evidence that extracellular vesicles released by human neutrophils infected with M. tuberculosis participate in the activation of the adaptive immune response against M. tuberculosis.

Convalescent Plasma to Treat COVID-19: A Two-Center, Randomized, Double-Blind Clinical Trial.

Background: The use of convalescent plasma (CP) has been considered for its immunological mechanisms that could benefit patients in moderate and severe stages of COVID-19. This study evaluated the safety and efficacy of the use of donor CP for COVID-19. Material and methods: A double-blind, randomized controlled clinical trial was conducted from May to October 2020. Thirty-nine participants with moderate (II) and severe (III) stages of COVID-19 confirmed by RT-PCR were included. The study randomization rate was set at 3:1. CPs were chosen for application with a neutralizing antibody titer of ≥1:32. Results: We observed a significantly lower 21-day post-transfusion mortality HR: 0.17 (95.0% CI [0.07−0.45, p < 0.001]) in the group receiving CP compared with the control group; protective units (PU) in the group receiving convalescent plasma after seven days were significantly higher (512 (32−16,384) vs. 96 (32−256), p = 0.01); the PAO2/FIO2 index showed a significant improvement in the group receiving CP (251.01 (109.4) vs. 109.2 (62.4), p < 0.001, in the control group). Conclusion: CP is safe and effective, as it decreased mortality in the CP group compared with the control group.

TLR2 Regulates Mast Cell IL-6 and IL-13 Production During Listeria monocytogenes Infection.

Listeria monocytogenes (L.m) is efficiently controlled by several cells of the innate immunity, including the Mast Cell (MC). MC is activated by L.m inducing its degranulation, cytokine production and microbicidal mechanisms. TLR2 is required for the optimal control of L.m infection by different cells of the immune system. However, little is known about the MC receptors involved in recognizing this bacterium and whether these interactions mediate MC activation. In this study, we analyzed whether TLR2 is involved in mediating different MC activation responses during L.m infection. We found that despite MC were infected with L.m, they were able to clear the bacterial load. In addition, MC degranulated and produced ROS, TNF-α, IL-1ß, IL-6, IL-13 and MCP-1 in response to bacterial infection. Interestingly, L.m induced the activation of signaling proteins: ERK, p38 and NF-κB. When TLR2 was blocked, L.m endocytosis, bactericidal activity, ROS production and mast cell degranulation were not affected. Interestingly, only IL-6 and IL-13 production were affected when TLR2 was inhibited in response to L.m infection. Furthermore, p38 activation depended on TLR2, but not ERK or NF-κB activation. These results indicate that TLR2 mediates only some MC activation pathways during L.m infection, mainly those related to IL-6 and IL-13 production.

Severe COVID-19 is marked by dysregulated serum levels of carboxypeptidase A3 and serotonin.

The immune response plays a critical role in the pathophysiology of SARS-CoV-2 infection ranging from protection to tissue damage and all occur in the development of acute respiratory distress syndrome (ARDS). ARDS patients display elevated levels of inflammatory cytokines and innate immune cells, and T and B cell lymphocytes have been implicated in this dysregulated immune response. Mast cells are abundant resident cells of the respiratory tract and are able to release different inflammatory mediators rapidly following stimulation. Recently, mast cells have been associated with tissue damage during viral infections, but their role in SARS-CoV-2 infection remains unclear. In this study, we examined the profile of mast cell activation markers in the serum of COVID-19 patients. We noticed that SARS-CoV-2-infected patients showed increased carboxypeptidase A3 (CPA3) and decreased serotonin levels in their serum when compared with symptomatic SARS-CoV-2-negative patients. CPA3 levels correlated with C-reactive protein, the number of circulating neutrophils, and quick SOFA. CPA3 in serum was a good biomarker for identifying severe COVID-19 patients, whereas serotonin was a good predictor of SARS-CoV-2 infection. In summary, our results show that serum CPA3 and serotonin levels are relevant biomarkers during SARS-CoV-2 infection. This suggests that mast cells and basophils are relevant players in the inflammatory response in COVID-19 and may represent targets for therapeutic intervention.

Extracellular vesicles released by J774A.1 macrophages reduce the bacterial load in macrophages and in an experimental mouse model of tuberculosis.

BACKGROUND: Tuberculosis is the leading cause of death by an infectious microorganism worldwide. Conventional treatment lasts at least six months and has adverse effects; therefore, it is important to find therapeutic alternatives that reduce the bacterial load and may reduce the treatment duration. The immune response against tuberculosis can be modulated by several mechanisms, including extracellular vesicles (EVs), which are nano-sized membrane-bound structures that constitute an efficient communication mechanism among immune cells. METHODS: The EVs released by the J774A.1 mouse macrophage cell line, both spontaneously (S-EV) and after infection with Mycobacterium tuberculosis H37Rv (Mtb-EV), were purified by ultra-centrifugation and size-exclusion chromatography. The size distribution and chemical composition of these EVs were evaluated, and their effect on the bacterial load and the production of cytokines was determined in both in vitro and in vivo models of M. tuberculosis infection. RESULTS: Mtb-EV are larger than S-EV, they contain M. tuberculosis-specific antigens (not detected in EVs released from M. fortuitum-infected J774A.1 cells) and are rich in phosphatidylserine, present in their outer membrane layer. S-EV, but not Mtb-EV, reduced the bacterial load and the production of MCP-1 and TNF-α in M. tuberculosis-infected macrophages, and these effects were reversed when phosphatidylserine was blocked with annexin V. Both S-EV and Mtb-EV significantly reduced the lung bacterial load in mice infected with M. tuberculosis after 60 days of treatment, but they had no effect on survival or on the lung pneumonic area of these mice. CONCLUSION: J774A.1 macrophages infected with M. tuberculosis H37Rv released EVs that differed in size and phosphatidylserine content from spontaneously released EVs, and these EVs also had different biological effects: S-EV reduced the mycobacterial load and the cytokine production in vitro (through a phosphatidylserine-dependent mechanism), while both EVs reduced the lung bacterial load in vivo. These results are the basis for further experiments to evaluate whether EVs improve the efficiency of the conventional treatment for tuberculosis.

Tilmicosin modulates the innate immune response and preserves casein production in bovine mammary alveolar cells during Staphylococcus aureus infection.

Tilmicosin is an antimicrobial agent used to treat intramammary infections against Staphylococcus aureus and has clinical anti-inflammatory effects. However, the mechanism by which it modulates the inflammatory process in the mammary gland is unknown. We evaluated the effect of tilmicosin treatment on the modulation of the mammary innate immune response after S. aureus infection and its effect on casein production in mammary epithelial cells. To achieve this goal, we used immortalized mammary epithelial cells (MAC-T), pretreated for 12 h or treated with tilmicosin after infection with S. aureus (ATCC 27543). Our data showed that tilmicosin decreases intracellular infection (P < 0.01) and had a protective effect on MAC-T reducing apoptosis after infection by 80% (P < 0.01). Furthermore, tilmicosin reduced reactive oxygen species (ROS) (P < 0.01), IL-1ß (P < 0.01), IL-6 (P < 0.01), and TNF-α (P < 0.05) production. In an attempt to investigate the signaling pathways involved in the immunomodulatory effect of tilmicosin, mitogen-activated protein kinase (MAPK) phosphorylation was measured by fluorescent-activated cell sorting. Pretreatment with tilmicosin increased ERK1/2 (P < 0.05) but decreased P38 phosphorylation (P < 0.01). In addition, the anti-inflammatory effect of tilmicosin helped to preserve casein synthesis in mammary epithelial cells (P < 0.01). This result indicates that tilmicosin could be an effective modulator inflammation in the mammary gland. Through regulation of MAPK phosphorylation, ROS production and pro-inflammatory cytokine secretion tilmicosin can provide protection from cellular damage due to S. aureus infection and help to maintain normal physiological functions of the bovine mammary epithelial cell.

Extracellular Vesicles Released from Mycobacterium tuberculosis-Infected Neutrophils Promote Macrophage Autophagy and Decrease Intracellular Mycobacterial Survival.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb). In the lungs, macrophages and neutrophils are the first immune cells that have contact with the infecting mycobacteria. Neutrophils are phagocytic cells that kill microorganisms through several mechanisms, which include the lytic enzymes and antimicrobial peptides that are found in their lysosomes, and the production of reactive oxygen species. Neutrophils also release extracellular vesicles (EVs) (100-1,000 nm in diameter) to the extracellular milieu; these EVs consist of a lipid bilayer surrounding a hydrophilic core and participate in intercellular communication. We previously demonstrated that human neutrophils infected in vitro with Mtb H37Rv release EVs (EV-TB), but the effect of these EVs on other cells relevant for the control of Mtb infection, such as macrophages, has not been completely analyzed. In this study, we characterized the EVs produced by non-stimulated human neutrophils (EV-NS), and the EVs produced by neutrophils stimulated with an activator (PMA), a peptide derived from bacterial proteins (fMLF) or Mtb, and observed that the four EVs differed in their size. Ligands for toll-like receptor (TLR) 2/6 were detected in EV-TB, and these EVs favored a modest increase in the expression of the co-stimulatory molecules CD80, a higher expression of CD86, and the production of higher amounts of TNF-α and IL-6, and of lower amounts of TGF-ß, in autologous human macrophages, compared with the other EVs. EV-TB reduced the amount of intracellular Mtb in macrophages, and increased superoxide anion production in these cells. TLR2/6 ligation and superoxide anion production are known inducers of autophagy; accordingly, we found that EV-TB induced higher expression of the autophagy-related marker LC3-II in macrophages, and the co-localization of LC3-II with Mtb inside infected macrophages. The intracellular mycobacterial load increased when autophagy was inhibited with wortmannin in these cells. In conclusion, our results demonstrate that neutrophils produce different EVs in response to diverse activators, and that EV-TB activate macrophages and promote the clearance of intracellular Mtb through early superoxide anion production and autophagy induction, which is a novel role for neutrophil-derived EVs in the immune response to Mtb.

Dialyzable leukocyte extracts activate TLR-2 on monocytes.

Dialyzable leukocyte extracts (DLE) transfer specific cell-mediated immune responses from sensitized donors to non-immune recipients. In addition, DLE have several immunomodulatory effects and are used for the treatment of several infectious and non-infectious diseases. Previous studies showed that human DLE obtained from virus-infected leukocytes and bovine DLE decrease the production of the pro-inflammatory cytokine TNF-alpha in response to bacterial lipopolysaccharide, in vitro and in vivo. In the present work, we inquire as to whether DLE from uninfected human leukocytes have the ability to regulate cytokine production in peripheral blood mononuclear cells (PBMC) in vitro. We observed that PBMC from healthy individuals were able to produce TNF-alpha, IL-12 and IL-10 after stimulation with DLE. Moreover, we identified monocytes as the main cell population that produced TNF-alpha after DLE stimulation. Interestingly, we found that DLE contain unidentified ligands that activate Toll-like receptor (TLR)-2. Finally, we observed that DLE directly activated monocytes through TLR-2. These results reveal a new biological activity of DLE, and suggest that part of the immunomodulatory properties of DLE could be attributed to TLR-2 activation on monocytes and to the induction of a pro-inflammatory environment that is crucial for control of infectious diseases.