lysX gene is differentially expressed among Mycobacterium tuberculosis strains with different levels of virulence.

Antimicrobial peptides (AMPs) are mainly produced by epithelial cells and macrophages to eliminate infecting mycobacteria through direct antimicrobial activity and immunomodulation. Indeed, it has been described that this line of defense is essential to control infection. However, Mycobacterium tuberculosis (Mtb) has developed mechanisms to avoid AMPs activity, for instance lysX adds lysine residues to surface phospholipids changing their net charge, leading to the repelling of the AMPs. In the present study, we determined that lysX gene is differentially expressed among Mtb strains. To achieve this aim we used several well-characterized Mtb clinical isolates, lysX mutated strains and reference strains. Our results showed that in the presence of AMPs, lysX expression increased significantly. Strains with higher lysX expression showed increased levels of intracellular survival in vivo and in vitro and induced more severe lesion related with pneumonia. Results showed that ability of Mtb to replicate intracellularly was directly correlated to the level of lysX expression showing that the amount of lysX produced by the bacterial cell is an important variable for the modulation of Mtb virulence.

Differences in Cytokine Production during Aging and Its Relationship with Antimicrobial Peptides Production.

Aging is a major health issue due to the increased susceptibility of elderly people to infectious, autoimmune, and cardiovascular diseases. Innate immunity is an important mechanism to avoid primary infections; therefore, decreasing of its activity may lead to development of infections. Antimicrobial peptides (AMPs) are effector molecules of innate immunity that can eliminate microbial invaders. The role that cytokines play in the regulation of these innate immune mechanisms needs to be explored. Serum determinations of Th1, Th2, and Th17 cytokines were performed in order to evaluate their association with AMPs human beta-defensin (HBD)-2 and LL-37 in young adults, elder adults, and elder adults with recurrent infections. Our results showed differences in interleukin (IL)-10 and IL-6 among the different groups. Inverse correlations in serum cytokine levels and HBD-2 production were identified for IL-10, IL-2, IL-4, tumor necrosis factor-α, and IL-6. Also inverse correlations were identified for IL-10, IL-4, and cathelicidin (LL-37). Such results could impact the development of immunomodulators that promote AMP production to prevent and/or contain infectious diseases in this population.

Glucose levels affect LL-37 expression in monocyte-derived macrophages altering the Mycobacterium tuberculosis intracellular growth control.

Diabetes mellitus (DM)-2 patients have an increased susceptibility to develop pulmonary tuberculosis; this is partly due to the impairment of the innate immunity because of their higher glucose concentrations. In the present study, we determined the effect of the glucose concentrations in the LL-37 expression in infected and non-infected macrophages. Our results showed that the increasing glucose concentrations correlates with the low cathelicidin expression in non-infected cells, however in Mycobacterium tuberculosis infected cells, LL-37 expression was substantially increased in higher glucose concentrations, nevertheless the mycobacterial burden also increased, this phenomena can be associated with the cathelicidin immunomodulatory activity. Further evaluation for LL-37 needs to be done to determine whether this peptide can be used as a biomarker of tuberculosis progression in DM2 patients.

LL-37 immunomodulatory activity during Mycobacterium tuberculosis infection in macrophages.

Tuberculosis is one of the most important infectious diseases worldwide. The susceptibility to this disease depends to a great extent on the innate immune response against mycobacteria. Host defense peptides (HDP) are one of the first barriers to counteract infection. Cathelicidin (LL-37) is an HDP that has many immunomodulatory effects besides its weak antimicrobial activity. Despite advances in the study of the innate immune response in tuberculosis, the immunological role of LL-37 during M. tuberculosis infection has not been clarified. Monocyte-derived macrophages were infected with M. tuberculosis strain H37Rv and then treated with 1, 5, or 15 µg/ml of exogenous LL-37 for 4, 8, and 24 h. Exogenous LL-37 decreased tumor necrosis factor alpha (TNF-α) and interleukin-17 (IL-17) while inducing anti-inflammatory IL-10 and transforming growth factor ß (TGF-ß) production. Interestingly, the decreased production of anti-inflammatory cytokines did not reduce antimycobacterial activity. These results are consistent with the concept that LL-37 can modulate the expression of cytokines during mycobacterial infection and this activity was independent of the P2X7 receptor. Thus, LL-37 modulates the response of macrophages during infection, controlling the expression of proinflammatory and anti-inflammatory cytokines.

1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

Characterization of antibacterial and hemolytic activity of synthetic pandinin 2 variants and their inhibition against Mycobacterium tuberculosis.

The contention and treatment of Mycobacterium tuberculosis and other bacteria that cause infectious diseases require the use of new type of antibiotics. Pandinin 2 (Pin2) is a scorpion venom antimicrobial peptide highly hemolytic that has a central proline residue. This residue forms a structural "kink" linked to its pore-forming activity towards human erythrocytes. In this work, the residue Pro14 of Pin2 was both substituted and flanked using glycine residues (P14G and P14GPG) based on the low hemolytic activities of antimicrobial peptides with structural motifs Gly and GlyProGly such as magainin 2 and ponericin G1, respectively. The two Pin2 variants showed antimicrobial activity against E. coli, S. aureus, and M. tuberculosis. However, Pin2 [GPG] was less hemolytic (30%) than that of Pin2 [G] variant. In addition, based on the primary structure of Pin2 [G] and Pin2 [GPG], two short peptide variants were designed and chemically synthesized keeping attention to their physicochemical properties such as hydrophobicity and propensity to adopt alpha-helical conformations. The aim to design these two short antimicrobial peptides was to avoid the drawback cost associated to the synthesis of peptides with large sequences. The short Pin2 variants named Pin2 [14] and Pin2 [17] showed antibiotic activity against E. coli and M. tuberculosis. Besides, Pin2 [14] presented only 25% of hemolysis toward human erythrocytes at concentrations as high as 100 µM, while the peptide Pin2 [17] did not show any hemolytic effect at the same concentration. Furthermore, these short antimicrobial peptides had better activity at molar concentrations against multidrug resistance M. tuberculosis than that of the conventional antibiotics ethambutol, isoniazid and rifampicin. Therefore, Pin2 [14] and Pin2 [17] have the potential to be used as an alternative antibiotics and anti-tuberculosis agents with reduced hemolytic effects.

Vitamin D supplementation promotes macrophages' anti-mycobacterial activity in type 2 diabetes mellitus patients with low vitamin D receptor expression.

The increasing number of people with type 2 diabetes (DM2) is alarming and if it is taken into account that the relative odds of developing tuberculosis in diabetic patients ranges from 2.44 to 8.33 compared with non-diabetic patients, thus in developing countries where these two diseases are encountering face to face, there is a need for prophylaxis strategies. The role of vitamin D has been widely implicated in growth control of Mycobacterium tuberculosis (Mtb) during primary infection mainly through the induction of certain antimicrobial peptides (AMPs). In this study we evaluated the vitamin D serum levels, CYP27B1-hydroxylase enzyme, vitamin D receptor (VDR) and AMPs gene expression in Healthy donors, DM2 and TB patients. Results showed that DM2 group has lower VDR and AMPs expression levels. When Monocytes Derived Macrophages (MDM) from DM2 patients with low VDR expression were supplemented with vitamin D, MDMs eliminate efficiently M. tuberculosis. This preliminary study suggests the use of vitamin D as prophylaxis for tuberculosis in high DM2 endemic countries.