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TLR2 activation
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Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation Junaid Khan, Praveen K. Sharma 1 , Arunika Mukhopadhaya ∗ Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector 81, SAS Nagar, Mohali, Punjab 140306, India
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Article history: Received 15 March 2015 Received in revised form 4 May 2015 Accepted 1 June 2015 Available online xxx Keywords: M1/M2 polarization Toll-like receptor OmpU Porin
a b s t r a c t Polarization of the monocytes and macrophages toward the M1 and M2 states is important for hosts’ defense against the pathogens. Moreover, it plays a crucial role to resolve the overwhelming inflammatory responses that can be harmful to the host. Polarization of macrophages/monocytes can be induced by pathogen-associated molecular patterns (PAMPs). PAMP-mediated monocyte/macrophage polarization is important during the infection, as pathogen can suppress host immune system by altering the polarization status of the macrophages/monocytes. OmpU, an outer membrane porin protein of Vibrio cholerae, possesses the ability to induce pro-inflammatory responses in monocytes/macrophages. It is also able to down-regulate the LPS-mediated activation of the monocytes/macrophages. Such observation leads us to believe that OmpU may induce a state that can be called as M1/M2-intermediate state. In the present study, we evaluated a set of M1 and M2 markers in RAW 264.7 murine macrophage cell line, and THP-1 human monocytic cell line, in response to the purified OmpU protein. We observed that OmpU, as a PAMP, induced M1-polarization by activating the Toll-like receptor (TLR) signaling pathway. OmpU induced formation of TLR1/TLR2-heterodimers. OmpU-mediated TLR-activation led to the MyD88 recruitment to the TLR1/TLR2 complex. MyD88, in turn, recruited IRAK1. Ultimately, OmpUmediated signaling led to the activation and subsequent nuclear translocation of the NFB p65 subunit. We also observed that blocking of the TLR1, TLR2, IRAK1, and NFB affected OmpU-mediated production of M1-associated pro-inflammatory cytokines such as TNF␣ and IL-6. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Modulation of the host’s immune status is very crucial in the host-pathogen relationship. Several molecules of the pathogen are responsible for host immunomodulation. Gram-negative pathogens carry an arsenal of antigens that can trigger a multitude of cellular responses (Fallon, 2009; Sakharwade et al., 2015). Some of these molecules act as the pathogen-associated molecular patterns (PAMPs) that are recognized by the pattern recognition receptors (PRRs) present on the host cells, mainly of immune origin, and thus generating innate immune responses in terms of pro-inflammatory responses (Kumar et al., 2011; Sorci and Faivre,
Abbreviations: PAMP, pathogen associated molecular pattern; PRR, pattern recognition receptors; TLR, toll-like receptor; MyD88, myeloid differentiation primary response 88; IRAK, interleukin 1-receptor associated kinase; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells. ∗ Corresponding author. Fax: +91 172 2240124. E-mail address: [email protected] (A. Mukhopadhaya). 1 Present address: Dr. Reddy’s Laboratories Ltd., Survey No 47, Bachupally, Qutbullapur, Hyderabad 500090, Telengana, India.
2009). Some of the PAMPs can dampen the host responses and help the pathogen to survive in the host system (Ashida et al., 2011). Outer membrane, an additional layer enveloping the gramnegative bacteria, consists of various components like lipopolysaccharide (LPS) and outer membrane proteins (OMPs) that play a crucial role in their interaction with the host organism (Sakharwade et al., 2015). Porins, a major group of bacterial OMPs, function as channels for solute transport. Other than channel property, porins have important roles in the pathogenesis and survival of the bacteria in the harsh environments (Nikaido, 2003). It has been shown in various studies, that porins are involved in the bacterial adherence processes and in the induction of apoptosis of the host cells (Bernardini et al., 1993; Muller et al., 1999; Negm and Pistole, 1999). Roles of porins in inducing pro-inflammatory responses, and their underlying signaling processes have been established in various reports (Biancone et al., 1997; Biswas, 2000; Galdiero et al., 2001; Gupta et al., 1999; Sakharwade et al., 2013; Sakharwade et al., 2015). Due to its immuno-modulatory property, porins are thought to be the potential vaccine candidate (Latz et al., 2004; Tabaraie et al., 1994).
http://dx.doi.org/10.1016/j.imbio.2015.06.009 0171-2985/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Khan, J., et al., Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.06.009
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The most widely studied PRRs, the Toll-like receptors (TLRs) are crucial for recognizing both surface-associated PAMPs and nucleic acids from invasive and non-invasive bacteria as well as viruses (Kawai and Akira, 2005; Kawai and Akira, 2007; West et al., 2006). TLRs have important roles in providing some degree of specificity to innate immune system and bridging it with the acquired immune responses (Werling and Jungi, 2003). TLR4 recognition is crucial for LPS-mediated pro-inflammatory responses. TLR1, TLR2, TLR4, TLR5, and TLR6 are the major surface-expressed PRRs. Upon activation TLR4 forms homodimer, and TLR2 forms heterodimer with either TLR1 or TLR6, whereas, TLR5 remains as monomer even on activation (West et al., 2006). TLR activation mainly leads to the activation of NFB transcription factor, which upon translocation to the nucleus up-regulates transcription of genes important for the induction of the pro-inflammatory responses (Hayden et al., 2006a,b; Tak and Firestein, 2001; West et al., 2006). Macrophages are one of the main targets for modulation of host innate and adaptive immune responses (Benoit et al., 2008; Martinez et al., 2009; Murray and Wynn, 2011; Sica and Mantovani, 2012). They can be activated through the TLR pathways to produce pro-inflammatory mediators, and activation of the Th1-subtypes of T helper cells. These types of macrophages are known as M1macrophages. M1 activation can happen with or without the presence of IFN␥ cytokines in the microenvironment. Interestingly, in the presence of IL-4/IL-13 cytokines, macrophages can get polarized towards an alternatively activated state, also known as the M2-state that is characterized by the release of cytokines and chemokines, which play major roles in immune-regulation, tissue healing and repair. In addition to cytokines, different PAMPs also possess the ability to polarize the macrophages towards either M1 or M2 state. OmpU, one of the major porins of the human pathogen Vibrio cholerae exhibits dual nature of host-immunomodulation (Khan et al., 2012; Sakharwade et al., 2013; Sakharwade et al., 2015). It can induce pro-inflammatory responses on one hand, and on the other it can down-regulate the LPS-mediated pro-inflammatory responses (Sakharwade et al., 2013). Pro-inflammatory responses such as, production of TNF␣, IL-6 and generation of nitric oxide (NO) suggested that OmpU can induce M1-polarization of macrophages, whereas, down-regulation of NO, TNF␣, IL-6 and IL-12 in OmpUpretreated LPS-activated cells suggest that OmpU may have the ability to induce polarization of the macrophages towards the M2 state (Murray and Wynn, 2011). In the present study we have investigated whether OmpU has the ability to induce M1-polarization or M2-polarization. We have also explored to delineate the cell-signaling pathways in OmpUmediated macrophage polarization. 2. Material and method 2.1. Purification of recombinant OmpU Recombinant V. cholerae OmpU protein was purified according to the protocol mentioned in our previous report (Khan et al., 2012). Briefly, V. cholerae El Tor O1 OmpU gene cloned in pET14b vector was transformed into Escherichia coli Origami B cells (EMD Millipore, Billerica, MA, USA) for OmpU expression. E. coli cells were cultured in Luria broth (HiMedia, Mumbai, India). At 0.5–0.6 OD600 , culture was induced with IPTG (HiMedia, Mumbai, India) for 3 h. The cells were centrifuged and resuspended in bacterial lysis buffer (GBiosciences, St. Louis, MO, USA) and were subjected to ultrasonication at 25 amplitude for 5 min, with 30 s pulses for lysis. Cell lysates were centrifuged at 18,500 × g at 4 ◦ C to isolate the inclusion bodies. Inclusion body was solubilized in denaturing buffer (PBS containing 8 M urea). Denatured protein in 8 M urea buffer supplemented with 20 mM imidazole was applied onto
manually packed Ni-NTA column (Qiagen GmbH, Hilden, Germany) equilibrated with the same buffer. The protein was eluted under the denaturing condition with 300 mM imidazole and 8 M urea in PBS. Eluted denatured protein was subjected to refolding. Protein was diluted into the refolding buffer [10% glycerol, 0.5% LDAO (N,N-dimethyldodecylamine N-oxide)] with constant stirring, and was incubated overnight at 4 ◦ C. The refolded protein was passed through the gel-filtration chromatography column of Sephacryl S200 (GE Healthcare, Piscataway, NJ, USA) equilibrated with a buffer containing 10 mM Tris–HCl, 10 mM NaCl and 0.5% LDAO. The protein was eluted with the same buffer, and was analyzed by SDSPAGE and Coomassie staining. Structural integrity and functionality of OmpU was confirmed by Far-UV circular dichorism (CD) spectroscopy and liposome-swelling assay respectively, as described earlier (Khan et al., 2012). 2.2. Cell lines and culture condition Mouse macrophage cell line RAW 264.7 (ATCC), and human monocytic cell line THP-1 (NCCS Pune, India) was sub-cultured and maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 100 Units/ml penicillin-100 Units/ml streptomycin (Invitrogen, Life Technologies, Carlsbad, CA, USA Life). Cells were incubated at 37 ◦ C in 5% CO2 environment. 2.3. Purification of human monocytes Human monocytes were purified from PBMCs using the Monocyte Isolation Kit II for human (MACS Miltenyi Biotec, Bergisch Gladbatch, Germany) according to the manufacturer’s protocol (Khilwani et al., 2015; Sakharwade et al., 2013). Work with human blood has been approved by the Institutional Bioethics Committee. 2.4. Semi-quantitative real time PCR for M1- and M2-associated genes and surface TLR genes expression RAW 264.7 and THP-1 cells were plated at a density of 2 × 106 cells/ml and treated with 3 g/ml OmpU or buffer (10 mM tris, 10 mM Nacl, 0.5% LDAO) as negative control, for 2 h, 4 h, 8 h, 12 h and 24 h. Cells were lysed with lysis buffer (RNAsure mini kit, Nucleopore, Genetix Biotech, New Delhi, India). Total RNA was isolated using the manufacturer protocol (RNAsure mini kit, Nucleo-pore, Genetix Biotech), and RNA was quantified by spectrophotometer (Thermo Fisher Scientific, Waltham, MA USA). Equal quantity (1 g) of total RNA of each sample was reverse transcribed using the maxima first strand cDNA synthesis kit, as directed by the manufacturer (Thermo Fisher Scientific, Waltham, MA USA). And relatively expressed quantity of each gene of interest was analysed using the Maxima Syber Green qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA USA) by Real Time PCR (Eppendorf, Hamburg, Germany). Sequences of all the primers were taken from the primer bank (Nucleic Acids Res 38:D792–D799. Doi:10.1093/nar/gkp1005 PubMed: (19,906,719). Results were expressed in terms of relative gene expression as mean-fold induction over the buffer treated controls. Normalization of relative gene expression was done by comparisons to either ˇ-actin, RPL13 or HPRT. 2.5. Flow cytometry for surface TLR expression RAW 264.7 and THP-1 cells at a density of 1 × 106 cells/ml were treated with 3 g/ml OmpU or reference buffer, and were incubated for 12 h and 24 h. Cells were harvested and washed in FACS buffer (PBS, 1% FBS), and then incubated with PE-conjugated anti-human TLR1, FITC-conjugated anti-human TLR2 or PE-conjugated antihuman TLR6 monoclonal antibody (MAb) (Biolegend, San Diego, CA, USA) for 30 min at 4 ◦ C. Cells after washing with the FACS buffer
Please cite this article in press as: Khan, J., et al., Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.06.009
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were acquired on a FACSCaliber (BD Bioscience, New Jersey, USA) flow cytometer.
2.10. siRNAs transfection of THP-1 cells for TLR2 and TLR6 knockdown
2.6. Neutralization of TLR with antibody to inhibit the cellular response
THP-1 cells were plated in serum-free media at a density of 7 × 104 cells for 6 h, and were then transfected with small interfering RNA (siRNA) (from GE Healthcare Dharmacon, Lafayette, CO, USA) with final concentration of 25 nM targeted against TLR2, TLR6, and also with the scrambled siRNA as the negative control. Transfection mixture was prepared by following the manufacturer’s protocol. Briefly, DharmaFECT (GE Healthcare Dharmacon) transfection reagent and siRNAs were mixed in serumfree media, and were incubated for 20 min before applying to the cells. Expression of TLRs for knockdown was analyzed by flow cytometry after 48 h post-transfection. Cells, subjected to siRNA-mediated knockdown, were treated with OmpU to study the inhibition of TNF␣ secretion upon TLR2- and TLR6-knockdown by ELISA.
THP1 cells and human monocytes isolated from PBMC were cultured at a density of 1 × 106 cells/ml, and were pre-incubated with anti-human TLR1 MAb, neutralizing antibody (Invivogen, San Diego, CA, USA), anti-human TLR2 MAb, neutralizing antibody (Biolegend), anti-human TLR6 Mab (Invivogen) or an isotype control IgG (Biolegend), one hour before the OmpU treatment (3 g/ml). Supernatants were collected for TNF␣ and IL-6 cytokines ELISA. ELISA Kits were from BD Bioscience. 2.7. Co-immunoprecipitation for the detection of MyD88 and IRAK1 recruitment downstream to the TLRs RAW 264.7 and THP-1 cells at a density of 1 × 106 cells/ml were plated and treated with 3 g/ml OmpU or control buffer for 1 h. Whole cell lysates were prepared using the lysis buffer (50 mM Tris–HCl, 137 mM NaCl, 2 mM EDTA, 1% Triton-X 100, 0.1% SDS, pH 7.6). 500 g of total cellular protein (estimated by Bradford Reagent from Sigma, St. Louis, USA) was incubated either with the antiTLR2 or anti-MyD88 antibody (Santa Cruz Biotechnology, Texas, USA) for 3 h at 4 ◦ C, and then protein A/G plus Agarose beads (Santa Cruz Biotechnology) were added and incubated for overnight at 4 ◦ C. Agarose bead–protein complex was washed three time with PBS and protein was eluted from beads by heating with 5 × SDS gel loading buffer at 100 ◦ C for 15 min. To detect the association of TLR1, TLR6, or MyD88 (Santa Cruz Biotechnology) with TLR2, and IRAK1 (Sigma–Aldrich) with MyD88 (Santa cruz Biotechnology) by immunoblotting, samples were subjected to SDS-PAGE, proteins was transferred on to the PVDF (polyvinylidene difluoride) membrane (Millipore, Billerica, Massachusetts, USA). Blots were developed by chemiluminescence-based method using Image Quant LAS 4000 (GE Healthcare life Sciences, Pittsburgh, USA). 2.8. Western blotting for NFB translocation RAW 264.7 and THP-1 cells were cultured at a density of 1 × 106 cells/ml and treated with 3 g/ml OmpU for 0 min, 30 min, 60 min. Cells were harvested, washed with PBS and subjected to buffer A (10 mM HEPES, 1.5 mM MgCl2 , 10 mM KCl, 0.5 mM DTT, 0.05% Igepal pH 7.9) and incubated on ice for 10 min and then centrifuged at 3000 rpm for 10 min. Supernatants were stored as cytoplasmic fraction and pellets were subjected to Buffer B (5 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol (v/v), 300 mM Nacl, pH 7.9), sonicated for few seconds and centrifuged at 24,000 × g to isolate the nuclear lystaes in the supernatant. Reagents for buffers preparation were obtained from Himedia, (Mumbai, India). To detect NFB (p65) translocation into the nucleus, both the fractions were subjected to SDS-PAGE, and proteins were transferred on to the PVDF membrane, and blot was developed by chemiluminescence-based method using Image Quant LAS 4000 (GE Healthcare life Sciences). 2.9. Inhibitor study to detect IRAK1 and NFB involvement RAW 264.7 and THP-1 cells were cultured in 6-well plate at the density of 1.5 × 106 cells/well. IRAK1/4 inhibitor (Sigma–Aldrich) at concentrations of 500 nM, 800 nM or 1000 nM, and NFB inhibitor Pyrrolidine dithiocarbamate (PDTC) (Sigma–Aldrich) at concentrations of 15 M or 25 M was added to the cultured cells 1 h before the treatment with OmpU (3 g/ml). Supernatants were collected for ELISA to check the inhibition of TNF␣ and IL-6 production by the corresponding inhibitors.
2.11. Statistical analysis Statistical analysis was done using Student’s two-sided t test. Data is represented as the mean ± SEM. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. OmpU induces M1-polarization of macrophages Monocytes and macrophages have the plasticity, which allows them to acquire classical (M1-polarized) and alternative form (M2-polarized) in response to various stimuli (Murray and Wynn, 2011). The groups of such stimuli, constituted by the pathogenassociated molecular patterns (PAMPs) possess the ability to determine the polarization pattern of the macrophages and monocytes (Hajishengallis and Lambris, 2011). To explore whether OmpU possesses the ability to induce M1 or M2-polarization, RAW 264.7 murine macrophage cells and THP-1 human monocytic cells were treated with OmpU and incubated for different time periods. After respective incubations, the cells were harvested and total RNA was isolated. Semi-quantitative real time PCR was done to assess the up-regulation of M1 and M2-associated gene expression. We observed that most of the M1-associated genes were upregulated at the transcriptional level, but none of the M2-associated genes showed any up-regulation in both the cell lines (Fig. 1A–D). 3.1.1. OmpU-treated cells showed transcriptional up-regulation of M1-associated genes Total RNA from OmpU-treated RAW 264.7 and THP-1 cells were analyzed for M1-associated chemokine genes that are important in causing the inflammatory responses. Chemokines, such as Mip-1␣, Mip-1 (macrophage inflammatory proteins), also called CCL3 and CCL4, respectively, are critical for granulocyte activation, whereas, CCL5 or RANTES is important in the recruitment of leucocytes such as, neutrophils (Gao et al., 1993; Guan et al., 2001; Jones et al., 2011). CXCL10 is important for the recruitment of the monocytes/macrophages, T cells, NK cells and dendritic cells (Liu et al., 2011). We observed that expression of all these M1-associated chemokine genes were upregulated in OmpU-treated RAW 264.7 cells (Fig. 1A). Similarly, total RNA from OmpU treated THP-1 cells were evaluated for the expression of mip-1˛, mip-1ˇ, and ccl5 genes, and we observed transcriptional up-regulation of all the said genes (Fig. 1C). Therefore, it is evident that OmpU is capable of inducing M1polarization.
Please cite this article in press as: Khan, J., et al., Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.06.009
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Fig. 1. OmpU induces M1-polarization of macrophages/monocytes. RAW 264.7 and THP-1 monocytes were plated at a density of 2 × 106 cells/ml, and were treated with OmpU or buffer. Treated cells were incubated for different time periods. After respective incubations, cells were harvested, total RNA was isolated and subjected to semi quantitative real time PCR. (±SEM values were determined from at least three biological replicates). (A) M1-associated chemokine genes such as, mip1a, mip1b, ccl5, cxcl10 showed transcriptional up-regulation in OmpU-treated RAW 264.7 cells, with maximum release at 4 h, 12 h, 12 hrs and 2 h, respectively. (B) M2-associated mediators such as arginase 1, il1ra, and ccl17 showed fold-changes around 1 or less than that, indicating no transcriptional up-regulation of the M2associated genes in OmpU-treated RAW 264.7 cells. (C) OmpU-treated THP-1 cells induced transcriptional up-regulation of M1-associated genes such as, mip1a, mip1b, ccl5 with maximum up-regulation at 12 h, 8 h, and 8 h, respectively. (D) M2-associated genes such as, arginase 1, ccl22 did not show any significant up-regulation in OmpU-treated THP-1 cells.
3.1.2. M2-associated genes are not expressed in OmpU-treated cells Total RNA from OmpU-treated RAW 264.7 and THP-1 cells were analyzed for M2-associated genes such as arginase 1, il1ra, ccl17 and ccl22. Arginase 1 is important for arginine metabolism to proline and polyamine, hence induce tissue repair and growth promotion (Martinez et al., 2009). It inhibits the activity of iNOS, and eventually M1-associated NO production from arginine. The interleukin-1 receptor antagonist (IL-1Ra) is a member of the IL1 family that binds to IL-1 receptors but does not induce any intracellular response (Arend et al., 1998; Taylor et al., 2005). In M2-polarised cells generally the surface expression of IL-1ra is high. CCL17 and CCL22 are Th2-recruiting chemokines and marker for
M2-associated macrophages (Mariani et al., 2004). We observed that OmpU-treated cells did not show transcriptional upregulation of any of these M2-associated genes (Fig. 1B and D).
3.2. OmpU mediates its signaling via TLR1/TLR2 pathway Pattern recognition receptors (PRRs) are crucial to recognize PAMPs that in turn decide the polarization patterns. TLRs are crucial to recognize the surface-associated PAMPs of non-invasive bacteria. Therefore, we wanted to check if at all TLRs are involved in the OmpU-mediated signaling, and if so, we wanted to identify the TLRs, important for the OmpU- mediated signaling.
Please cite this article in press as: Khan, J., et al., Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.06.009
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Fig. 2. OmpU-treated cells showed transcriptional up-regulation of TLR1, TLR2, TLR6; however, increased surface expressions were observed for TLR1 and TLR2 only. RAW 264.7 and THP-1 cells were treated with OmpU. After incubation for different time points cells were harvested and total RNA was isolated and subjected to semi-quantitative real time PCR to probe gene expression of TLR1, TLR2, TLR4, and TLR6. (±SEM values were determined from at least three biological replicates). In a different setup cells were treated with OmpU, and after incubation cells were harvested and stained with anti-TLR1, anti-TLR2 and anti-TLR6 and analyzed by flow cytometry. (Data are shown as representative plots for 3 individual experiments). (A) TLR1, TLR2 and TLR6 genes showed more than 2-fold transcriptional upregulation with maximum at around 12 h, 2 h and 12 h, respectively in OmpU-treated RAW 264.7 cells. (B) In OmpU-treated THP-1 cells, TLR1, TLR2 and TLR6 genes showed around 2-fold or more than 2-fold up-regulation at time points of 12 h, 24 h and 12 h, respectively. (C) There are increased expression of TLR1 and TLR2, but not that of TLR6 in OmpU-treated RAW 264.7 cells. (Shaded curves, buffer-treated controls; solid lines, OmpU-treated cells). (D) TLR1 and TLR2 showed increased surface expression in OmpU-treated THP-1 cells. (Shaded curves, buffer-treated controls; solid lines, OmpU-treated cells; dashed lined, OmpU-treated cells stained with isotype antibody control).
3.2.1. OmpU-treated cells showed transcriptional up-regulation of tlr1, tlr2, and tlr6 genes Firstly, we checked the transcriptional up-regulation of the important cell surface TLRs upon the OmpU treatment. RAW 264.7 and THP-1 cells were treated with OmpU or buffer and incubated for different time points up to 24 h. After the respective incubations, cells were harvested and total RNA was extracted and analyzed by semi-quantitative real time PCR. In case of RAW 264.7 cells, we observed, over 2-fold up-regulation in the transcriptional level of tlr1 and tlr2 genes (Fig. 2A). Additionally, we observed that in case of tlr6 there was around 2-fold up-regulation, whereas, in case of tlr4, upregulation was less than 2 fold (Fig. 2A). Same set of TLR genes were analyzed in THP-1 cell lines. Similar to RAW 264.7, in THP-
1 as well, we observed transcriptional up-regulation of tlr1, tlr2, and tlr6 in response to the OmpU treatment, but not that of tlr4 (Fig. 2B). This observation at the mRNA level indicated that OmpU may induce hetero-dimerization of TLR1/TLR2 or TLR2/TLR6.
3.2.2. OmpU induces surface expression TLR1 and TLR2 but not that of TLR6 RAW 264.7 and THP-1 cells were incubated with OmpU or buffer for different time points and analyzed for surface expression of TLRs by flow cytometry. We observed an increase in surface expression of TLR1 and TLR2 in OmpU-treated cells at some specific time points, but we did not observe surface expression of TLR6 at any
Please cite this article in press as: Khan, J., et al., Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.06.009
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Fig. 3. OmpU mediates its signaling via TLR1 and TLR2, but not through TLR6. THP-1 cells or purified monocytes from human PBMCs were plated and treated with neutralizing antibody against TLR1, TLR2, TLR6, or the isotype control followed by treatment with OmpU. After incubation, supernatants were collected and analyzed for TNF␣ or IL-6 production. (Each experiment was done at least in triplicates; *p < 0.05, **p < 0.01, ***p < 0.001). (A) TNF␣ production and (B) IL-6 production was suppressed in OmpU-treated THP-1 cells when prior to OmpU-treatment TLR1 and TLR2 were neutralized with the corresponding neutralizing antibodies. There was no effect with TLR6 neutralizing antibody or isotype control-pretreated OmpU-activated cells. There was less production of TNF␣ (C) and IL-6 (D) when primary human monocytes were treated with TLR1 or TLR2 neutralizing antibodies followed by OmpU-treatment.
time points (Fig 2C and D). This observation indicated that TLR1 and TLR2 might be involved in the OmpU-mediated signaling.
3.2.3. Blocking of TLR1 and TLR2 affects TNF˛ and IL-6 secretion from OmpU-treated cells THP-1 cells were plated and treated with anti-TLR1, antiTLR2, anti-TLR6 neutralizing antibodies or isotype IgG monoclonal antibody, and incubated for 60 min. Further, neutralizing antibodytreated cells and control IgG-treated cells were activated with OmpU-treatment. We observed that in response to OmpU, TLR1, and TLR2-neutralized cells showed significantly reduced cellular activation compared to non-neutralized cells in terms of TNF␣ and IL-6 secretion (Fig. 3A and B). In contrast, in response to OmpU, TLR6-neutralized cells and isotype IgG-treated cells showed similar level of TNF␣ and IL-6 as the only-OmpU-treated cells (Fig. 3A and B).
To verify the above observation in different cell types, we purified monocyte population from human PBMCs, and treated the cells with TLR1, TLR2, or TLR6 neutralizing antibodies, or the isotype control. Similar to THP-1, we observed that production of both TNF␣ and IL-6 were significantly reduced in TLR1- and TLR2neutralized cells, whereas, there was no reduction observed in the cytokines production in TLR6-neutralized and isotype IgG-treated cells (Fig. 3C). These observations clearly indicated that TLR1 and TLR2 are probably involved in the OmpU-mediated signaling.
3.2.4. TLR2 knock-down by siRNA affects OmpU-mediated production of TNF˛ and IL-6 THP-1 cells were plated and transfected with siRNA against TLR2 and TLR6 or scrambled siRNA control. We observed down-regulation of cell-surface TLR2 and TLR6 in specific siRNA-
Please cite this article in press as: Khan, J., et al., Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.06.009
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Fig. 4. siRNA-mediated TLR2 knock-down in THP-1 cells showed decreased production of TNF␣ in response to OmpU treatment. (A) There is down regulation of TLR2 and TLR6 surface expression in THP-1 cells when transfected with specific siRNA (shaded histogram for mock transfected cells, dashed histogram for the scrambled siRNA transfected cells, line histogram for specific siRNA transfected cells) (Representative plot of 3 individual experiments). (B) In response to OmpU treatment, there is significantly decreased production of TNF␣ upon TLR2 knock-down in THP1 cells as compared to the TLR6 knock-down in the THP-1 cells. (Representative of 3 individual experiments; ±SEM calculated from 3 technical triplicates; ***p < 0.001).
transfected cells but not in cells transfected with the scrambled siRNA (Fig. 4A). Further TLR2-siRNA, TLR6-siRNA or scrambled siRNAtransfected cells were activated with OmpU and checked for TNF␣ and IL-6 production. We observed that upon TLR2-knockdown there was less production of both the cytokines as compared to those in the scrambled siRNA-transfected cells, whereas, TLR6 knock-down allowed production of comparable extent of cytokines as observed in the scrambled siRNA-transfected cells (Fig. 4B). These observations clearly indicate that the OmpU-mediated signaling involves TLR2 but not TLR2/TLR6-heterodimerization.
3.2.5. OmpU-mediated signaling involve TLR1 and TLR2 heterodimerization RAW 264.7 and THP-1 cells were plated at 1.5 × 106 cells/ml density, and were treated with OmpU and incubated for 1 h. Whole cell lysates were prepared, and immunoprecipitation were done with anti-TLR2 antibody or anti-TLR1 antibody. We observed that in both the cases, TLR2 co-immunoprecipitated with TLR1, whereas, TLR6 did not (Fig. 5A–D). This further confirmed that the OmpU-mediated signaling involved formation of TLR2 heterodimer with TLR1, but not with TLR6. We also observed that OmpU directly interacted with TLR2/TLR1 heterodimer as it co-immunoprecipitated with both TLR1 and TLR2 upon pull down (Fig. 5A–D).
Fig. 5. OmpU directly interacts with TLR1 and TLR2, and MyD88 gets recruited in the receptor complex upon OmpU treatment. RAW264.7 (A and B) and THP-1 (C and D) cells were treated with OmpU, and were subjected to co-immunoprecipitation with anti-TLR2 (A and C) or anti-TLR1 (B and D) antibodies. In both the cases, TLR1 and TLR2 are found to be associated with each other, and MyD88 is recruited to the complex. OmpU is also found to be co-immunoprecipitated with the TLR1/TLR2 receptor complex. TLR6 is not found to be co-immunoprecipitated with TLR2, thus it does not appears to be involved in the OmpU-mediated activation pathway. (All the blots are representative of at least 3 individual experiments).
3.3. OmpU induces recruitment of MyD88 and IRAK1 downstream to TLR- activation MyD88 (myeloid differentiation primary response 88), and IRAK1 (Interleukin 1 receptor-associated kinase 1) are the important signal transducers in the TLR signaling pathways (Janssens and Beyaert, 2002; Kawai and Akira, 2007; West et al., 2006). Upon TLR activation MyD88 gets recruited at the cytoplasmic-TIR domain of activated TLRs. MyD88 as one of the crucial adaptor protein of the TLR-signaling complex recruits IRAK1, a serine/threonine-protein kinase. Once associated with MyD88, IRAK1 undergoes phosphorylation by IRAK4. This process is crucial for further down-stream signaling involving TRAF6 and NFB activation that leads to proinflammatory cytokine production. 3.3.1. MyD88 is involved in OmpU mediated signaling To check whether upon activation with OmpU, MyD88 is recruited with TLR1/2 complex, whole cell lysates were prepared from OmpU-treated RAW 264.7 and THP-1 cells followed by immunoprecipitation with anti-TLR2 or anti-TLR1 antibody. We observed
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Fig. 6. IRAK-1 is involved in OmpU-mediated signaling. RAW 264.7(A) and THP-1 (B) cells were treated with different doses of IRAK-1 inhibitor and then stimulated with OmpU. We observed suppression of TNF␣ and IL-6 responses in the inhibitor-treated cells. (Data are represented as an average of 3 individual experiments; p value was calculated on SEM values; *p < 0.05, **p < 0.01, ***p < 0.001). (C) RAW 264.7 and THP-1 cells were treated with OmpU and immunoprecipitated with anti-MyD88 antibody. We observed that IRAK-1 is associated with MyD88 in the OmpU-treated cells. (Representative data of 3 independent experiments).
that MyD88 were co-immunoprecipitated along with TLR2 and TLR1 (Fig. 5A–D), thus clearly indicating its involvement in OmpUmediated signaling.
3.3.2. Blocking of IRAK1-phosphorylation inhibits TNF˛ and IL-6 production To probe whether IRAK1 is involved in OmpU-mediated signaling, RAW 264.7 and THP-1 cells were plated and treated with different doses of IRAK1/4 inhibitor for 1 h. Following incubation, inhibitor-treated and untreated cells were activated with OmpU. We observed that there was less production of cytokines with increasing doses of IRAK1/4 inhibitor compared to cells not pretreated with the inhibitor (Fig. 6A–B). This result indicated the involvement of IRAK1 in the OmpU-mediated signaling.
3.3.3. IRAK1 is recruited downstream to MyD88 in OmpU-treated cells As we know that MyD88 association as adaptor molecule is crucial for IRAK1 association with the receptor complex. To check whether IRAK1 is associated with MyD88 in OmpUmediated signaling, we prepared whole cell lysate from OmpU or buffer-treated RAW 264.7 and THP-1 cells and immunoprecipitated with anti-MyD88 antibody. We observed that IRAK1 is co-immunoprecipitated with MyD88 proving that IRAK1 was associated with MyD88 in OmpU-treated cells (Fig. 6C). 3.4. NFB activation is involved in OmpU-mediated signaling NFB (nuclear factor kappa-light-chain-enhancer of activated B cells), a nuclear transcription factor, is important in regulating
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Fig. 7. OmpU-mediated signaling involves NFB activation. (A) RAW 264.7 and (B) THP-1 cells were treated with NFB inhibitor PDTC, and subsequently the cells were treated with OmpU and assessed for TNF␣ and IL-6 production. There is suppression of TNF␣ and IL-6 production in NFB-treated cells. (±SEM was calculated on 3 individual experiments; *p < 0.05, **p < 0.01, ***p < 0.001). (C) RAW 264.7 and (D) THP-1 cells were treated with OmpU, and cytoplasmic fractions were analyzed for IB and NFB and nuclear lysates were analyzed for NFB by immunoblotting. In cytoplasmic fraction, there is reduction of both IB and NFB signal with time, and corresponding increase in NFB is observed in the nuclear fraction. (Representative data of 3 individual experiments).
expression of genes critical for inflammation (Hayden et al., 2006; Li and Verma, 2002). In its inactive form, NFB remains in the cytoplasm forming an inhibitory complex with IB family of inhibitor proteins. Upon activation of TLRs, subsequent downstream signaling activates kinases such as, IKKs that phosphorylates IB leading to its degradation. Release of IB exposes the nuclear localization signals (NLS) of NF-B subunits leading to their translocation to the nucleus. In the nucleus, NF-B binds to the upstream of various genes with a consensus sequence activating their transcription. 3.4.1. PDTC, an NFB inhibitor reduces TNF˛ and IL-6 production To check whether activation of NFB is critical in OmpUmediated signaling, THP-1 and RAW 264.7 cells were plated and treated with PDTC, an inhibitor for NFB, followed by OmpU or buffer treatment. After incubation, supernatants were collected and quantitated for TNF␣ and IL-6 by ELISA. We observed that TNF␣ and IL-6 productions were significantly inhibited upon the use of the NFB inhibitor, suggesting involvement of NFB in OmpUmediated signaling (Fig 7A and B). 3.4.2. NFB translocates to nucleus in response to OmpU treatment NFB activation leads to translocation of NFB from cytosol to the nucleus. To confirm the involvement of NFB in OmpUmediated signaling, THP-1 and RAW 264.7 cells were plated and treated with OmpU and incubated for 30 min or 1 h. Following incubation cells were harvested and cytoplasmic fractions and nuclear fractions were subjected to immunoblot analysis. We observed a decrease in IB and NFB signal over the time from the cyto-
plasm, and a corresponding increase in NFB in the nuclear lysate of OmpU-treated cells (Fig. 7C and D). Hence, the above result indicates that OmpU-mediated signaling involves NFB activation and its translocation to nucleus. 4. Discussion Macrophage heterogeneity plays a crucial role in determining the innate immune as well as adaptive immune responses. Classically activated macrophages are known as M1-polarized macrophages and are able to secrete cytokines and chemokines that induce pro-inflammatory responses. Alternatively, activated macrophages or M2-polarized macrophages have the ability to suppress the inflammatory responses. Sometimes macrophages may attain a state that is intermediate between M1 or M2, which is called M1/M2 state. In this M1/M2 state macrophage has the ability to produce pro-inflammatory mediators, as well as mediators to suppress the pro-inflammatory responses. Previous work by Sakharwade et al. (2013), (2015) from our lab showed that V. cholerae OmpU has the ability to induce pro-inflammatory responses, in terms of production of NO, TNF␣, and IL-6. Sakharwade et al. (2013), (2015) also reported that V. cholerae OmpU does not induce suppressor cytokines such as, IL-10 and TGF-production in cell lines. Hence, it seems quite possible that V. cholerae OmpU is able to induce M1-polarization. But in that very study Sakharwade et al. (2013), (2015) showed quite convincingly that V. cholerae OmpU possesses the ability to suppress the production of NO, TNF␣, IL6, and IL-12 from cells activated with LPS. This raised a doubt in our mind as to whether OmpU really has the ability to polarize macrophage/monocyte towards M1-polarization state.
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In this background, the aim of present study was to explore whether V. cholerae OmpU could really induce M1-polarization, or it could trigger M2-polarization, or in other words, whether V. cholerae OmpU could induce an M1/M2 polarization state. In this direction, we treated RAW 264.7, murine macrophage cell line and THP-1 human monocytic cell line with recombinant OmpU, and checked for a set of M1-associated and M2-associated markers. We observed the increased of expression of all the M1-associated genes at some time point or the others, whereas, we did not observe expression of any M2-associated genes. Above observations and previous work by Sakharwade et al. (2013), (2015) showing production of NO, TNFa, IL-6 and no production of TGF and IL-10, together suggested that V. cholerae OmpU induces M1-polarization. Further, we wanted to understand the underlying mechanism of V. cholerae OmpU-mediated M1-polarization. PAMPs are recognized by pattern recognition receptors such as TLRs, activating the downstream signaling culminating mostly in inflammatory responses. TLR-mediated response may be MyD88-dependent or independent (Kawai and Akira, 2007; West et al., 2006). Porins from several gram-negative bacteria were investigated in earlier studies for their roles as PAMPs, and their ability to induce proinflammatory responses after recognition by TLRs. In the present study, neutralization of surface TLRs and their knock-down by siRNA in THP-1 cells, suggested that V. cholerae OmpU, presumably as a PAMP, is recognized by TLR2/TLR1 hetero-dimer receptor complex. Co-immunoprecipitation data also suggested involvement of TLR2/TLR1 dimer, and also OmpU, as a ligand, directly interacting to the receptor complex. In addition, we observed that OmpUmediated signaling via TLR involved MyD88 and IRAK-1. Generally, signaling via TLR ultimately leads to the activation of the transcription factors that in turn activate genes important for cellular functions. One such important transcription factor is NFkB. In inactive form, NFB remains bound with the inhibitor IB. This IB-NFB complex stays in the cytoplasm. Once activated, NFB translocates to the nucleus and modulate the transcription of the genes with effector functions. There are 5 members in the NFB sub-family, and they generally remain as homo- or heterodimer, and as such bind to the DNA after translocating into the nucleus following activation (Hayden et al., 2006a,b; Tak and Firestein, 2001). These 5 members are P65, P52, P50, cRel and RelB. Among these 5 members, various combinations of heteroor homo-dimers determine the fate of the cell function. P65 forms hetero-dimer with P50 or P52, and upregulates the transcription of pro-inflammatory genes, whereas, P52/P50 homodimer or P50/P50 homodimer upregulate transcription of suppressor cytokine genes. In our study, we observed that in response to OmpU-treatment p65 was translocated from cytoplasm to nucleus indicating its involvement in the OmpU-mediated M1-polarization. Altogether, the present study reveals that V. cholerae OmpU is able to induce M1-polarization of macrophages and monocytes by activating TLR2/TLR1. Activated TLR2/1 heterodimer recruits MyD88 that in turn recruits IRAK1 to the receptor complex. This signaling ultimately leads to the degradation of IB, and activation and translocation of NFB to the nucleus.
Conflict of interest There is no conflict of interest.
Acknowledgement This work was supported by a grant (to AM) from the Department of Biotechnology (DBT), India (DBT Grant No. BT/PR1205/MED/29/318/2011) and funding from IISER Mohali.
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Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation Junaid Khan, Praveen K. Sharma 1 , Arunika Mukhopadhaya ∗ Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector 81, SAS Nagar, Mohali, Punjab 140306, India
a r t i c l e
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Article history: Received 15 March 2015 Received in revised form 4 May 2015 Accepted 1 June 2015 Available online xxx Keywords: M1/M2 polarization Toll-like receptor OmpU Porin
a b s t r a c t Polarization of the monocytes and macrophages toward the M1 and M2 states is important for hosts’ defense against the pathogens. Moreover, it plays a crucial role to resolve the overwhelming inflammatory responses that can be harmful to the host. Polarization of macrophages/monocytes can be induced by pathogen-associated molecular patterns (PAMPs). PAMP-mediated monocyte/macrophage polarization is important during the infection, as pathogen can suppress host immune system by altering the polarization status of the macrophages/monocytes. OmpU, an outer membrane porin protein of Vibrio cholerae, possesses the ability to induce pro-inflammatory responses in monocytes/macrophages. It is also able to down-regulate the LPS-mediated activation of the monocytes/macrophages. Such observation leads us to believe that OmpU may induce a state that can be called as M1/M2-intermediate state. In the present study, we evaluated a set of M1 and M2 markers in RAW 264.7 murine macrophage cell line, and THP-1 human monocytic cell line, in response to the purified OmpU protein. We observed that OmpU, as a PAMP, induced M1-polarization by activating the Toll-like receptor (TLR) signaling pathway. OmpU induced formation of TLR1/TLR2-heterodimers. OmpU-mediated TLR-activation led to the MyD88 recruitment to the TLR1/TLR2 complex. MyD88, in turn, recruited IRAK1. Ultimately, OmpUmediated signaling led to the activation and subsequent nuclear translocation of the NFB p65 subunit. We also observed that blocking of the TLR1, TLR2, IRAK1, and NFB affected OmpU-mediated production of M1-associated pro-inflammatory cytokines such as TNF␣ and IL-6. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Modulation of the host’s immune status is very crucial in the host-pathogen relationship. Several molecules of the pathogen are responsible for host immunomodulation. Gram-negative pathogens carry an arsenal of antigens that can trigger a multitude of cellular responses (Fallon, 2009; Sakharwade et al., 2015). Some of these molecules act as the pathogen-associated molecular patterns (PAMPs) that are recognized by the pattern recognition receptors (PRRs) present on the host cells, mainly of immune origin, and thus generating innate immune responses in terms of pro-inflammatory responses (Kumar et al., 2011; Sorci and Faivre,
Abbreviations: PAMP, pathogen associated molecular pattern; PRR, pattern recognition receptors; TLR, toll-like receptor; MyD88, myeloid differentiation primary response 88; IRAK, interleukin 1-receptor associated kinase; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells. ∗ Corresponding author. Fax: +91 172 2240124. E-mail address: [email protected] (A. Mukhopadhaya). 1 Present address: Dr. Reddy’s Laboratories Ltd., Survey No 47, Bachupally, Qutbullapur, Hyderabad 500090, Telengana, India.
2009). Some of the PAMPs can dampen the host responses and help the pathogen to survive in the host system (Ashida et al., 2011). Outer membrane, an additional layer enveloping the gramnegative bacteria, consists of various components like lipopolysaccharide (LPS) and outer membrane proteins (OMPs) that play a crucial role in their interaction with the host organism (Sakharwade et al., 2015). Porins, a major group of bacterial OMPs, function as channels for solute transport. Other than channel property, porins have important roles in the pathogenesis and survival of the bacteria in the harsh environments (Nikaido, 2003). It has been shown in various studies, that porins are involved in the bacterial adherence processes and in the induction of apoptosis of the host cells (Bernardini et al., 1993; Muller et al., 1999; Negm and Pistole, 1999). Roles of porins in inducing pro-inflammatory responses, and their underlying signaling processes have been established in various reports (Biancone et al., 1997; Biswas, 2000; Galdiero et al., 2001; Gupta et al., 1999; Sakharwade et al., 2013; Sakharwade et al., 2015). Due to its immuno-modulatory property, porins are thought to be the potential vaccine candidate (Latz et al., 2004; Tabaraie et al., 1994).
http://dx.doi.org/10.1016/j.imbio.2015.06.009 0171-2985/© 2015 Elsevier GmbH. All rights reserved.
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The most widely studied PRRs, the Toll-like receptors (TLRs) are crucial for recognizing both surface-associated PAMPs and nucleic acids from invasive and non-invasive bacteria as well as viruses (Kawai and Akira, 2005; Kawai and Akira, 2007; West et al., 2006). TLRs have important roles in providing some degree of specificity to innate immune system and bridging it with the acquired immune responses (Werling and Jungi, 2003). TLR4 recognition is crucial for LPS-mediated pro-inflammatory responses. TLR1, TLR2, TLR4, TLR5, and TLR6 are the major surface-expressed PRRs. Upon activation TLR4 forms homodimer, and TLR2 forms heterodimer with either TLR1 or TLR6, whereas, TLR5 remains as monomer even on activation (West et al., 2006). TLR activation mainly leads to the activation of NFB transcription factor, which upon translocation to the nucleus up-regulates transcription of genes important for the induction of the pro-inflammatory responses (Hayden et al., 2006a,b; Tak and Firestein, 2001; West et al., 2006). Macrophages are one of the main targets for modulation of host innate and adaptive immune responses (Benoit et al., 2008; Martinez et al., 2009; Murray and Wynn, 2011; Sica and Mantovani, 2012). They can be activated through the TLR pathways to produce pro-inflammatory mediators, and activation of the Th1-subtypes of T helper cells. These types of macrophages are known as M1macrophages. M1 activation can happen with or without the presence of IFN␥ cytokines in the microenvironment. Interestingly, in the presence of IL-4/IL-13 cytokines, macrophages can get polarized towards an alternatively activated state, also known as the M2-state that is characterized by the release of cytokines and chemokines, which play major roles in immune-regulation, tissue healing and repair. In addition to cytokines, different PAMPs also possess the ability to polarize the macrophages towards either M1 or M2 state. OmpU, one of the major porins of the human pathogen Vibrio cholerae exhibits dual nature of host-immunomodulation (Khan et al., 2012; Sakharwade et al., 2013; Sakharwade et al., 2015). It can induce pro-inflammatory responses on one hand, and on the other it can down-regulate the LPS-mediated pro-inflammatory responses (Sakharwade et al., 2013). Pro-inflammatory responses such as, production of TNF␣, IL-6 and generation of nitric oxide (NO) suggested that OmpU can induce M1-polarization of macrophages, whereas, down-regulation of NO, TNF␣, IL-6 and IL-12 in OmpUpretreated LPS-activated cells suggest that OmpU may have the ability to induce polarization of the macrophages towards the M2 state (Murray and Wynn, 2011). In the present study we have investigated whether OmpU has the ability to induce M1-polarization or M2-polarization. We have also explored to delineate the cell-signaling pathways in OmpUmediated macrophage polarization. 2. Material and method 2.1. Purification of recombinant OmpU Recombinant V. cholerae OmpU protein was purified according to the protocol mentioned in our previous report (Khan et al., 2012). Briefly, V. cholerae El Tor O1 OmpU gene cloned in pET14b vector was transformed into Escherichia coli Origami B cells (EMD Millipore, Billerica, MA, USA) for OmpU expression. E. coli cells were cultured in Luria broth (HiMedia, Mumbai, India). At 0.5–0.6 OD600 , culture was induced with IPTG (HiMedia, Mumbai, India) for 3 h. The cells were centrifuged and resuspended in bacterial lysis buffer (GBiosciences, St. Louis, MO, USA) and were subjected to ultrasonication at 25 amplitude for 5 min, with 30 s pulses for lysis. Cell lysates were centrifuged at 18,500 × g at 4 ◦ C to isolate the inclusion bodies. Inclusion body was solubilized in denaturing buffer (PBS containing 8 M urea). Denatured protein in 8 M urea buffer supplemented with 20 mM imidazole was applied onto
manually packed Ni-NTA column (Qiagen GmbH, Hilden, Germany) equilibrated with the same buffer. The protein was eluted under the denaturing condition with 300 mM imidazole and 8 M urea in PBS. Eluted denatured protein was subjected to refolding. Protein was diluted into the refolding buffer [10% glycerol, 0.5% LDAO (N,N-dimethyldodecylamine N-oxide)] with constant stirring, and was incubated overnight at 4 ◦ C. The refolded protein was passed through the gel-filtration chromatography column of Sephacryl S200 (GE Healthcare, Piscataway, NJ, USA) equilibrated with a buffer containing 10 mM Tris–HCl, 10 mM NaCl and 0.5% LDAO. The protein was eluted with the same buffer, and was analyzed by SDSPAGE and Coomassie staining. Structural integrity and functionality of OmpU was confirmed by Far-UV circular dichorism (CD) spectroscopy and liposome-swelling assay respectively, as described earlier (Khan et al., 2012). 2.2. Cell lines and culture condition Mouse macrophage cell line RAW 264.7 (ATCC), and human monocytic cell line THP-1 (NCCS Pune, India) was sub-cultured and maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 100 Units/ml penicillin-100 Units/ml streptomycin (Invitrogen, Life Technologies, Carlsbad, CA, USA Life). Cells were incubated at 37 ◦ C in 5% CO2 environment. 2.3. Purification of human monocytes Human monocytes were purified from PBMCs using the Monocyte Isolation Kit II for human (MACS Miltenyi Biotec, Bergisch Gladbatch, Germany) according to the manufacturer’s protocol (Khilwani et al., 2015; Sakharwade et al., 2013). Work with human blood has been approved by the Institutional Bioethics Committee. 2.4. Semi-quantitative real time PCR for M1- and M2-associated genes and surface TLR genes expression RAW 264.7 and THP-1 cells were plated at a density of 2 × 106 cells/ml and treated with 3 g/ml OmpU or buffer (10 mM tris, 10 mM Nacl, 0.5% LDAO) as negative control, for 2 h, 4 h, 8 h, 12 h and 24 h. Cells were lysed with lysis buffer (RNAsure mini kit, Nucleopore, Genetix Biotech, New Delhi, India). Total RNA was isolated using the manufacturer protocol (RNAsure mini kit, Nucleo-pore, Genetix Biotech), and RNA was quantified by spectrophotometer (Thermo Fisher Scientific, Waltham, MA USA). Equal quantity (1 g) of total RNA of each sample was reverse transcribed using the maxima first strand cDNA synthesis kit, as directed by the manufacturer (Thermo Fisher Scientific, Waltham, MA USA). And relatively expressed quantity of each gene of interest was analysed using the Maxima Syber Green qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA USA) by Real Time PCR (Eppendorf, Hamburg, Germany). Sequences of all the primers were taken from the primer bank (Nucleic Acids Res 38:D792–D799. Doi:10.1093/nar/gkp1005 PubMed: (19,906,719). Results were expressed in terms of relative gene expression as mean-fold induction over the buffer treated controls. Normalization of relative gene expression was done by comparisons to either ˇ-actin, RPL13 or HPRT. 2.5. Flow cytometry for surface TLR expression RAW 264.7 and THP-1 cells at a density of 1 × 106 cells/ml were treated with 3 g/ml OmpU or reference buffer, and were incubated for 12 h and 24 h. Cells were harvested and washed in FACS buffer (PBS, 1% FBS), and then incubated with PE-conjugated anti-human TLR1, FITC-conjugated anti-human TLR2 or PE-conjugated antihuman TLR6 monoclonal antibody (MAb) (Biolegend, San Diego, CA, USA) for 30 min at 4 ◦ C. Cells after washing with the FACS buffer
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were acquired on a FACSCaliber (BD Bioscience, New Jersey, USA) flow cytometer.
2.10. siRNAs transfection of THP-1 cells for TLR2 and TLR6 knockdown
2.6. Neutralization of TLR with antibody to inhibit the cellular response
THP-1 cells were plated in serum-free media at a density of 7 × 104 cells for 6 h, and were then transfected with small interfering RNA (siRNA) (from GE Healthcare Dharmacon, Lafayette, CO, USA) with final concentration of 25 nM targeted against TLR2, TLR6, and also with the scrambled siRNA as the negative control. Transfection mixture was prepared by following the manufacturer’s protocol. Briefly, DharmaFECT (GE Healthcare Dharmacon) transfection reagent and siRNAs were mixed in serumfree media, and were incubated for 20 min before applying to the cells. Expression of TLRs for knockdown was analyzed by flow cytometry after 48 h post-transfection. Cells, subjected to siRNA-mediated knockdown, were treated with OmpU to study the inhibition of TNF␣ secretion upon TLR2- and TLR6-knockdown by ELISA.
THP1 cells and human monocytes isolated from PBMC were cultured at a density of 1 × 106 cells/ml, and were pre-incubated with anti-human TLR1 MAb, neutralizing antibody (Invivogen, San Diego, CA, USA), anti-human TLR2 MAb, neutralizing antibody (Biolegend), anti-human TLR6 Mab (Invivogen) or an isotype control IgG (Biolegend), one hour before the OmpU treatment (3 g/ml). Supernatants were collected for TNF␣ and IL-6 cytokines ELISA. ELISA Kits were from BD Bioscience. 2.7. Co-immunoprecipitation for the detection of MyD88 and IRAK1 recruitment downstream to the TLRs RAW 264.7 and THP-1 cells at a density of 1 × 106 cells/ml were plated and treated with 3 g/ml OmpU or control buffer for 1 h. Whole cell lysates were prepared using the lysis buffer (50 mM Tris–HCl, 137 mM NaCl, 2 mM EDTA, 1% Triton-X 100, 0.1% SDS, pH 7.6). 500 g of total cellular protein (estimated by Bradford Reagent from Sigma, St. Louis, USA) was incubated either with the antiTLR2 or anti-MyD88 antibody (Santa Cruz Biotechnology, Texas, USA) for 3 h at 4 ◦ C, and then protein A/G plus Agarose beads (Santa Cruz Biotechnology) were added and incubated for overnight at 4 ◦ C. Agarose bead–protein complex was washed three time with PBS and protein was eluted from beads by heating with 5 × SDS gel loading buffer at 100 ◦ C for 15 min. To detect the association of TLR1, TLR6, or MyD88 (Santa Cruz Biotechnology) with TLR2, and IRAK1 (Sigma–Aldrich) with MyD88 (Santa cruz Biotechnology) by immunoblotting, samples were subjected to SDS-PAGE, proteins was transferred on to the PVDF (polyvinylidene difluoride) membrane (Millipore, Billerica, Massachusetts, USA). Blots were developed by chemiluminescence-based method using Image Quant LAS 4000 (GE Healthcare life Sciences, Pittsburgh, USA). 2.8. Western blotting for NFB translocation RAW 264.7 and THP-1 cells were cultured at a density of 1 × 106 cells/ml and treated with 3 g/ml OmpU for 0 min, 30 min, 60 min. Cells were harvested, washed with PBS and subjected to buffer A (10 mM HEPES, 1.5 mM MgCl2 , 10 mM KCl, 0.5 mM DTT, 0.05% Igepal pH 7.9) and incubated on ice for 10 min and then centrifuged at 3000 rpm for 10 min. Supernatants were stored as cytoplasmic fraction and pellets were subjected to Buffer B (5 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol (v/v), 300 mM Nacl, pH 7.9), sonicated for few seconds and centrifuged at 24,000 × g to isolate the nuclear lystaes in the supernatant. Reagents for buffers preparation were obtained from Himedia, (Mumbai, India). To detect NFB (p65) translocation into the nucleus, both the fractions were subjected to SDS-PAGE, and proteins were transferred on to the PVDF membrane, and blot was developed by chemiluminescence-based method using Image Quant LAS 4000 (GE Healthcare life Sciences). 2.9. Inhibitor study to detect IRAK1 and NFB involvement RAW 264.7 and THP-1 cells were cultured in 6-well plate at the density of 1.5 × 106 cells/well. IRAK1/4 inhibitor (Sigma–Aldrich) at concentrations of 500 nM, 800 nM or 1000 nM, and NFB inhibitor Pyrrolidine dithiocarbamate (PDTC) (Sigma–Aldrich) at concentrations of 15 M or 25 M was added to the cultured cells 1 h before the treatment with OmpU (3 g/ml). Supernatants were collected for ELISA to check the inhibition of TNF␣ and IL-6 production by the corresponding inhibitors.
2.11. Statistical analysis Statistical analysis was done using Student’s two-sided t test. Data is represented as the mean ± SEM. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. OmpU induces M1-polarization of macrophages Monocytes and macrophages have the plasticity, which allows them to acquire classical (M1-polarized) and alternative form (M2-polarized) in response to various stimuli (Murray and Wynn, 2011). The groups of such stimuli, constituted by the pathogenassociated molecular patterns (PAMPs) possess the ability to determine the polarization pattern of the macrophages and monocytes (Hajishengallis and Lambris, 2011). To explore whether OmpU possesses the ability to induce M1 or M2-polarization, RAW 264.7 murine macrophage cells and THP-1 human monocytic cells were treated with OmpU and incubated for different time periods. After respective incubations, the cells were harvested and total RNA was isolated. Semi-quantitative real time PCR was done to assess the up-regulation of M1 and M2-associated gene expression. We observed that most of the M1-associated genes were upregulated at the transcriptional level, but none of the M2-associated genes showed any up-regulation in both the cell lines (Fig. 1A–D). 3.1.1. OmpU-treated cells showed transcriptional up-regulation of M1-associated genes Total RNA from OmpU-treated RAW 264.7 and THP-1 cells were analyzed for M1-associated chemokine genes that are important in causing the inflammatory responses. Chemokines, such as Mip-1␣, Mip-1 (macrophage inflammatory proteins), also called CCL3 and CCL4, respectively, are critical for granulocyte activation, whereas, CCL5 or RANTES is important in the recruitment of leucocytes such as, neutrophils (Gao et al., 1993; Guan et al., 2001; Jones et al., 2011). CXCL10 is important for the recruitment of the monocytes/macrophages, T cells, NK cells and dendritic cells (Liu et al., 2011). We observed that expression of all these M1-associated chemokine genes were upregulated in OmpU-treated RAW 264.7 cells (Fig. 1A). Similarly, total RNA from OmpU treated THP-1 cells were evaluated for the expression of mip-1˛, mip-1ˇ, and ccl5 genes, and we observed transcriptional up-regulation of all the said genes (Fig. 1C). Therefore, it is evident that OmpU is capable of inducing M1polarization.
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Fig. 1. OmpU induces M1-polarization of macrophages/monocytes. RAW 264.7 and THP-1 monocytes were plated at a density of 2 × 106 cells/ml, and were treated with OmpU or buffer. Treated cells were incubated for different time periods. After respective incubations, cells were harvested, total RNA was isolated and subjected to semi quantitative real time PCR. (±SEM values were determined from at least three biological replicates). (A) M1-associated chemokine genes such as, mip1a, mip1b, ccl5, cxcl10 showed transcriptional up-regulation in OmpU-treated RAW 264.7 cells, with maximum release at 4 h, 12 h, 12 hrs and 2 h, respectively. (B) M2-associated mediators such as arginase 1, il1ra, and ccl17 showed fold-changes around 1 or less than that, indicating no transcriptional up-regulation of the M2associated genes in OmpU-treated RAW 264.7 cells. (C) OmpU-treated THP-1 cells induced transcriptional up-regulation of M1-associated genes such as, mip1a, mip1b, ccl5 with maximum up-regulation at 12 h, 8 h, and 8 h, respectively. (D) M2-associated genes such as, arginase 1, ccl22 did not show any significant up-regulation in OmpU-treated THP-1 cells.
3.1.2. M2-associated genes are not expressed in OmpU-treated cells Total RNA from OmpU-treated RAW 264.7 and THP-1 cells were analyzed for M2-associated genes such as arginase 1, il1ra, ccl17 and ccl22. Arginase 1 is important for arginine metabolism to proline and polyamine, hence induce tissue repair and growth promotion (Martinez et al., 2009). It inhibits the activity of iNOS, and eventually M1-associated NO production from arginine. The interleukin-1 receptor antagonist (IL-1Ra) is a member of the IL1 family that binds to IL-1 receptors but does not induce any intracellular response (Arend et al., 1998; Taylor et al., 2005). In M2-polarised cells generally the surface expression of IL-1ra is high. CCL17 and CCL22 are Th2-recruiting chemokines and marker for
M2-associated macrophages (Mariani et al., 2004). We observed that OmpU-treated cells did not show transcriptional upregulation of any of these M2-associated genes (Fig. 1B and D).
3.2. OmpU mediates its signaling via TLR1/TLR2 pathway Pattern recognition receptors (PRRs) are crucial to recognize PAMPs that in turn decide the polarization patterns. TLRs are crucial to recognize the surface-associated PAMPs of non-invasive bacteria. Therefore, we wanted to check if at all TLRs are involved in the OmpU-mediated signaling, and if so, we wanted to identify the TLRs, important for the OmpU- mediated signaling.
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Fig. 2. OmpU-treated cells showed transcriptional up-regulation of TLR1, TLR2, TLR6; however, increased surface expressions were observed for TLR1 and TLR2 only. RAW 264.7 and THP-1 cells were treated with OmpU. After incubation for different time points cells were harvested and total RNA was isolated and subjected to semi-quantitative real time PCR to probe gene expression of TLR1, TLR2, TLR4, and TLR6. (±SEM values were determined from at least three biological replicates). In a different setup cells were treated with OmpU, and after incubation cells were harvested and stained with anti-TLR1, anti-TLR2 and anti-TLR6 and analyzed by flow cytometry. (Data are shown as representative plots for 3 individual experiments). (A) TLR1, TLR2 and TLR6 genes showed more than 2-fold transcriptional upregulation with maximum at around 12 h, 2 h and 12 h, respectively in OmpU-treated RAW 264.7 cells. (B) In OmpU-treated THP-1 cells, TLR1, TLR2 and TLR6 genes showed around 2-fold or more than 2-fold up-regulation at time points of 12 h, 24 h and 12 h, respectively. (C) There are increased expression of TLR1 and TLR2, but not that of TLR6 in OmpU-treated RAW 264.7 cells. (Shaded curves, buffer-treated controls; solid lines, OmpU-treated cells). (D) TLR1 and TLR2 showed increased surface expression in OmpU-treated THP-1 cells. (Shaded curves, buffer-treated controls; solid lines, OmpU-treated cells; dashed lined, OmpU-treated cells stained with isotype antibody control).
3.2.1. OmpU-treated cells showed transcriptional up-regulation of tlr1, tlr2, and tlr6 genes Firstly, we checked the transcriptional up-regulation of the important cell surface TLRs upon the OmpU treatment. RAW 264.7 and THP-1 cells were treated with OmpU or buffer and incubated for different time points up to 24 h. After the respective incubations, cells were harvested and total RNA was extracted and analyzed by semi-quantitative real time PCR. In case of RAW 264.7 cells, we observed, over 2-fold up-regulation in the transcriptional level of tlr1 and tlr2 genes (Fig. 2A). Additionally, we observed that in case of tlr6 there was around 2-fold up-regulation, whereas, in case of tlr4, upregulation was less than 2 fold (Fig. 2A). Same set of TLR genes were analyzed in THP-1 cell lines. Similar to RAW 264.7, in THP-
1 as well, we observed transcriptional up-regulation of tlr1, tlr2, and tlr6 in response to the OmpU treatment, but not that of tlr4 (Fig. 2B). This observation at the mRNA level indicated that OmpU may induce hetero-dimerization of TLR1/TLR2 or TLR2/TLR6.
3.2.2. OmpU induces surface expression TLR1 and TLR2 but not that of TLR6 RAW 264.7 and THP-1 cells were incubated with OmpU or buffer for different time points and analyzed for surface expression of TLRs by flow cytometry. We observed an increase in surface expression of TLR1 and TLR2 in OmpU-treated cells at some specific time points, but we did not observe surface expression of TLR6 at any
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Fig. 3. OmpU mediates its signaling via TLR1 and TLR2, but not through TLR6. THP-1 cells or purified monocytes from human PBMCs were plated and treated with neutralizing antibody against TLR1, TLR2, TLR6, or the isotype control followed by treatment with OmpU. After incubation, supernatants were collected and analyzed for TNF␣ or IL-6 production. (Each experiment was done at least in triplicates; *p < 0.05, **p < 0.01, ***p < 0.001). (A) TNF␣ production and (B) IL-6 production was suppressed in OmpU-treated THP-1 cells when prior to OmpU-treatment TLR1 and TLR2 were neutralized with the corresponding neutralizing antibodies. There was no effect with TLR6 neutralizing antibody or isotype control-pretreated OmpU-activated cells. There was less production of TNF␣ (C) and IL-6 (D) when primary human monocytes were treated with TLR1 or TLR2 neutralizing antibodies followed by OmpU-treatment.
time points (Fig 2C and D). This observation indicated that TLR1 and TLR2 might be involved in the OmpU-mediated signaling.
3.2.3. Blocking of TLR1 and TLR2 affects TNF˛ and IL-6 secretion from OmpU-treated cells THP-1 cells were plated and treated with anti-TLR1, antiTLR2, anti-TLR6 neutralizing antibodies or isotype IgG monoclonal antibody, and incubated for 60 min. Further, neutralizing antibodytreated cells and control IgG-treated cells were activated with OmpU-treatment. We observed that in response to OmpU, TLR1, and TLR2-neutralized cells showed significantly reduced cellular activation compared to non-neutralized cells in terms of TNF␣ and IL-6 secretion (Fig. 3A and B). In contrast, in response to OmpU, TLR6-neutralized cells and isotype IgG-treated cells showed similar level of TNF␣ and IL-6 as the only-OmpU-treated cells (Fig. 3A and B).
To verify the above observation in different cell types, we purified monocyte population from human PBMCs, and treated the cells with TLR1, TLR2, or TLR6 neutralizing antibodies, or the isotype control. Similar to THP-1, we observed that production of both TNF␣ and IL-6 were significantly reduced in TLR1- and TLR2neutralized cells, whereas, there was no reduction observed in the cytokines production in TLR6-neutralized and isotype IgG-treated cells (Fig. 3C). These observations clearly indicated that TLR1 and TLR2 are probably involved in the OmpU-mediated signaling.
3.2.4. TLR2 knock-down by siRNA affects OmpU-mediated production of TNF˛ and IL-6 THP-1 cells were plated and transfected with siRNA against TLR2 and TLR6 or scrambled siRNA control. We observed down-regulation of cell-surface TLR2 and TLR6 in specific siRNA-
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Fig. 4. siRNA-mediated TLR2 knock-down in THP-1 cells showed decreased production of TNF␣ in response to OmpU treatment. (A) There is down regulation of TLR2 and TLR6 surface expression in THP-1 cells when transfected with specific siRNA (shaded histogram for mock transfected cells, dashed histogram for the scrambled siRNA transfected cells, line histogram for specific siRNA transfected cells) (Representative plot of 3 individual experiments). (B) In response to OmpU treatment, there is significantly decreased production of TNF␣ upon TLR2 knock-down in THP1 cells as compared to the TLR6 knock-down in the THP-1 cells. (Representative of 3 individual experiments; ±SEM calculated from 3 technical triplicates; ***p < 0.001).
transfected cells but not in cells transfected with the scrambled siRNA (Fig. 4A). Further TLR2-siRNA, TLR6-siRNA or scrambled siRNAtransfected cells were activated with OmpU and checked for TNF␣ and IL-6 production. We observed that upon TLR2-knockdown there was less production of both the cytokines as compared to those in the scrambled siRNA-transfected cells, whereas, TLR6 knock-down allowed production of comparable extent of cytokines as observed in the scrambled siRNA-transfected cells (Fig. 4B). These observations clearly indicate that the OmpU-mediated signaling involves TLR2 but not TLR2/TLR6-heterodimerization.
3.2.5. OmpU-mediated signaling involve TLR1 and TLR2 heterodimerization RAW 264.7 and THP-1 cells were plated at 1.5 × 106 cells/ml density, and were treated with OmpU and incubated for 1 h. Whole cell lysates were prepared, and immunoprecipitation were done with anti-TLR2 antibody or anti-TLR1 antibody. We observed that in both the cases, TLR2 co-immunoprecipitated with TLR1, whereas, TLR6 did not (Fig. 5A–D). This further confirmed that the OmpU-mediated signaling involved formation of TLR2 heterodimer with TLR1, but not with TLR6. We also observed that OmpU directly interacted with TLR2/TLR1 heterodimer as it co-immunoprecipitated with both TLR1 and TLR2 upon pull down (Fig. 5A–D).
Fig. 5. OmpU directly interacts with TLR1 and TLR2, and MyD88 gets recruited in the receptor complex upon OmpU treatment. RAW264.7 (A and B) and THP-1 (C and D) cells were treated with OmpU, and were subjected to co-immunoprecipitation with anti-TLR2 (A and C) or anti-TLR1 (B and D) antibodies. In both the cases, TLR1 and TLR2 are found to be associated with each other, and MyD88 is recruited to the complex. OmpU is also found to be co-immunoprecipitated with the TLR1/TLR2 receptor complex. TLR6 is not found to be co-immunoprecipitated with TLR2, thus it does not appears to be involved in the OmpU-mediated activation pathway. (All the blots are representative of at least 3 individual experiments).
3.3. OmpU induces recruitment of MyD88 and IRAK1 downstream to TLR- activation MyD88 (myeloid differentiation primary response 88), and IRAK1 (Interleukin 1 receptor-associated kinase 1) are the important signal transducers in the TLR signaling pathways (Janssens and Beyaert, 2002; Kawai and Akira, 2007; West et al., 2006). Upon TLR activation MyD88 gets recruited at the cytoplasmic-TIR domain of activated TLRs. MyD88 as one of the crucial adaptor protein of the TLR-signaling complex recruits IRAK1, a serine/threonine-protein kinase. Once associated with MyD88, IRAK1 undergoes phosphorylation by IRAK4. This process is crucial for further down-stream signaling involving TRAF6 and NFB activation that leads to proinflammatory cytokine production. 3.3.1. MyD88 is involved in OmpU mediated signaling To check whether upon activation with OmpU, MyD88 is recruited with TLR1/2 complex, whole cell lysates were prepared from OmpU-treated RAW 264.7 and THP-1 cells followed by immunoprecipitation with anti-TLR2 or anti-TLR1 antibody. We observed
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Fig. 6. IRAK-1 is involved in OmpU-mediated signaling. RAW 264.7(A) and THP-1 (B) cells were treated with different doses of IRAK-1 inhibitor and then stimulated with OmpU. We observed suppression of TNF␣ and IL-6 responses in the inhibitor-treated cells. (Data are represented as an average of 3 individual experiments; p value was calculated on SEM values; *p < 0.05, **p < 0.01, ***p < 0.001). (C) RAW 264.7 and THP-1 cells were treated with OmpU and immunoprecipitated with anti-MyD88 antibody. We observed that IRAK-1 is associated with MyD88 in the OmpU-treated cells. (Representative data of 3 independent experiments).
that MyD88 were co-immunoprecipitated along with TLR2 and TLR1 (Fig. 5A–D), thus clearly indicating its involvement in OmpUmediated signaling.
3.3.2. Blocking of IRAK1-phosphorylation inhibits TNF˛ and IL-6 production To probe whether IRAK1 is involved in OmpU-mediated signaling, RAW 264.7 and THP-1 cells were plated and treated with different doses of IRAK1/4 inhibitor for 1 h. Following incubation, inhibitor-treated and untreated cells were activated with OmpU. We observed that there was less production of cytokines with increasing doses of IRAK1/4 inhibitor compared to cells not pretreated with the inhibitor (Fig. 6A–B). This result indicated the involvement of IRAK1 in the OmpU-mediated signaling.
3.3.3. IRAK1 is recruited downstream to MyD88 in OmpU-treated cells As we know that MyD88 association as adaptor molecule is crucial for IRAK1 association with the receptor complex. To check whether IRAK1 is associated with MyD88 in OmpUmediated signaling, we prepared whole cell lysate from OmpU or buffer-treated RAW 264.7 and THP-1 cells and immunoprecipitated with anti-MyD88 antibody. We observed that IRAK1 is co-immunoprecipitated with MyD88 proving that IRAK1 was associated with MyD88 in OmpU-treated cells (Fig. 6C). 3.4. NFB activation is involved in OmpU-mediated signaling NFB (nuclear factor kappa-light-chain-enhancer of activated B cells), a nuclear transcription factor, is important in regulating
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Fig. 7. OmpU-mediated signaling involves NFB activation. (A) RAW 264.7 and (B) THP-1 cells were treated with NFB inhibitor PDTC, and subsequently the cells were treated with OmpU and assessed for TNF␣ and IL-6 production. There is suppression of TNF␣ and IL-6 production in NFB-treated cells. (±SEM was calculated on 3 individual experiments; *p < 0.05, **p < 0.01, ***p < 0.001). (C) RAW 264.7 and (D) THP-1 cells were treated with OmpU, and cytoplasmic fractions were analyzed for IB and NFB and nuclear lysates were analyzed for NFB by immunoblotting. In cytoplasmic fraction, there is reduction of both IB and NFB signal with time, and corresponding increase in NFB is observed in the nuclear fraction. (Representative data of 3 individual experiments).
expression of genes critical for inflammation (Hayden et al., 2006; Li and Verma, 2002). In its inactive form, NFB remains in the cytoplasm forming an inhibitory complex with IB family of inhibitor proteins. Upon activation of TLRs, subsequent downstream signaling activates kinases such as, IKKs that phosphorylates IB leading to its degradation. Release of IB exposes the nuclear localization signals (NLS) of NF-B subunits leading to their translocation to the nucleus. In the nucleus, NF-B binds to the upstream of various genes with a consensus sequence activating their transcription. 3.4.1. PDTC, an NFB inhibitor reduces TNF˛ and IL-6 production To check whether activation of NFB is critical in OmpUmediated signaling, THP-1 and RAW 264.7 cells were plated and treated with PDTC, an inhibitor for NFB, followed by OmpU or buffer treatment. After incubation, supernatants were collected and quantitated for TNF␣ and IL-6 by ELISA. We observed that TNF␣ and IL-6 productions were significantly inhibited upon the use of the NFB inhibitor, suggesting involvement of NFB in OmpUmediated signaling (Fig 7A and B). 3.4.2. NFB translocates to nucleus in response to OmpU treatment NFB activation leads to translocation of NFB from cytosol to the nucleus. To confirm the involvement of NFB in OmpUmediated signaling, THP-1 and RAW 264.7 cells were plated and treated with OmpU and incubated for 30 min or 1 h. Following incubation cells were harvested and cytoplasmic fractions and nuclear fractions were subjected to immunoblot analysis. We observed a decrease in IB and NFB signal over the time from the cyto-
plasm, and a corresponding increase in NFB in the nuclear lysate of OmpU-treated cells (Fig. 7C and D). Hence, the above result indicates that OmpU-mediated signaling involves NFB activation and its translocation to nucleus. 4. Discussion Macrophage heterogeneity plays a crucial role in determining the innate immune as well as adaptive immune responses. Classically activated macrophages are known as M1-polarized macrophages and are able to secrete cytokines and chemokines that induce pro-inflammatory responses. Alternatively, activated macrophages or M2-polarized macrophages have the ability to suppress the inflammatory responses. Sometimes macrophages may attain a state that is intermediate between M1 or M2, which is called M1/M2 state. In this M1/M2 state macrophage has the ability to produce pro-inflammatory mediators, as well as mediators to suppress the pro-inflammatory responses. Previous work by Sakharwade et al. (2013), (2015) from our lab showed that V. cholerae OmpU has the ability to induce pro-inflammatory responses, in terms of production of NO, TNF␣, and IL-6. Sakharwade et al. (2013), (2015) also reported that V. cholerae OmpU does not induce suppressor cytokines such as, IL-10 and TGF-production in cell lines. Hence, it seems quite possible that V. cholerae OmpU is able to induce M1-polarization. But in that very study Sakharwade et al. (2013), (2015) showed quite convincingly that V. cholerae OmpU possesses the ability to suppress the production of NO, TNF␣, IL6, and IL-12 from cells activated with LPS. This raised a doubt in our mind as to whether OmpU really has the ability to polarize macrophage/monocyte towards M1-polarization state.
Please cite this article in press as: Khan, J., et al., Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.06.009
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In this background, the aim of present study was to explore whether V. cholerae OmpU could really induce M1-polarization, or it could trigger M2-polarization, or in other words, whether V. cholerae OmpU could induce an M1/M2 polarization state. In this direction, we treated RAW 264.7, murine macrophage cell line and THP-1 human monocytic cell line with recombinant OmpU, and checked for a set of M1-associated and M2-associated markers. We observed the increased of expression of all the M1-associated genes at some time point or the others, whereas, we did not observe expression of any M2-associated genes. Above observations and previous work by Sakharwade et al. (2013), (2015) showing production of NO, TNFa, IL-6 and no production of TGF and IL-10, together suggested that V. cholerae OmpU induces M1-polarization. Further, we wanted to understand the underlying mechanism of V. cholerae OmpU-mediated M1-polarization. PAMPs are recognized by pattern recognition receptors such as TLRs, activating the downstream signaling culminating mostly in inflammatory responses. TLR-mediated response may be MyD88-dependent or independent (Kawai and Akira, 2007; West et al., 2006). Porins from several gram-negative bacteria were investigated in earlier studies for their roles as PAMPs, and their ability to induce proinflammatory responses after recognition by TLRs. In the present study, neutralization of surface TLRs and their knock-down by siRNA in THP-1 cells, suggested that V. cholerae OmpU, presumably as a PAMP, is recognized by TLR2/TLR1 hetero-dimer receptor complex. Co-immunoprecipitation data also suggested involvement of TLR2/TLR1 dimer, and also OmpU, as a ligand, directly interacting to the receptor complex. In addition, we observed that OmpUmediated signaling via TLR involved MyD88 and IRAK-1. Generally, signaling via TLR ultimately leads to the activation of the transcription factors that in turn activate genes important for cellular functions. One such important transcription factor is NFkB. In inactive form, NFB remains bound with the inhibitor IB. This IB-NFB complex stays in the cytoplasm. Once activated, NFB translocates to the nucleus and modulate the transcription of the genes with effector functions. There are 5 members in the NFB sub-family, and they generally remain as homo- or heterodimer, and as such bind to the DNA after translocating into the nucleus following activation (Hayden et al., 2006a,b; Tak and Firestein, 2001). These 5 members are P65, P52, P50, cRel and RelB. Among these 5 members, various combinations of heteroor homo-dimers determine the fate of the cell function. P65 forms hetero-dimer with P50 or P52, and upregulates the transcription of pro-inflammatory genes, whereas, P52/P50 homodimer or P50/P50 homodimer upregulate transcription of suppressor cytokine genes. In our study, we observed that in response to OmpU-treatment p65 was translocated from cytoplasm to nucleus indicating its involvement in the OmpU-mediated M1-polarization. Altogether, the present study reveals that V. cholerae OmpU is able to induce M1-polarization of macrophages and monocytes by activating TLR2/TLR1. Activated TLR2/1 heterodimer recruits MyD88 that in turn recruits IRAK1 to the receptor complex. This signaling ultimately leads to the degradation of IB, and activation and translocation of NFB to the nucleus.
Conflict of interest There is no conflict of interest.
Acknowledgement This work was supported by a grant (to AM) from the Department of Biotechnology (DBT), India (DBT Grant No. BT/PR1205/MED/29/318/2011) and funding from IISER Mohali.
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Please cite this article in press as: Khan, J., et al., Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.06.009