Rommel Chacón-Salinas

Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes.

It has been shown recently that different genotypes of Mycobacterium tuberculosis induce distinct immune responses in the host, as reflected by variations in cytokine and iNOS expression. Because these molecules are probably regulated by multiple factors in vivo this complex phenomenon was partially analysed by assessing cytokine and iNOS expression by real‐time polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA) in an in vitro model of bone marrow‐derived macrophages infected with three different M. tuberculosis genotypes: Canetti, H37 Rv and Beijing. Although the three genotypes induced production of iNOS and the different cytokines tested at 24 h post‐infection, macrophages infected with the Beijing isolate expressed the highest levels of mRNA for iNOS, interleukin (IL)‐1β, tumour necrosis factor (TNF)‐α, IL‐12 cytokines and lower levels of IL‐10 compared with cells infected with other genotypes. This expression pattern has been associated with infection control, but during infection in vivo with the Beijing genotype it is lost upon progression to chronic phase. The failure to control infection is likely to be influenced by cytokines produced by other cell types and bacterial molecules expressed during the course of disease. Results presented in this work show that each genotype has the ability to induce different levels of cytokine expression that could be related to its pathogenesis during infection.

Mycobacterium tuberculosis lipids regulate cytokines, TLR-2/4 and MHC class II expression in human macrophages

The interaction of macrophages with Mycobacterium tuberculosis through Toll-like receptors is critical in defining the cytokine profile that may or may not control disease progression. Cell-wall lipids are the main pathogen-associated molecular ligands of mycobacteria, in this paper, we analysed how lipid fractions of three different strains of the M. tuberculosis complex (genotypes Canetti, Beijing and H37Rv) affected the innate immunity by regulating TNF-alpha and IL-10 secretion, TLR2, TLR4, and MHC class II expression of human monocyte-derived macrophages. Of note, lipid fractions from the Beijing genotype (hypervirulent phenotype) preferentially induced macrophages to secrete high amounts of TNF-alpha and IL-10, but downregulated TLR2, TLR4 and MHC class II expression. In contrast, lipids from M. tuberculosis Canetti induced lower amounts of TNF-alpha and IL-10, upregulated TLR2 and TLR4 without modifying MHC class II expression. These results indicate that the virulent mycobacterial genotype Beijing expresses lipids that negatively modified cytokine, TLR and MHC class II expression. These findings may help to unravel the complex mechanisms used by virulent mycobacteria to evade and subvert the immune response.

The Effect of Iron on the Expression of Cytokines in Macrophages Infected with Mycobacterium tuberculosis

Iron is known to play an important role in different bacterial infections and, in particular, in their development. One example is infection with Mycobacterium tuberculosis where iron contributes to growth and survival of the bacteria within the host cell. The majority of studies performed on tuberculosis have focused on the direct effect of iron on bacterial growth; however, little is known about how iron modifies the mycobacterial–host interaction. In order to address this, we have investigated the effect of iron on intracellular growth of M. tuberculosis in J774 macrophages and the molecular mechanisms that are affected during this interaction. We observed that iron modifies intracellular growth of the mycobacteria and that their growth kinetics was modified from that observed for the extracellular situation in the presence of iron. Similarly, when iron was present during the infection, there was a reduced release of tumour necrosis factor‐α and it was related to a higher number of bacilli inside the host cell and low expression of interleukin‐1 (IL‐1) and IL‐6 mRNA. Hence, this work demonstrates that iron, besides promoting mycobacterial growth, also regulates the relationship between macrophage and bacteria.

Identification of peptides presented by HLA class I molecules on cervical cancer cells with HPV-18 infection.

In this work we eluted peptides from purified class I MHC molecules, isolated from a novel human cervical carcinoma cell line (INBL), generated in our laboratory and positive for HPV-18 infection. A fraction of these peptides was capable of stimulating T lymphocytes obtained from a donor matched for HLA-Cw4 and who was also HPV-18+. Direct N-terminal Edman degradation of these peptides, revealed the sequence (XQFPIFLQF) that matched 85% with the sequence NVFPIFLQM localized in between the 54 and 62 residues of the HPV-18 L1 protein. After stimulation with the synthetic peptide NVFPIFLQM, T lymphocytes from the donor were capable to lyse INBL cells. Our results provide evidence of the existence of naturally occurring viral epitopes presented on cervical cancer cells by the HLA-Cw4 allele, that could be useful for immunotherapy on this type of patient.

Recognition of Candida albicans by Dectin-1 induces mast cell activation.

Mast cells are crucial elements of the innate immune response. They reside in tissues that are commonly exposed to the external environment, such as the skin and mucosae, where they can rapidly detect the presence of pathogens and mount a potent inflammatory response that recruits other cellular effectors of the immune response. The contribution of mast cells to the immune response to viruses, bacteria, protozoa and multicellular parasites is well established, but there is scarce information about the role of these cells in fungal infections. In this study, we analyzed if mast cells are activated by Candida albicans and if the C-type lectin receptor Dectin-1 is involved in its recognition. We found that both yeasts and hyphae of C. albicans-induced mast cell degranulation and production of TNF-α, IL-6, IL-10, CCL3 and CCL4, while only yeasts were able to induce IL-1β. Mast cells also produced ROS after stimulation with both dimorphic phases of C. albicans. When mast cells were activated with yeasts and hyphae, they showed decreased expression of IκBα and increased presence of phosphorylated Syk. Blockade of the receptor Dectin-1, but not Toll-like receptor 2, decreased TNF-α production by mast cell in response to C. albicans. These results indicate that mast cells are capable of sensing the two phases of C. albicans, and suggest that mast cells participate as an early inductor of inflammation during the early innate immune response to this fungus.

Listeria monocytogenes induces mast cell extracellular traps

Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when β-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.

Mycobacterium Tuberculosis Beijing Genotype Induces Differential Cytokine Production by Peripheral Blood Mononuclear Cells of Healthy BCG Vaccinated Individuals

Members of the Mycobacterium tuberculosis (Mtb) Beijing genotype are a major concern due to their high prevalence in tuberculosis patients and their high rate of multi-drug resistance. Although it has been shown that Beijing modifies macrophage behavior, little is known about how this genotype could affect the cellular immune response. In order to address this issue, peripheral blood mononuclear cells (PBMC) from healthy BCG vaccinated individuals were stimulated with protein extracts from three Mycobacterium tuberculosis genotypes: Canetti, H37Rv and Beijing evaluating T cell proliferation and cytokine production. In this system both CD4+ and CD8+ proliferated in a similar manner independently of the Mtb genotype used for stimulation. Regarding cytokines, all strains induced similar levels of IFN-γ, but were unable to induce IL-4 and TGF-β. Contrasting, Canetti strain induced lower production of IL-10, TNF-α and IL-12 compared to H37Rv and Beijing. Interestingly, PBMC stimulated with the Beijing strain produced the highest levels of IL-12 and IL-10 than those stimulated with other strains. This differential cytokine expression could affect the pathogenesis induced by Beijing strain through the modulation of inflammatory process in the host, but the precise mechanisms by which this cytokine environment affects the Beijing strain pathogenesis needs further characterization.

Production of IL-10, TNF and IL-12 by Peripheral Blood Mononuclear Cells in Mexican Workers Exposed to a Mixture of Benzene–Toluene–Xylene

BACKGROUND AND AIMS Occupational exposure to low-level benzene and the joint action of toluene-xylene probably cause effects on circulating monocytes immune response. We undertook this study to determine relationship between occupational exposure to benzene-toluene-xylene mixture (BTX) and IL-10, TNF and IL-12 production by peripheral blood mononuclear cells. METHODS Exposure was estimated in 54 workers from a paint company in Mexico City through BTX accumulated potential dose (BTX-APD). Two exposure groups were formed: high and low BTX-APD established with a cutoff point at ≥1.0 of BTX-APD, as a function of the geometric mean of the estimators value distribution and the higher agreement between BTX-APD ≥1.0 and the areas referred as using (or not) organic solvents in the work process. IL-10, TNF and IL-12 concentrations were measured with ELISA. Through multiple linear regression models, the production of each of the proposed cytokines and of the whole set was assessed. RESULTS Workers with high BTX-APD showed a significant reduction in TNF production (β = -1,196.0 pg/mL; p = 0.01); a reduction for IL-10 (β = -520.3; p = 0.13) and IL-12 (β = -843.3; p = 0.09) was also observed, although without statistical significance. CONCLUSIONS TNF production assessed in workers with a high BTX-APD is lower than in those with a low BTX-APD, but not in IL-10 and IL-12 production.

Differential activation of dendritic cells by Mycobacterium tuberculosis Beijing genotype.

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.

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-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.