MicroRNA 26a (miR-26a)/KLF4 and CREB-C/EBP beta regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection

For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBP beta signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBP beta signaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions

C/EBPβ regulates M1, M2 markers in Mtb-infected macrophages as well as the survival of Mtb in macrophages.

<p>Cells were transfected with control siRNA or <i>C/ebpbeta</i> siRNA (A-K; M); or with control siRNA or <i>Creb</i>siRNA (L) for 48 h prior to infection. (A-I) After infection of RAW264.7 cells with Mtb, relative expression of various M2 or M1 markerswas measured by qRT-PCR (A-E) or cytokine release was measured by ELISA (F-I) in the supernatants at different time points. (J-M) BMDMs (J) or RAW264.7 (K,L, M) were transfected as indicated, followed by infection with Mtb (K,L). In panel M, cells were treated with L-NAME before infection. Bacterial counts were determined at different time points. Data represent the means ± SEM (n = 3 for panels A-I, K-M; n = 2 for panel J). *<i>p</i>< 0.05; ** <i>p<</i>0.01; *** <i>p<</i>0.001. NS, not significant; BDL, below detection limit.</p

KLF4 regulates bacterial survival in macrophages.

<p>RAW264.7 cells (A, C) or BMDMs (B) were transfected with control siRNA or <i>Klf4</i> siRNA. After 48 h, cells were infected with Mtb (A,B) or pretreated with L-NAME (C) prior to infection with Mtb. Bacterial counts were determined at different time points post-infection. Results are means ± SEM (n = 3). *<i>p</i>< 0.05; ** <i>p<</i>0.01.</p

miR-26a and KLF4 regulate trafficking of Mtb to macrophages.

<p>(A,B) RAW264.7 (A) or BMDMs (B) were transfected with control mimic or miR-26a mimic for 24 h (C,D) RAW264.7 (C) or BMDMs (D) were transfected with control siRNA or <i>Klf4</i> siRNA for 48 h. Cells were infected with FITC-labelled Mtb (green). After 24 h, cells were fixed and stained with LAMP1 antibody and Alexa-546 conjugated secondary antibody (red), and visualized by confocal microscopy. Nuclei were stained with DAPI. Colocalization of red and green fluorescence indicates that the mycobacteria reside in the lysosomal compartment. The panels on the right represent quantification of the results. The data are representative of three independent experiments in RAW264.7 and two independent experiments in BMDMs. ** <i>p<</i>0.01; *** <i>p<</i>0.001.</p

KLF4 and miR-26a regulate arginase and iNOS activities in Mtb-infected macrophages.

<p>(A-F) RAW264.7 cells (A,B,D) or BMDMs (C,E) were transfected with either control siRNA or <i>Klf4</i> siRNA for 48 h prior to infection with Mtb for different periods of time, followed by measurement of arginase activity (B,C), nitrite production (D,E) or Western blotting of lysates for KLF4(A) or iNOS (F). For B-E, results are means ± SEM, n = 3. For A and F, blots are representative of results obtained in three separate experiments. (G-L) RAW264.7 cells (G,I) or BMDMs (H,J) were transfected with control mimic or miR-26a mimic; or RAW264.7 cells were transfected with control inhibitor or miR-26a inhibitor (K,L) prior to infection with Mtb. Arginase activity (G,H,L) or nitrite production (I-K) was measured at different periods of time after infection. Results are means ± SEM, n = 3. ** <i>p<</i>0.01; *** <i>p<</i>0.001. For (F), intensities of bands were measured by densitometric scanning. The fold change in iNOS was calculated with respect to uninfected cells.</p

miR-26a regulates the expression of KLF4 in Mtb-infected macrophages.

<p>(A-F) RAW264.7 (A, B, E, F) or hMDMs (C), or BMDMs (D) were transfected with control mimic or with miR-26a mimic (A-D, F); or with control inhibitor or miR-26a inhibitor (E) prior to infection. (F) RAW264.7 cells were transfected with control mimic or with miR-26a mimic, then treated with MG132; prior to infection with Mtb. Time-dependent expression of KLF4 was analyzed by Western blotting. The data shown in panels A, B, D-F are representative of three independent experiments; the data shown in panel C is representative of two independent experiments. Intensities of bands were measured by densitometric scanning. The fold change was calculated with respect to uninfected cells.</p

miR-26a is downregulated during Mtb infection and regulates the survival of Mtb in macrophages.

<p>(A-E) RAW264.7 cells (A, D, E) or BMDMs (B) or hMDMs (C) were infected with Mtb for different periods of time as indicated in the figure. RNA was isolated and expression of miR-26a (A-C) or pri-miR-26a-1 or pri-miR-26a-2 (D,E) was assessed by Northern blotting (A-C) using U6 for normalization; or by semi-quantitative RT-PCR (D, E) using <i>Gapdh</i> for normalization. (F-I) The effect of miR-26a on bacterial survival was analyzed by transfecting RAW264.7 (F,I) or BMDMs (G) or hMDMs (H) with control mimic or miR-26a mimic (F-H), or with control inhibitor or miR-26a inhibitor (I) prior to infection with Mtb. CFUs were determined at different time periods post-infection as indicated. Results represent the means ± SEM (n = 3); *<i>p</i>< 0.05; ** <i>p<</i>0.01; *** <i>p<</i>0.001. (J-L) Expression of miR-26a was analysed by Northern blotting in lungs (J), spleen (K) or lymph nodes (L). The numbers on the horizontal axes of panels J-L represent individual mice (uninfected: 1–3; infected: 4–8). Intensities of bands were measured by densitometric scanning. The data is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006410#ppat.1006410.s002" target="_blank">S2A–S2C Fig</a>.</p

Schematic representation of the post-transcriptional and transcriptional regulation of M1/M2 polarization and autophagy in Mtb-infected macrophages.

<p>Mtb infection is associated with the downregulation of miR-26a which in turn upregulates its target KLF4; and CREB-mediated transcriptional upregulation of C<i>/ebpbeta</i>. This is associated with decreased expression of M1 markers such as TNF-α, IL-12, IL-6, and iNOS with a concomitant increase in the expression of M2 markers such as IL-10, CCL24, arginase and Msr1. Further, upregulation of KLF4 favours increased expression of Mcl1 which in turn inhibits autophagosome formation and consequently, lysosomal trafficking of Mtb. Mtb infection thus tilts the innate immune response of the host towards pathways that favor the survival of the bacterium.</p

KLF4 regulates autophagy in Mtb-infected macrophages.

<p>(A, B) BMDMs or RAW264.7 (C,D) were transfected with control siRNA or <i>Klf4</i> siRNA prior to infection with Mtb. After 6 h, the cells were fixed and incubated with LC3 antibody, followed by staining with Alexa-488-conjugated secondary antibody. LC3 puncta (green) formation was detected by fluorescence microscopy. The experiments were done in triplicate and at least 100 cells were counted for each condition. The data are representative of results obtained in three independent experiments in RAW264.7 and two independent experiments in BMDMs. ** <i>p<</i>0.01; *** <i>p<</i>0.001. (E-G) Mcl-1 levels were analyzed after infection of RAW264.7 with Mtb. Cells were treated with control siRNA or <i>Klf4</i> siRNA (E) or control mimic or miR-26a mimic (F) or combinations of control mimic, miR-26a mimic, empty vector or HA-KLF4 plasmid as indicated (G), prior to infection. Blots are representative of the results obtained in two independent experiments. (H) Cells were treated with either control siRNA or <i>Klf4</i> siRNA for 48 h. Where indicated, cells were treated with 3-MA prior to infection. Bacterial counts were determined at the indicated time points. Results represent the means ± SEM (n = 3) ** <i>p<</i>0.01; *** <i>p<</i>0.001. Bar = 20 μm.</p

KLF4 and C/EBPβ are differentially regulated in <i>Mycobacterium tuberculosis</i> (Mtb)-infected macrophages and KLF4 is targeted by miR-26a.

<p>(A,B) RAW264.7 cells were infected with Mtb at an MOI of 10 for 24 h, RNA was isolated and the relative expression of <i>Klf4</i> (A) and <i>C/ebpbeta</i> (B) were analyzed with respect to uninfected cells (normalized to 1). (C) Time-dependent expression of KLF4 was analyzed by Western blotting in Mtb-infected RAW264.7. Intensities of bands were measured by densitometric scanning. Each data point is represented as ratio of intensities of KLF4 and GAPDH (indicated between the two blots). (D)Schematic representation of <i>Klf4</i> mRNA showing the relative positions of coding sequence and 3′-UTR regions (not to scale). (E,F) HEK293 cells were co-transfected with plasmids expressing <i>Klf4</i> 3′UTR (wild type, WT) (E) or mutant, MUT) (F) and β-galactosidase, along with miR-26a mimic (or control mimic). After 24 h of transfection, luciferase assays were performed. Readings were normalized to β-galactosidase activities. (G) Association of miR-26a with <i>Klf4</i>. RAW 264.7 cells were cotransfected with c-<i>myc</i>-Ago2 in the presence or absence of miR-26a mimic for 24 h followed by lysis of cells and immunoprecipitation with either anti-c-<i>myc</i>-agarose or protein A/G-agarose (ctrl). qRT-PCR was performed to confirm the presence of <i>Klf4</i> in the immunoprecipitates of cells transfected with miR-26a. Results in panels A,B, E-G represent means ± SEM (n = 3). The blot in (C) is representative of three experiments. ** <i>p<</i>0.01; *** <i>p<</i>0.001.</p