Innate immunity is the first line of defense against invading microorganisms in vertebrates and triggers an antigen-specific adaptive immune response (38
). AMPs are thought to be significant effectors of innate immunity through their immunomodulatory activity and direct killing of microorganisms (2
). AMPs are divided into two families: defensins and cathelicidins (1
). Both kinds of AMPs have been involved in the immunopathogenesis of several infectious diseases (3
). Abnormalities in HBD-2 production, have been involved in lung infections exhibited by patients with cystic fibrosis (3
). On the other hand, LL-37 is the only cathelicidin identified in humans so far and has been linked to skin and infectious bowel diseases (1
). LL-37 can be induced by different molecules and has been used as a treatment for shigellosis in a rabbit model (27
Scant information is available on the role of AMPs in TB. We showed that during mycobacterial infection of lung ECs, there is high expression of HBD-2 (30
). Moreover, studies with experimental models revealed that mice with uncontrolled infection produced scant quantities of mBD-3 and -4, while animals that controlled mycobacterial growth produced abundant quantities of both defensins (29
Whether cathelicidin LL-37 is involved in innate immunity against intracellular pathogens such as M. tuberculosis
has not been determined in the human lung. To our knowledge, this is the first report showing gene induction and production of LL-37 in human cells after infection with M. tuberculosis
. We demonstrated that when lung ECs were infected with M. tuberculosis
, high levels of LL-37 were produced, primarily after 18 h, in a dose-dependent manner, and the same response was induced by S. enterica
serovar Typhi. This result is in agreement with our previous study, which demonstrated the highest HBD-2 production by the same lung EC cell line after 18 h of mycobacterial infection (30
), suggesting that both peptides, and probably other molecules, may act synergistically against M. tuberculosis
during primary lung epithelial infection (9
Neutrophils are one of the first cells to arrive at the infection site during tuberculosis, thus preventing early mycobacterial blood dissemination (4
). Moreover, it has been reported that phagocytosis of apoptotic neutrophils and granules by macrophages results in decreased viability of intracellular M. tuberculosis
). Therefore, the transfer of AMPs from neutrophils to macrophages provides a cooperative defense strategy between innate immune cells against intracellular pathogens. We infected neutrophils with M. tuberculosis
to assess whether these cells produced LL-37 during in vitro infection and compared it to salmonella infection, which has been reported to induce the production of this peptide (32
). Our results showed that neutrophils efficiently produced LL-37 when infected with M. tuberculosis
. and may indicate early bactericidal activity of this peptide against M. tuberculosis
It has been reported that AMs and MDMs are important participants in the innate immune response against M. tuberculosis
). We investigated the production of LL-37 in AMs and MDMs during M. tuberculosis
infection. Our results showed that MDMs infected with M. tuberculosis
expressed LL-37 at high levels, and this expression was related to the MOI. Gene expression in MDMs correlated with the percentage of immunostained cells. Intriguingly, infected monocytes did not show such expression; thus, it appears that the level of macrophage differentiation is associated with the ability to produce LL-37.
We sought to detect the production of LL-37 in infected AMs, considering that this cell type is the main effector cell in M. tuberculosis
destruction and antigen presentation in the lung (41
). Our immunocytochemistry data showed that AMs infected with M. tuberculosis
produced more LL-37 than any other cell type analyzed in our study. Indeed, the immunostaining was also stronger in AMs than in any of the other cells. In fact, all AMs positive for LL-37 showed phagocytosed mycobacteria as determined by Ziehl-Nielsen staining. This observation was corroborated and extended by immunoelectron microscopy, which showed LL-37 on the mycobacterial surface and cytoplasm and also in phagosomal vesicles. Interestingly, scarce bacilli were observed after 18 h of infection, but double immunolabeling of LAM and LL-37 was seen inside AM phagosomal vesicles. This observation suggests that at this time point of infection, many bacteria were killed and LL-37 could participate in this process.
Liu et al. showed that TLR activation of MDMs induced killing of M. tuberculosis
through LL-37 participation and this induction is in part due to the vitamin D receptor and vitamin D hydroxylase gene activation (18a
). During our experiments, no vitamin D supplementation was used to induce LL-37 production. However, we used human serum, which is a source of vitamin D, so we could not determine the real participation of this factor in LL-37 production. This interesting point will be explored in future experiments.
The histopathological hallmark of tuberculosis is granulomas, whose main function is to prevent dissemination (15
). There is no information available about LL-37 production in tuberculous granulomas. Our immunohistochemistry results showed that LL-37 was not detected in lung granulomas. Considering that AMPs are produced during early infection and TB granulomas are typical of a chronic infection, this finding is not surprising. In contrast, pneumonia caused by gram-negative organisms showed strong LL-37 immunostaining in neutrophils and macrophages, as well as bronchial ECs. These results suggested that LL-37 is mainly produced during acute infection and probably early M. tuberculosis
infection and has limited or no participation in the bactericidal activity in chronic granulomatous inflammation.
During the innate immune response, TLR activation triggers direct antimicrobial activity (41
). Therefore, we studied LL-37 production after TLR stimulation. We stimulated MDMs with different TLR ligands, including TLR-9. In agreement with previous publications (37
), we found that LPS induced strong production of LL-37. However, other authors reported that LPS had minimal capacity to stimulate cathelicidin production after blood mononuclear cell activation (33
). This difference could be explained by the different kinds of cells used in our experiments and also by the inherent responses produced by the study subjects. As previously reported, cathelicidin blocks and inactivates LPS, thus inhibiting septic shock (18
), and it has also been reported that some subjects are more susceptible to septicemia than others.
Interestingly, when cells were stimulated with purified DNA from M. tuberculosis
, a ligand for TLR-9, cells responded with high LL-37 production. Some AMPs, such as HBD-2, can be induced through TLR-9 ligands in lung ECs (24
). On the other hand, several reports suggested that TLR-9 is a very important receptor for optimal innate immune responses against M. tuberculosis
). We found that AMs and MDMs expressed high levels of TLR-9, and this could be related to the inherent immune response against M. tuberculosis
in some individuals (our unpublished data).
In summary, our results showed that AMs are most efficient at producing LL-37 after infection with M. tuberculosis, suggesting that cathelicidin from AMs may be an important participant in the innate immune response during early infection in humans, probably after TLR-9 activation with M. tuberculosis DNA. Other significant issues, such as the role of cathelicidin in the immunopathogenesis of TB, its direct bactericidal activity, and its participation in immunoregulation, should be studied in the future.