MicroRNA-155 knockout mice are susceptible to Mycobacterium tuberculosis infection

Accepted Manuscript MicroRNA-155 knockout mice are susceptible to Mycobacterium tuberculosis infection Hiroki Iwai, Keiji Funatogawa, Kazunori Matsumura, Masako Kato-Miyazawa, Fumiko Kirikae, Kotaro Kiga, Chihiro Sasakawa, Tohru Miyoshi-Akiyama, Teruo Kirikae PII:

S1472-9792(14)20671-8

DOI:

10.1016/j.tube.2015.03.006

Reference:

YTUBE 1313

To appear in:

Tuberculosis

Received Date: 1 December 2014 Accepted Date: 15 March 2015

Please cite this article as: Iwai H, Funatogawa K, Matsumura K, Kato-Miyazawa M, Kirikae F, Kiga K, Sasakawa C, Miyoshi-Akiyama T, Kirikae T, MicroRNA-155 knockout mice are susceptible to Mycobacterium tuberculosis infection, Tuberculosis (2015), doi: 10.1016/j.tube.2015.03.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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MicroRNA-155 knockout mice are susceptible to Mycobacterium tuberculosis infection

2 Hiroki Iwaia, Keiji Funatogawab, Kazunori Matsumuraa, Masako Kato-Miyazawaa, Fumiko Kirikaea,

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Kotaro Kigad, Chihiro Sasakawac,d,e, Tohru Miyoshi-Akiyamaa and Teruo Kirikae*a

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a

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Medicine, Toyama, Shinjuku, Tokyo 162-8655, Japan

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b

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108-8639, Japan.

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Department of Infectious Diseases, Research Institute, National Center for Global Health and

Tochigi Prefectural Institute of Public Health, Tochigi 329-1196, Japan.

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Nippon Institute for Biological Science, Tokyo 198-0024, Japan.

Division of Bacterial Infection, The Institute of Medical Science, The University of Tokyo, Tokyo

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Medical Mycology Research Center, Chiba University, Chiba 260-8673, Japan.

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*Corresponding author at: Department of Infectious Diseases, Research Institute, National Center for

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Global Health and Medicine, Toyama, Shinjuku, Tokyo 162-8655, Japan

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Phone: +81 3 3202-7181; Fax: +81 3 3202-7364

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E-mail address: [email protected] (T. Kirikae)

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KEYWORDS

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Mycobacterium tuberculosis, microRNA, miR-155 1

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ABSTRACT

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MicroRNAs (miRNAs) are short, conserved, non-coding RNA molecules that repress translation,

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followed by the decay of miRNA-targeted mRNAs that encode molecules involved in cell

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differentiation, development, immunity and apoptosis. At least six miRNAs, including microRNA-155

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(miR-155), were up-regulated when born marrow-derived macrophages from C57BL/6 mice were

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infected with Mycobacterium tuberculosis Erdman. C57BL/6 mice intravenously infected with Erdman

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showed up-regulation of miR-155 in livers and lungs. Following infection, miR-155-deficient C57BL/6

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mice died significantly earlier and had significantly higher numbers of CFU in lungs than wild-type

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mice. Moreover, fewer CD4+ T cells, but higher numbers of monocytes and neutrophils, were present in

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the lungs of Erdman-infected miR-155 knockout (miR-155-/-) than of wild-type mice. These findings

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indicated that miR-155 plays a critical role in immune responses to M. tuberculosis.

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1. Introduction MicroRNAs (miRNAs) are small non-coding RNAs expressed by eukaryotic cells that regulate gene expression at the levels of transcription, RNA processing, and translation [1]. The expression of some

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miRNAs affects immune responses to bacterial pathogens [2]. For example, miR-155 was shown to be

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up-regulated in Helicobacter pylori-infected bone marrow-derived macrophages and to inhibit

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DNA-damage induced apoptosis [3]; moreover miR-155 was up-regulated in the gastric mucosae of H.

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pylori-infected mice [4]. Conversely, miR-155 knockout mice (miR-155-/-) failed to control H. pylori

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infection [4], showed impaired CD8+ T cell responses to infection with Listeria monocytogenes [5],

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were more susceptible to Citrobacter rodentium infection [6], and showed ability to clear Streptococcus

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pneumoniae from the nasopharynx [7], but showed greater resistance to Pseudomonas aeruginosa

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keratitis [8].

Several studies [9-12] have suggested that miR-155 plays an important role in the activities of

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monocytes/macrophages during mycobacterial infection. The expression of miR-155 was found to be

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higher in peripheral blood mononuclear cells of tuberculosis patients than of healthy controls [9]. Both

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Mycobacterium smegmatis and M. tuberculosis induced miR-155 expression in human macrophages

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[10]. In addition, M. bovis BCG induced miR-155 expression in a TLR2-dependent manner in murine

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macrophages, both in vivo and in vitro [10]. M. tuberculosis H37Rv induced miR-155 expression in

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cultured murine macrophages in a manner dependent on the production of early secreted antigenic

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target protein 6 (ESAT-6) [11]. In addition, miR-155 promoted autophagy, eliminating intracellular

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H37Rv and BCG, by targeting Ras homologue enriched in brain (Rheb) [12].

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Many studies (reviewed in [13]) have shown that miR-155 is highly expressed within lymphocytes

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(both B and T cells) and mediates a number of lymphocyte responses. miR-155 plays a crucial role in

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the regulation of lymphocyte subsets, including B cells and CD8+ and CD4+ T cells, such as T helper 3

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type 1 (Th1), Th2, Th17 and regulatory T cells [13]. miR-155 regulates a wide spectrum of genes in

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CD4+ T cells, including genes that encode cytokines, chemokines, and transcription factors for normal

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immune function [14]. miR-155 is also involved in regulating T helper cell differentiation and germinal

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center reactions, optimizing T cell–dependent antibody responses [15].

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This study was performed to assess the role of miR-155 in M. tuberculosis infection in mice,

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showing that miR-155-/- mice were highly susceptible to M. tuberculosis infection and had impaired

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CD4 and IFN-γ responses.

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2. Materials and Methods

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2.1 Bacterial strains and culture conditions

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M. tuberculosis Erdman (NIHJ1640), the kind gift of Toshio Yamazaki of the National Institute of

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Infectious Diseases (Tokyo, Japan), was grown on egg-based Ogawa slant medium (Kyokuto

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Pharmaceuticals, Japan) for 2-3 weeks, until abundant growth was observed. The bacteria were

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transferred to 12 mL glass tubes containing 4 mL of MycoBroth (modified Middlebrook 7H9 broth,

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Kyokuto) and incubated at 37 ˚C without shaking for an additional 1-2 weeks. Bacteria floating in the

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culture medium were decanted, transferred to 50 mL conical tubes, collected by centrifugation at 3,100

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rpm for 20 minutes, washed twice with PBS and filtered with a 5 µm Acrodisc (Nihon Pall, Japan) to

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remove bacterial clumps. The filtered bacterial suspensions were used for infection experiments.

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2.2 Mice strains and infection

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Male miR-155-/- mice (B6.Cg-mir155tm1.1Rsky/J), aged 7-9 weeks, were purchased from The Jackson

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Laboratories (Bar Harbor, ME, USA). As described by the supplier, these miR-155-/- mice were on a 4

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C57BL/6 (sub-strain C57BL/6N) background. Age-matched male C57BL/6 (sub-strain C57BL/6JJcl)

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mice were from Clea Japan; this sub-strain had been obtained by Clea Japan from The Jackson

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Laboratory at F166 in 1989. The filtered bacterial suspensions were adjusted to an OD (530 nm) of 0.3

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(equivalent to approximately 1×107 CFU/mL) using a spectrophotometer (ViSpec II; Kyokuto, Japan),

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and mice were intravenously injected with 200 µl of suspension. Numbers of bacteria were confirmed

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by determining CFU/mL using the standard plate method. Mouse survival was monitored; in some

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experiments, M. tuberculosis CFU in organs was determined 90 days after infection.

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Bone marrow derived-macrophages from C57BL/6 mice were infected with M. tuberculosis at a

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multiplicity of infection (MOI) of 10:1. Five days later, total RNA was extracted using miRNeasy Mini

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Kits (Qiagen, Japan) according to the manufacturer’s instructions. miRNA microarray experiments

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were performed using mouse miRNA ver12.1 chips (Toray Industries, Japan), with the data processed

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using the global mean normalization method.

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2.4 Quantitative real-time PCR

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Total RNA was extracted from tissues using Trizol (Life Technologies, Japan) and reverse transcribed

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using a Transcriptor First Strand Synthesis Kit (Roche Diagnostics, Japan). PCR was performed with

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SYBR-Green (Roche Diagnostics) using HT7900 (Life Technologies). miRNA real-time PCR was

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performed to detect miR-155 and Rnu6 (U6 small nuclear RNA) using miScript SYBR Green PCR

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Kits (Qiagen). Levels of miRNA were normalized relative to those of U6.

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2.5 Flow cytometry

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Lungs were obtained from the infected mice, cut into small pieces (~1mm3) and digested for 1 h at

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37°C in PBS (Nacalai, Japan) containing 1 mg/mL collagenase type II (Worthington Biochemical,

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USA) and 0.5 mg/mL DNase I (Roche). The digests were passed through a 40 µm strainer (BD

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Bioscience, Japan) using the plunger of a 1 mL syringe. Red blood cells were hemolyzed, and the cell

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suspensions were washed with PBS and incubated with anti-mouse CD16/CD32 (BioLegend, Japan) to

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block non-specific binding. The cell suspensions were subsequently incubated with the following

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antibodies: CD3e-FITC (clone: 145-2C11), CD4-PE/Cy7 (RM4-5), CD8a-APC (53-6.7), CD19-PE

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(6D5), CD11b-FITC (M1/70), CD11c-PE (N418) and Ly-6G/Ly-6C (Gr-1)-APC (RB6-8C5) (all

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BioLegend, Japan). The stained cells were fixed with 4% paraformaldehyde for 1 h and analyzed by

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flow cytometry (Cytomics FC500 cell analyzer, Beckman Coulter, Japan).

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Seven weeks after infection, the mice were anesthetized with sevoflurane and euthanized by cervical

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dislocation. Their spleens were excised and homogenized, and the cells were harvested using 100 µm

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cell strainers. Erythrocytes were lysed using ACK buffer (Life Technologies) and washed in RPMI

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(Nacalai). Cell suspensions were prepared in RPMI supplemented with 10% FBS, NaHCO3,

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L-glutamine, non-essential amino acids, sodium pyruvate and 2-mercaptoethanol and plated at 2×105

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cells/well in 96-well round-bottomed culture plates. The cells were cultured in triplicate at 37˚C for 48

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h in 5% CO2with or without 2 × 105/well of M. tuberculosis Erdman. The supernatants were harvested,

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filtered with 0.2 µm filters and stored at -30˚C. Cytokine concentrations in the supernatants were

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determined with Luminex200 (Luminex, Japan), using MILLIPLEX MAP Mouse Cytokine/Chemokine

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Magnetic Bead Panel-Premixed 25 Plex (Merck, Japan), according to the manufacturer’s instructions.

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3. Results and Discussion Of the 1,274 miRNAs tested, only seven showed different levels of expression in M. tuberculosis Erdman infected and uninfected macrophages (Figure 1A). Of these seven miRNAs, six, including

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miR-21, miR-21*, miR-146a, miR-146b, miR210 and miR-155, were up-regulated in Erdman-infected

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macrophages, whereas one, miR-223, was down-regulated. miR-155 and miR-223 have been reported

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to affect tuberculosis immunity [9-12, 16], with miR-155 up-regulated in cultured macrophages

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infected with M. tuberculosis H37Rv [11], M. bovis BCG [10, 12] and M. smegmatis [9], and miR-223

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either down-regulated [17] or up-regulated [16] in patients with tuberculosis. miR-223-/- mice were

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susceptible to tuberculosis by regulating lung neutrophil recruitment [16]. Consistent with previous findings [10, 12], the levels of expression of miR-155 were significantly

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higher in lungs (7.3-fold) of Erdman-infected than uninfected mice (Figure 1B). Moreover, the levels of

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miR-155 expression were much higher in livers of Erdman infected than uninfected mice (Figure 1B).

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High levels of miR-155 expression in livers were also detected in mice infected with Francisella

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novicida [18]. Similar levels of miR-155 expression were observed in spleens of uninfected and

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infected mice (Figure 1B). Since M. bovis BCG was reported to up-regulate miR-155 in the spleens of

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C57BL/6 mice [10], our findings suggest that M. tuberculosis and M. bovis BCG differ in their ability

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to induce miR-155 expression in spleens.

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miR-155-/- mice were more susceptible than wild-type (WT) mice to M. tuberculosis Erdman (Figure

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2A), with mean survival of the two infected strains being 74.5 and 153.0 days, respectively (Figure 2A).

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In addition, the numbers of CFU per organ 90 days after infection were significantly higher in

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miR-155-/- than in WT mice (Figure 2B), with the difference especially marked in the lungs (691-fold). 7

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Although these results suggest that miR-155-deficient mice are more susceptible to M. tuberculosis

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infection than WT mice, the difference in susceptibility may have been due to genes other than

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miR-155. The miR-155-/- mice, on a genetic background of the C57BL/6N sub-strain, may have several

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genetic differences in levels of SNPs than WT mice of sub-strain C57BL/6J.

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miR-155 induction was apparently correlated with M. tuberculosis growth in mouse lungs, but not in

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mouse livers. Other factor(s) may also contribute to M. tuberculosis growth in livers. The levels of

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miR-155 induction were much higher in livers than in lungs of WT mice infected with Erdman

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(97.7-fold vs. 7.3-fold) (Figure 1B), although differences in CFUs in the livers of WT and miR-155-/-

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mice were not as large as the differences in lungs (Figure 2B). Intravenous infection with M.

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tuberculosis H37Rv was reported to induce a 2.5-fold increase in miR-155 expression in mouse lungs

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[12], similar to our findings. These increases in miR-155 expression may result from infiltration of

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immune cells into lungs of infected mice rather than from miR-155 induction in these cells. To test this,

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the proportions of miR-155-expressing cells obtained from lungs of mice infected with M. tuberculosis

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must be analyzed using cell sorting techniques.

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Seven weeks after infection with Erdman, the numbers of CD4+ T cells were significantly lower,

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while the numbers of monocytes/macrophages and neutrophils were significantly higher, in the lungs of

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miR-155-/- than of WT mice (Figure 3A). The reduced CD4+ T cell response in miR-155-/- mice was

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accompanied by impaired M. tuberculosis clearance (Figure 2B).

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Cytokine/chemokine profiles in response to Erdman infection differed markedly in miR-155-/- and

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WT mice 7 weeks after infection (Figure 3B). Erdman-infected miR-155-/- mice showed significantly

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lower concentrations of IFN-γ than did infected WT mice. In contrast, infected miR-155-/- mice

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showed significantly higher concentrations of granulocyte-colony stimulating factor (G-CSF), 8

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macrophage inflammatory proteins (MIP)-1α, MIP-1β and MIP-2 than WT mice (Figure 3B). The

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levels of interleukin (IL)-1α, IL-2, IL-6, IL-10, IL-17, IFN-γ-inducible protein-10 kDa (IP-10),

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cytokine-induced neutrophil chemoattractant (KC), monocyte chemoattractant protein (MCP)-1,

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regulated upon activation, normal T cell expressed and secreted (RANTES) and TNF-α were similar in

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the two strains following Erdman infection.

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The present study showed that miR-155 promotes Th1 (IFN-γ) responses to M. tuberculosis

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infection, while inhibiting the production of Th2 cytokines, such as IL-6 and IL-10, and neutrophil

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recruitment. M. tuberculosis-specific-CD4+ Th1 cells are thought to play a central role in tuberculosis

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immunity by producing IFN-γ, which contributes to the recruitment and activation of

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monocytes/macrophages [19]. During early stages of infection, Th17 cells participate in protection

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against TB by recruiting monocytes and Th1 lymphocytes to the site of granuloma formation [19]. It is

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unclear, however, whether neutrophils can kill internalized mycobacteria, in particular virulent M.

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tuberculosis strains [20].

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It is also unclear whether miR-155 directly affects the anti-M. tuberculosis activities of macrophages, including their anti-bactericidal activity and cell surface expression of MHC class II molecules. Several

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studies [9-12], including ours, showed that M. tuberculosis and other Mycobacterium spp. induced

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miR-155 expression in cultured macrophages. Down-regulation of miR-155 by transfection of a

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miR-155 inhibitor was found to enhance the anti-bactericidal activity of macrophages against M.

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tuberculosis [11] and, conversely, to reduce the anti bactericidal activity of macrophages against M.

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bovis BCG [12]. miR-155 expression did not seem to affect macrophage-mediated phagocytosis against

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BCG [12]. miR-155 knockdown in Kupffer cells decreased MHC class II expression in a mouse liver

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allograft model [21].

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In conclusion, our findings indicate that miR-155 is essential for mouse immunity to M. tuberculosis infection. miR-155 regulates CD4+ T cell protective responses to M. tuberculosis, mediated by IFN-γ.

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miR-155 has been reported to regulate mouse immunity to a number of bacterial pathogens [4-7],

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suggesting that it plays important and complex roles in both innate and adaptive immune responses to

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these pathogens [2]. miR-155 is essential for the T cell-mediated control of Helicobacter pylori

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infection and for the induction of chronic gastritis and colitis [4]. miR-155-/- mice are required for

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optimal CD8+ T cell responses to Listeria monocytogenes [5]. Germinal center formation and humoral

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immune responses against Citrobacter rodentium were impaired in infected miR-155-/- mice [6].

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miR-155-/- mice have lower levels of Th1 and Th17 cells and a reduced capacity to induce Th1 cells

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over the course of Streptococcus pneumoniae colonization of the nasopharynx [7].

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Funding: This study was supported by Grants for International Health Research (26A-103 and

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24A-93103) from the Ministry of Health, Labor, and Welfare of Japan.

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Competing interests: None declared.

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Ethical approval: Protocols of animal experiments were reviewed and approved by the Animal Care

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and Use Committee of the NCGM Research Institute (permit number 12027) and the ethical committee

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of Tochigi Prefectural Institute of Public Health and Environmental Science (permit number 000720)

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based on the “Basic Guidelines for Proper Conduct of Animal Testing and Related Activities in the

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Research Institutions under the Jurisdiction of the Ministry of Health, Labour and Welfare of JAPAN”.

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Figure legends

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Figure 1 M. tuberculosis up-regulates miR-155 expression.

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(A) Scatter plot of differential expression of microRNAs (miRNAs) in mouse bone marrow

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macrophages (BMDMs) that were and were not infected with M. tuberculosis. Open circles indicate

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up-regulated miRNAs, open squares indicate down-regulated miRNAs and filled squares indicate

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similar levels of expression.

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(B) Expression of miRNAs in the liver, lungs and spleen of C57BL/6 mice 7 weeks after M.

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tuberculosis injection (1.9×106 CFU/mouse). Expression of miR-155 was assayed by real-time

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quantitative RT-PCR. *P<0.01, **P<0.005 by Student’s t-tests. Error bars indicate the means ± SD.

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Figure 2 Absence of miR-155 results in susceptibility to M. tuberculosis infection.

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(A) Survival curves of miR-155-/- (n=10) and WT (n=8) mice injected i.v. with M. tuberculosis (3.8×

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106 CFU/mouse). Differences between miR-155-/- and WT were statistically significant. *P<0.0001 by

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the log-rank test.

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(B) Bacterial burdens, as measured by CFUs in the liver, lungs and spleen, 90 days after intravenous

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infection with 2.2×106 CFU/mouse. *P<0.05, **P<0.01 by Student’s t-tests.

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Figure 3 miR-155-/- mice show impaired immune responses to M. tuberculosis infection.

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(A) C57BL/6 (WT) and miR-155-/- mice were infected with M. tuberculosis (1.9×106 CFU/mouse) for

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7 weeks. The absolute numbers of the indicated subsets in the individual mice were calculated from the

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relative percentage of each subset and the total lung cells. *P<0.05, **P<0.01, ***P<0.005 by

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Student’s t-tests. Error bars indicate the means ± SD. 14

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(B) Cytokine and chemokine concentrations in the supernatants of spleen cell cultures of C57BL/6 and

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miR-155-/- mice seven weeks after infection with M. tuberculosis (1.9×106 CFU/mouse) and

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stimulated in vitro with M. tuberculosis. *P<0.05, ***P<0.001 by one-way ANOVA. Error bars

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indicate the means ± SD.

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