In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 19 ( 2012-05-08)
Abstract:
In conclusion, we found that the H. pylori T4SS protein can increase the expression of miR-155 in immune cells independently of the known regulation via TLRs ( Fig. P1 ). Because evidence now suggests that TLR signaling in general is dampened at the gastric surface, our findings may be of particular relevance for persistent H. pylori infections in the human stomach, where it is known that strains containing the T4SS cause more severe disease. Future work is needed to resolve the precise roles of miR-155 and macrophages in H. pylori disease. MicroRNAs inhibit the expression of their gene targets. We also wanted to identify gene targets of miR-155 in macrophages during H. pylori infection. We used microarray analysis, a method of measuring the expression of large numbers of genes, to screen the gene activity in response to H. pylori in WT macrophages and also macrophages that had been depleted of miR-155. We compared microarray datasets to determine previously unidentified miR-155 gene targets, which were down-regulated during infection. Many of the gene targets identified are known to be associated with cell-death pathways and DNA damage responses. Of particular interest, among the 35 miR-155 down-regulated gene targets with predicted miR-155-binding sites were the genes Pmaip1 , Tspan14 , and Lpin1 , which have been variously linked to cell-death or apoptotic pathways ( Fig. P1 ). We found that H. pylori -infected macrophages lacking miR-155 were more sensitive to cell death induced by the DNA-damaging agent cisplatin. This result suggested that miR-155 is induced during H. pylori infection and protects macrophages against cell death. It is possible that prolonged macrophage survival contributes to H. pylori disease. Certainly, depletion of macrophages protected against H. pylori pathology in a mouse model of gastritis. Furthermore, depletion of miR-155 in T cells, another immune cell, also protected mice against such pathology ( 4 , 5 ). In both examples, bacterial loads remained high, although pathology was reduced, presenting an intriguing conundrum for H. pylori research. Using quantitative PCR (qPCR) techniques, a method to measure gene expression, we quantified the levels of miR-155 expressed during infection with different H. pylori strains and in the different macrophage cell types. We found that all bacterial strains increased miR-155 expression via activation of TLRs. More interestingly, we observed that strains containing the T4SS up-regulated miR-155 more strongly (when some other signaling pathways were blocked) than strains that lacked the T4SS. Thus, we concluded that, in addition to previous observations implicating TLRs in miR-155 regulation, miR-155 up-regulation also is dependent partly on the direct interaction of the T4SS with macrophages. Moreover, we found that this up-regulation occurred independently of the CagA protein and via the activation of another inflammatory signaling pathway, the NF-κB pathway. Thus, we identified a role for the prototypical H. pylori T4SS pathogenicity factor independent of its major effector protein, both of which are encoded on the bacterial cag PAI island. We wanted to know which bacterial factors are responsible for inducing the expression of miR-155, an miRNA mediator of inflammation that recently was discovered to be up-regulated during H. pylori infection ( 2 ). We studied its expression in macrophages and the consequences of this expression during H. pylori infection. To do so, we infected either WT mouse macrophages or macrophages that were mutated to lack Toll-like receptors (TLRs). TLRs are key receptors for bacterial/viral components, such as LPS, that stimulate responses from host immune cells. Macrophages, in particular, are rich in these receptors. For example, when LPS interacts with TLRs in macrophages, these macrophages are stimulated to mount a powerful immune response. Because miR-155 already has been demonstrated to be up-regulated when TLR2 and TLR4 ( 3 ) are stimulated in this manner, we used macrophages that lacked TLR2 and TLR4 or key TLR signaling adaptor proteins (MyD88/Trif) to identify other factors that might be responsible for inducing miR-155. We also used different H. pylori strains that either naturally lack some of the bacterial pathogenicity factors or contain antibiotic resistance cassettes in place of some of the pathogenicity factors. Bacterial factors implicated in H. pylori pathogenicity are expressed largely by the bacterial cag PAI island, which encodes the functional type four secretion system (T4SS) and the CagA effector protein. The innate immune responses of the host also are implicated as factors in H. pylori disease; for example, the inflammatory response itself may be potentially damaging to tissues ( 1 ). Macrophage immune cells are key mediators of inflammatory responses, but their precise role in H. pylori pathology is undetermined. MicroRNAs (miRNAs) are short sequences of genetic material that can inhibit genetic activity and which may play a role in macrophage-induced pathogenicity. The bacterium Helicobacter pylori infects ∼50% of the human population worldwide and is a major cause of gastritis, peptic ulcer, and gastric cancer. However, researchers have not yet discovered which factors determine whether infections progress from being relatively benign, and often symptomless, to causing severe disease (in 10–20% of cases). It is evident, however, that bacterial, host, and environmental factors are involved. Here, we report a unique role for the key H. pylori pathogenicity factor, the cag PAI pathogenicity island, a segment in the pathogen’s genome, in modulating an immune response during infection of host immune cells.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1116125109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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