MicroRNA profiling reveals opposing expression patterns for miR-511 in alternatively and classically activated macrophages

<div><p></p><p><i>Background</i>: Macrophages are heterogeneous cells, which possess pleotropic effector and immunoregulatory functions. The phenotypic diversity of macrophages is best exemplified by the ability of IL-4 or IL-13, two key cytokines in asthma to promote macrophages into a suppressive/anti-inflammatory phenotype (e.g. alternatively activated or M2) whereas exposure to IFN-γ followed by microbial trigger renders macrophages pro-inflammatory (e.g. classically activated or M1). Intriguingly, only limited data exists regarding the expression of miRNA in M2 macrophages. <i>Objective</i>: To define the miRNA profile of M2 and M1 macrophages. <i>Methods</i>: Bone marrow-derived macrophages were activated to classically and alternatively activated states using IL-4, IL-13 or IFN-γ followed by <i>Escherichia coli</i> stimulation. Thereafter, an unbiased miRNA “mining” approach was utilized and the expression of several miRNAs was validated following <i>in-vitro</i> and <i>in-vivo</i> macrophage activation (qPCR). miR-511 over-expression was performed followed by global transcriptional and bioinformatic analyses. <i>Results</i>: We report unique miRNA expression profiles in M2 and M1 macrophages involving multiple miRNAs. Among these miRNAs, we established that miR-511 is increased in macrophages following IL-4- and IL-13-stimulation and decreased in M1 macrophages both <i>in-vitro</i> and <i>in-vivo</i>. Increased miR-511 expression was sufficient to induce marked transcriptional changes in macrophages. Interestingly, bioinformatics analyses revealed that miR-511 altered the expression of gene products that are associated with hallmark alternatively activated macrophage functions, such as cellular proliferation, wound healing responses and inflammation. <i>Conclusions</i>: Our data establish miR-511 as a bona fide M2-associated miRNA. These data may have significant implications in asthma where the expression of IL-4 and IL-13 are highly increased.</p></div

Natural Killer Receptor 1 Dampens the Development of Allergic Eosinophilic Airway Inflammation

<div><p>The function of NCR1 was studied in a model of experimental asthma, classified as a type 1 hypersensitivity reaction, in mice. IgE levels were significantly increased in the serum of OVA immunized NCR1 deficient (<i>NCR1</i>^{<i>gfp/gfp</i>}) mice in comparison to OVA immunized wild type <i>(NCR1</i>^<i>+/+</i>) and adjuvant immunized mice. Histological analysis of OVA immunized <i>NCR1</i>^{<i>gfp/gfp</i>} mice revealed no preservation of the lung structure and overwhelming peribronchial and perivascular granulocytes together with mononuclear cells infiltration. OVA immunized <i>NCR</i>^<i>+/+</i> mice demonstrated preserved lung structure and peribronchial and perivascular immune cell infiltration to a lower extent than that in <i>NCR1</i>^{<i>gfp/gfp</i>} mice. Adjuvant immunized mice demonstrated lung structure preservation and no immune cell infiltration. OVA immunization caused an increase in PAS production independently of NCR1 presence. Bronchoalveolar lavage (BAL) revealed NCR1 dependent decreased percentages of eosinophils and increased percentages of lymphocytes and macrophages following OVA immunization. In the OVA immunized <i>NCR1</i>^{<i>gfp/</i>gfp} mice the protein levels of eosinophils’ (CCL24) and Th2 CD4⁺ T-cells’ chemoattractants (CCL17, and CCL24) in the BAL are increased in comparison with OVA immunized <i>NCR</i>^<i>+/+</i> mice. In the presence of NCR1, OVA immunization caused an increase in NK cells numbers and decreased NCR1 ligand expression on CD11c⁺GR1⁺ cells and decreased NCR1 mRNA expression in the BAL. OVA immunization resulted in significantly increased IL-13, IL-4 and CCL17 mRNA expression in <i>NCR1</i>^<i>+/+</i> and <i>NCR1</i>^{<i>gfp/</i>gfp} mice. IL-17 and TNFα expression increased only in OVA-immunized <i>NCR1</i>^<i>+/+</i>mice. IL-6 mRNA increased only in OVA immunized <i>NCR1</i>^{<i>gfp/gfp</i>} mice. Collectively, it is demonstrated that NCR1 dampens allergic eosinophilic airway inflammation.</p></div

Increased inflammation in the lungs of <i>NCR1</i>^{<i>gfp/gfp</i>} mice compared to <i>NCR1</i>^<i>+/+</i> mice (H&E+PAS x340).

<p>Mice were immunized and samples were process as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160779#pone.0160779.g002" target="_blank">Fig 2</a>. (A) OVA immunized <i>NCR</i>^{<i>gfp/gfp</i>} (H&E+PAS x340). (B) OVA/Alum immunized <i>NCR</i>^<i>+/+</i> (H&E+PAS x340). A thick arrow with a short tail identifies mononuclear cells and thin arrow with a long tail identifies polymorphonuclear cells.</p

A decrease in NCR1 mRNA expression and increase of NCR1 ligand expression in the BAL of OVA immunized mice.

<p><i>NCR</i>^<i>+/+</i> and <i>NCR</i>^{<i>gfp/gfp</i>} mice were immunized with either adjuvant alone or with OVA/alum as described in the Material and Methods section. The mice were euthanized and the lung tissue was dissociated into single cells. (A) Total number of live immune cell. (B) NK cell number as determined by anti NK1.1 antibody. (C) qRT PCR performed with <i>NCR1</i> appropriate primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160779#pone.0160779.s006" target="_blank">S1 Table</a>). (D) Five populations of cells carrying or lacking CD11c or GR1 were determined and stained with LY94-Ig fusion protein. *p > 0.05.</p

Increased inflammation in the lungs of <i>NCR1</i>^{<i>gfp/gfp</i>} mice H&E+PAS x180).

<p><i>NCR1</i>^{<i>gfp/gfp</i>} C57Bl/6 mice were intraperitoneally (i.p) immunized with either OVA/alum or adjuvant only on days 0 and 14. Ten days after the second immunization mice were challenged twice intranasally with OVA at days 24 and 27. Twenty-four hours following the second immunization mice were euthanized and the left lungs were harvested for histology and RT-PCR. Representative Hematoxylin& Eosin (H&E) and PAS stained sections from each group are shown. Inflammation severity was assessed blindly by the pathologist. (A) OVA/alum immunized <i>NCR</i>^{<i>gfp/gfp</i>} (H&E+PAS x180). (B) Alum immunized <i>NCR</i>^{<i>gfp/gfp</i>} (H&E+PAS x180).</p

NCR1 involvement in chemokine and Th2, type immune cytokines in response to allergic airway inflammation.

<p><i>NCR1</i>^<i>+/+</i> and <i>NCR1</i>^{<i>gfp/gfp</i>} mice were immunized with either OVA or adjuvant as described in Material and Methods, and 24 h following the last challenge the lungs were harvested and taken for RT-PCR. The bar graph represents levels of cytokine mRNA following either adjuvant or OVA immunization. Levels of cytokine mRNA were analyzed by RT-PCR and calibrated to mRNA level of HPRT (n = 3–7). (A) IL-4, (B) IL-13, (C) CCL17 (D) IL-17; (E). TNFα; (F) IL-6. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the adjuvant immunized <i>NCR1</i>^<i>+/+</i> group ± SD (two-tailed Student <i>t</i>-test).</p

NCR1 is involved in eosinophil and macrophage infiltration to the lung.

<p><i>NCR1</i>^<i>+/+</i> (Adjuvant n = 8, OVA n = 10) and <i>NCR1</i>^{<i>gfp/gfp</i>} (adjuvant n = 10, OVA n = 11) mice were immunized with either OVA or adjuvant as described in the Materials and Methods section. (A) The BAL was lavaged from each mouse, stained with PI and analyzed by flow cytometry for live cell count. The bar graph represents normalized number to the adjuvant immunized <i>NCR1</i>^<i>+/+</i> group whose average was considered as 1 of live cells in the BAL. (B-E) The bar graph represents an average percentage of eosinophils (B), macrophages (C), lymphocytes (D), and neutrophils (E) in the lung BAL (percentage of cells ± SEM; two-tailed Student <i>t</i>-test). These are the combined results of 2 experiments performed at different time points. * p < 0.05, **p < 0.01 *** p < 0.001.</p

Increased inflammation in the lungs of <i>NCR1</i>^<i>+/+</i> mice (H&E+PAS x180).

<p><i>NCR1</i>^<i>+/+</i> mice were immunized and samples were process as describe in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160779#pone.0160779.g002" target="_blank">Fig 2</a>. (A) OVA/Alum immunized <i>NCR</i>^<i>+/+</i> (H&E+PAS x180). (B) Alum immunized <i>NCR</i>^<i>+/+</i> (H&E+PAS x270).</p

Increased IgE production in the absence of NCR1.

<p><i>NCR</i>1^<i>+/+</i> and <i>NCR1</i>^{<i>gfp/gfp</i>} C57Bl/6 mice were i.p. immunized with either OVA/Alum or adjuvant only on days 0 and 14. Ten days after the second immunization, mice were challenged twice intranasally with OVA at days 24 and 27. Twenty four h following the second challenge, serum was drawn for measurement of the total IgE levels (n = 8 to 11). Results are the summary of 2 independent experiments and were normalized according to the OVA immunized <i>NCR1</i>^<i>+/+</i> group average that was considered as 1 in each experiment. *p < 0.05; **p < 0.01 1 way ANOVA multiple comparisons, GraphPad)</p