Introduction
miRNAs are short RNAs (19–25 nucleotides) typically involved in the downregulation of gene expression. They are initially transcribed as long, primary miRNAs (pri-miRNAs) that vary greatly in length. pri-miRNA is cleaved by the protein Microprocessor, leaving a shorter hairpin loop structure known as precursor miRNA (pre-miRNA). Once the pre-miRNA is exported from the nucleus, the hairpin loop is removed by Dicer, leaving a double-stranded miRNA structure known as the miRNA/miRNA* complex. One strand (mature miRNA) of this complex will be loaded onto the RNA-induced silencing complex (RISC). The other miRNA strand, denoted with an asterisk, is called the minor mature miRNA, and may also have a biological function. Mature miRNA anneal and bind to their mRNA target of interest via RISC, leading to mRNA degradation or translation inhibition. Since miRNAs do not require an exact nucleotide match to bind to their target, each miRNA can potentially downregulate hundreds of targets.
miRNAs play an important role in immune system development and function, and also regulate the innate immune response. For example,
E. coli LPS activates the human toll-like receptor pathway via TLR4, which increases production of cytokines including TNFα. A strong LPS response can lead to sepsis, a dangerous, life-threatening medical condition. Expression of miR-146a is upregulated during stimulation of human THP-1 cells by LPS (2). In addition, LPS stimulation of murine macrophages causes upregulation of miR-147, let-7e, and miR-181c, as well as downregulation of miR-155 and miR-125b (3, 4), and miR-601 has been shown to inhibit NFκB, a key transcription factor involved in the innate immune response (5). Understanding how miRNAs regulate the immune response may lead to more effective treatments for immune system disorders.
Yersinia pestis is a gram-negative pathogen that infects both humans and fleas and causes plague.
Y. pestis growing within the flea produces lipooligosaccharide (LOS) instead of LPS due to a mutation in the O-antigen gene cluster (see Temperature-dependent LOS structures). The type of LOS made by
Y. pestis following a flea infection produces a strong agonistic TLR4 response, similar to LPS from
E. coli. These conditions can be mimicked by growing
Y. pestis in culture at 21–25° (6). However, when
Y. pestis is introduced to humans via a flea bite, the LOS structure is modified and causes an antagonistic TLR4 response and evasion of the innate immune system (see
Temperature-dependent LOS structures). This response can be replicated by growing
Y. pestis in culture at 37°C.
To identify an effect of the different forms of LOS on the innate immune response of cells, the expression of both mRNA and miRNA was analyzed using the Human Toll-Like Receptor Signaling Pathway RT
2 Profiler PCR Array and the Human miRNA Genome V2.0 Complete (384 well) RT
2 PCR Array, respectively, following exposure of two cell lines to LOS derived from
Y. pestis grown at 21–25°C or 37°C.
Results
The secretion of TNFα, a key cytokine upregulated during the TLR4-mediated innate immune response, was measured to identify the effect of LOS purified from cultures grown at 21°C compared with LOS purified from cultures grown at 37°C on peripheral blood mononuclear cells (PBMCs) (see
PBMC response to Y. pestis LOS). LOS purified from cells grown at a low temperature (i.e., mimicking flea infection) elicited a greater TLR4-mediated response, similar to the response caused by LPS purified from
E. coli cells (not shown).
LOS purified from cultures grown at 25°C or 37°C was added to THP-1 or U-937 cells to identify an effect on gene expression using the Human Toll-Like Receptor Signaling Pathway RT
2 Profiler PCR Array (see
Toll-like receptor gene expression responses to Y. pestis LOS). As expected, a greater TLR signaling response was elicited from LOS purified from cultures grown at 25°C (this produces an equivalent result to LOS purified from cells grown at 21°C; data not shown). The expression of key genes including TNF, NFκB family members, and toll-like receptors was also altered (see
Toll-like receptor gene expression responses to Y. pestis LOS, part C).
LOS purified from cultures grown at 25°C or 37°C was added to THP-1 cells (see
miRNA expression responses to LOS in THP-1 cells) to identify an effect on miRNA expression using the Human miRNA Genome V2.0 Complete (384 well) RT
2 PCR Array. As expected, greater miRNA expression changes are seen following stimulation from LOS purified from cells grown at 25°C. Since miR-146a is reported to be upregulated during LPS stimulation of THP-1 cells, it is interesting to note upregulation of the minor species miR-146a* in this experiment following stimulation with LOS purified from cells grown at 37°C, indicating some similarity between the innate immune response to
E. coli LPS and this form of
Y. pestis LOS. In addition, miR-155 is reported to be downregulated during murine macrophage LPS stimulation, whereas here miR-155* is upregulated. However, it is possible that these differences are due to the model systems used.
LOS purified from cultures grown at 25°C or 37°C was also added to THP-1 cells (see
miRNA expression responses to LOS in U-937 cells) to identify an effect on miRNA expression using the Human miRNA Genome V2.0 Complete (384 well) RT
2 PCR Array. As expected, greater miRNA expression changes are seen following stimulation with LOS purified from cells grown at 25°C. However, similar to the Toll-like receptor signaling response (see figure
Toll-like receptor gene expression responses to Y. pestis LOS), U-937 cells show a smaller miRNA expression response compared to THP-1 cells.
Since miR-125b is reported to be downregulated during murine macrophage LPS stimulation, it is interesting to note that the minor species miR-125b-1* is upregulated, although miR-125b is not. These data suggest a biological function for this minor miRNA species. In addition, it is intriguing to note that multiple miRNAs are differentially regulated following stimulation with LOS purified from cells grown at 37°C. These include miR-601, which has been shown to inhibit NFκB, a key transcription factor involved in the innate immune response.
Conclusions
In this study, the Human Toll-Like Receptor Signaling Pathway RT
2 Profiler PCR Array and the Human miRNA Genome V2.0 Complete (384 well) RT
2 PCR Array were used to identify how different forms of
Y. pestis LOS affect the innate immune response of THP-1 and U-937 cells. A greater immune response, measured by upregulation or downregulation of mRNA, and a greater number of differentially regulated miRNA were observed in THP-1 cells following stimulation by
Y. pestis LOS. Additionally, stimulation of cells with LOS purified from cells grown at 25°C (mimicking flea infection) elicited a greater immune response and differential regulation of miRNAs compared to stimulation with LOS purified from cells grown at 37°C (mimicking human infection). Further study of changes in miR-125b and miR-125b-1* as well as miR-155 and miR-155* regulation following LOS treatment may identify mechanisms that bring about differential responses to LOS, as well as LPS, stimulation.
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