Iris Estrada-García

Salmonella is a Gram-negative bacterium known to be the major cause of gastrointestinal diseases and systemic infections. During infection of murine B cells, Salmonella activates the PI3K/Akt pathway through its effector, SopB. This signaling pathway induces the downregulation of NLRC4 transcription, resulting in reduced secretion of IL-1β. Thus, Salmonella-infected B cells do not progress to pyroptosis; consequently, the bacteria can survive inside these cells. However, the mechanism by which Salmonella evades the control of B cells has not yet been elucidated. In this study, we found that SopB activates mTORC1, which is necessary for bacterial survival, since B cells cultured with the mTORC1 inhibitor rapamycin and B cells lacking raptor can control Salmonella infection. A similar result was observed in B cells when they were infected with the Salmonella SopB mutant (Δsopb). Salmonella also promoted the phosphorylation of the ULK1 complex at serine 757 (Ser757) by mTORC1, resulting in decreased levels of LC3-II in infected B cells. In this study, we did not observe these results when B cells were infected with Δsopb Salmonella. Our results demonstrated that Salmonella survival within B cells depends on the inhibition of autophagy by mTORC1 activation.

Ursolic and oleanolic acids are natural isomeric triterpenes known for their anticancer activity. Here, we investigated the effect of triterpenes on the viability of A549 human lung cancer cells and the role of autophagy in their activity. The induction of autophagy, the mitochondrial changes and signaling pathway stimulated by triterpenes were systematically explored by confocal microscopy and western blotting. Ursolic and oleanolic acids induce autophagy in A549 cells. Ursolic acid activates AKT/mTOR pathways and oleanolic acid triggers a pathway independent on AKT. Both acids promote many mitochondrial changes, suggesting that mitochondria are targets of autophagy in a process known as mitophagy. The PINK1/Parkin axis is a pathway usually associated with mitophagy, however, the mitophagy induced by ursolic or oleanolic acid is just dependent on PINK1. Moreover, both acids induce an ROS production. The blockage of autophagy with wortmannin is responsible for a decrease of mitochondrial membrane potential (Δψ) and cell death. The wortmannin treatment causes an over-increase of p62 and Nrf2 proteins promote a detoxifying effect to rescue cells from the death conducted by ROS. In conclusion, the mitophagy and p62 protein play an important function as a survival mechanism in A549 cells and could be target to therapeutic control.

Brucellosis, also known as “undulant fever” is a zoonotic disease caused by Brucella, which is a facultative intracellular bacterium. Despite efforts to eradicate this disease, infection in uncontrolled domestic animals persists in several countries and therefore transmission to humans is common. Brucella evasion of the innate immune system depends on its ability to evade the mechanisms of intracellular death in phagocytic cells. The BvrR-BvrS two-component system allows the bacterium to detect adverse conditions in the environment. The BvrS protein has been associated with genes of virulence factors, metabolism, and membrane transport. In this study, we predicted the DNA sequence recognized by BvrR with Gibbs Recursive Sampling and identified the three-dimensional structure of BvrR using I-TASSER suite, and the interaction mechanism between BvrR and DNA with Protein-DNA docking and molecular dynamics (MD) simulation. Based on the Gibbs recursive Sampling analysis, we found the motif AAHTGC (H represents A, C, and T nucleotides) as a possible sequence recognized by BvrR. The docking and EMD simulation results showed that C-terminal effector domain of BvrR protein is likely to interact with AAHTGC sequence. In conclusion, we predicted the structure, recognition motif, and interaction of BvrR with DNA.

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.

Valproic acid (VPA) is widely recognized for its use in the control of epilepsy and other neurological disorders in the past 50 years. Recent evidence has shown the potential of VPA in the control of certain cancers, owed in part to its role in modulating epigenetic changes through the inhibition of histone deacetylases, affecting the expression of genes involved in the cell cycle, differentiation, and apoptosis. The direct impact of VPA in cells of the immune system has only been explored recently. In this review, we discuss the effects of VPA in the suppression of some activation mechanisms in several immune cells that lead to an anti-inflammatory response. As expected, immune cells are not exempt from the effect of VPA, as it also affects the expression of genes of the cell cycle and apoptosis through epigenetic modifications. In addition to inhibiting histone deacetylases, VPA promotes RNA interference, activates histone methyltransferases, or represses the activation of transcription factors. However, during the infectious process, the effectiveness of VPA is subject to the biological nature of the pathogen and the associated immune response; this is because VPA can promote the control or the progression of the infection. Due to its various effects, VPA is a promising alternative for the control of autoimmune diseases and hypersensitivity and needs to be further explored.

Sepsis, one of the leading causes of death in intensive care units, is caused by a dysregulated host response to infection that leads to life-threatening organ dysfunction. The proinflammatory and anti-inflammatory responses activated by the infecting microorganism become systemic, and the sustained anti-inflammatory response induces a state of immunosuppression that is characterized by decreased expression of HLA-DR on monocytes, T cell apoptosis, and reduced production of TNF-α by monocytes and macrophages in response to TLR ligands. Innate lymphoid cells (ILCs) are lymphocytes that lack Ag-specific receptors and lineage-specific markers; they express HLA-DR and are activated by cytokines and by direct recognition of microbial molecules. In this study, we evaluated if ILCs are affected by the anti-inflammatory response during sepsis. We found that the number of peripheral blood ILCs was decreased in septic patients compared with healthy volunteers; this decrease was caused by a reduction in ILC1 and ILC3 and is associated with apoptosis, because ILCs from septic patients expressed active caspase 3. ILCs from septic patients had decreased HLA-DR expression but increased expression of the activating receptors NKp46 and NKp44; they also showed a sustained expression of CD127 (IL-7R α-chain) and retained their capacity to produce TNF-α in response to TLR ligands. These results indicate that during sepsis, ILCs have decreased HLA-DR expression and die via apoptosis, similar to monocytes and T cells, respectively. However, other effector functions of ILCs (activation through NKp46 and NKp44, TNF-α production) may remain unaffected by the immunosuppressive environment prevailing in septic patients.

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-β, 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.

Neutrophils are one the earliest, crucial innate defenses against innumerable pathogens. Their main microbicidal activities include phagocytosis and degranulation, with many pharmacologically active molecules contributing to inflammation. Recently, a novel antimicrobial mechanism was discovered; the Neutrophil Extracelullar Traps (NETs) formed by extrusion of DNA and associated molecules (histones, elastase, antimicrobial peptides, among others) which trap and kill microorganisms. Since NETs were recently described, research has focused on their induction and microbicidal properties, and recently on disease involvement. However, the functional consequences of NETs interacting with other immune cells, either resident or recruited during early inflammation, have not been assessed. We therefore investigated the consequences of exposing two major APCs, macrophages (Mfs) and conventional Dendritic Cells (cDCs) to NETs. Our data revealed that at early times (30 min), both Antigen Presenting Cells (APCs) showed induction of important costimulatory molecules (CD80, CD86). Unexpectedly, however, at later times (6 and 24 hours) NETs apparently triggered a cell death process in these APCs by a caspase- and Apoptosis induced factor (AIF)-dependent pathway, suggesting mitochondrial damage. By rhodamine-123 labelling we found that in both APCs, relatively prolonged exposure to NETs or their components importantly decreased the mitochondrial membrane potential. Ultrastructural analysis confirmed mitochondrial alterations in both APCs. Our results would suggest that early in inflammation, NETs can activate the two main APCs (Mfs and cDCs), but as the process continues, NETs can then initiate apoptosis of these cells through mitochondrial harm. Conceivable, this “late” induction of cell death in these two APCs might start limiting an ongoing inflammatory process to control it.

Mycobacterium tuberculosis (Mtb) inhibits dendritric cells (DC) function in order to delay T cell response. Furthermore, there is increasing evidence that genetic diversity of Mtb strains can affect their interaction with the immune system. Beijing genotype has attracted attention because of its high prevalence and multi-drug resistance. Although it is known that this genotype is hypervirulent and differentially activates macrophages when compared to other genotypes, little is known about its interaction with DC. In order to address this issue, murine bone marrow derived DC (BMDC) were stimulated with soluble extracts (SE) from BCG, H37Rv, Canetti and Beijing genotypes. We observed that unlike other mycobacteria strains, SE-Beijing was unable to induce maturation of DC as assessed by cell surface MHC-II expression. DC stimulated with SE-Beijing failed to produce IL-12 and TNF-α, but did secrete IL-10. Interestingly, SE-Beijing induced CCR7 and PDL-1 on BMDC, but did not induce the expression of CD86. When BMDC stimulated with SE-Beijing were used to activate CD4+ cells they were unable to induce a Th1 response when compared with less virulent genotypes. These results indicate that Beijing is able to modulate DC activation and function, which may be related to the pathogenesis induced by this genotype.