Ficolin-3 activity towards the opportunistic pathogen, Hafnia alvei

Immunobiology 220 (2015) 117–123

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Ficolin-3 activity towards the opportunistic pathogen, Hafnia alvei Mateusz Michalski a,b , Anna St. Swierzko a,∗ , Jolanta Lukasiewicz c , Aleksandra Man-Kupisinska c , Iwona Karwaciak c , Patrycja Przygodzka a , Maciej Cedzynski a a b c

Institute of Medical Biology, Polish Academy of Sciences, Lodowa 106, 93-232 Lodz, Poland Institute of Microbiology, Biotechnology and Immunology, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland

a r t i c l e

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Article history: Received 5 June 2014 Received in revised form 11 August 2014 Accepted 11 August 2014 Available online 19 August 2014 Keywords: Complement Ficolin-3 H-ficolin Hafnia Innate immunity Lipopolysaccharide (LPS) Phagocytosis

a b s t r a c t Ficolin-3 (also called H-ficolin or Hakata antigen) is a complement-activating pattern recognition molecule, possessing a fibrinogen-like domain involved in carbohydrate binding. Amongst human ficolins, Ficolin-3 has the highest concentration in serum and is the most potent lectin pathway activator in vitro. Evidence for its physiological function is sparse, although its deficiency has been suggested to increase susceptibility to infections. The specificity of Ficolin-3 is poorly characterized and currently few ligands are known. Here we report agglutination of Hafnia alvei, a Gram-negative enteric commensal bacterium and opportunist pathogen, in the presence of recombinant Ficolin-3 and calcium. Ficolin-3 also augmented phagocytosis of H. alvei by macrophages and displayed bactericidal activity. Additionally, Ficolin-3 inhibited host cells’ response to TLR4/MD-2/CD14-LPS dependent NF-␬B activation. This is the first demonstration of protective activity of Ficolin-3 against a human bacterial pathogen. Although human Ficolin-3 does not recognise and bind to common pathogenic bacteria, it could be an important component of innate immunity providing protection, for example, from commensal flora that can cause extraintestinal, opportunistic infections. © 2014 Elsevier GmbH. All rights reserved.

Introduction Ficolin-3 (H-ficolin, Hakata antigen, thermolabile ␤-2 macroglobulin) was first detected as an autoantigen precipitated by antibodies in sera of some systemic lupus erythematosus (SLE) patients (Yae et al. 1991). It is an oligomer (tetra- to octamer) of basic subunits, each consisting of three identical 34 kDa polypeptide chains (Sugimoto et al. 1998; Hummelshoj et al. 2008). Like other ficolins, Ficolin-3 possesses an N-terminal cysteine-rich collagen-like domain and a C-terminal fibrinogen-like domain (Sugimoto et al. 1998). The collagen-like region forms complexes with Mannose-binding lectin (MBL)-associated serine proteases (MASP), which, after interaction of the fibrinogen-like domain with target structures, become activated and initiate the lectin pathway of complement activation (Matsushita et al. 2002). Although the related human Ficolin-1 and Ficolin-2 are >80% mutually identical

Abbreviations: MASP, Mannose-binding lectin-associated serine protease; MBL, mannose-binding lectin. ∗ Corresponding author. Tel.: +48 42 2723607; fax: +48 42 2723630. E-mail address: [email protected] (A. St. Swierzko). http://dx.doi.org/10.1016/j.imbio.2014.08.012 0171-2985/© 2014 Elsevier GmbH. All rights reserved.

in sequence, Ficolin-3 has only 48% sequence identity to the others (Sugimoto et al. 1998). In contrast to Ficolins-1 and -2 (and the collectins), Ficolin-3 is resistant to bacterial collagenases, possibly supporting its potential importance at sites of infection (Hummelshoj et al. 2008). Ficolin-3 was shown to be synthesized by hepatocytes, bile duct epithelial cells, ciliated bronchial- and type II alveolar epithelial cells. The level of its synthesis in lung exceeds that in the liver (Akaiwa et al. 1999; Hummelshoj et al. 2008). A frame-shift mutation in exon 5 (+1637delC) of the FCN3 gene causes Ficolin-3 deficiency and was suggested to increase susceptibility to severe recurrent infections and neonatal invasive enterocolitis (MuntheFog et al. 2009; Schlapbach et al. 2011). Although Ficolin-3 has been implicated in innate immunity (Matsushita et al. 2002), it does not seem to recognize common bacterial pathogens (Krarup et al. 2005). It binds avidly to an extracellular polysaccharide (EPS) of one strain of Aerococcus viridans (Tsujimura et al. 2001), isolated from contaminated human plasma. It also binds certain lipopolysaccharides (LPS) including those from Salmonella typhimurium, S. minnesota and Escherichia coli O111. Beside complement activation upon binding to isolated A. viridans EPS, bactericidal activity of Ficolin-3 was demonstrated

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(Tsujimura et al. 2002). Previously, we reported the interaction of Ficolin-3 with the O-specific polysaccharides of Hafnia alvei LPS (strains 23, PCM 1200, 1203, 1205) (Swierzko et al. 2012), a Gramnegative commensal of the human gut and a rare but significant opportunistic pathogen (Gunthard and Pennekamp 1996). We used this model to obtain insight into the possible biological role(s) of Ficolin-3 in innate immunity. Specifically, we investigated the ability of Ficolin-3 to agglutinate bacteria, its bactericidal effect, and its contribution to phagocytosis as well as its modifying effect on LPS-dependent cell activation. Materials and methods Normal human serum Normal human serum (NHS) was used as a source of human Ficolin-3. Serum was obtained from a healthy volunteer with normal Ficolin-3 and -2 levels (25 ␮g/ml and 3.9 ␮g/ml, respectively) and low Ficolin-1 and MBL concentrations (0.6 ␮g/ml and 0.4 ␮g/ml, respectively). Bacterial strains and culture conditions H. alvei strains PCM 1200 and PCM 1222 came from the Polish Collection of Microorganisms (Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland). Bacteria were cultured in LB medium. Some experiments (flow cytometry) were performed after inactivation of bacteria with 0.5% phenol for 30 min at 37 ◦ C. Preparation of LPS LPS was extracted from bacterial cells by the hot phenol/water method (Westphal and Jann 1965) and purified as described previously (Petersson et al. 1997). GFP-expressing bacteria To obtain the green fluorescent protein (GFP)-tagged H. alvei PCM 1200 and PCM 1222 bacteria, the pGLO plasmid was used. Since H. alvei strains are naturally resistant to ampicillin, the kanamycin resistance gene was cloned into pGLO. It was amplified by PCR, using the following primers: Forward: 5 -GCCAAGCTTTCAGAAGAACT-3 Reverse: 5 -ATGAATTCGATGATTGAACAAGA-3 . The product of amplification was cloned into the pGLO plasmid and then subjected to a double restriction digestion with EcoRI and HindIII enzymes (Thermo Scientific, USA). The ligation was used to transform the competent H. alvei cells (CaCl2 method). Positive clones were screened on solid LB medium containing 50 ␮g/ml kanamycin and 0.05% arabinose. Cell lines and culture conditions The human macrophage-like cell line, U-937, came from the collection of the Laboratory of Immunobiology of Infections (IMB PAS, Lodz, Poland). The cells were routinely grown in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), l-glutamine/penicillin/streptomycin (Gibco). HEK293/TLR4-MD2CD14 and HEK293/null cells (Invivogen, USA) were grown as described above, in DMEM additionally supplemented with blasticidin, hygromycin B and normocin (Invivogen), according to the manufacturer’s protocol.

Agglutination assay The agglutination assay was, in general, performed as described by Ariki et al. (2011) GFP-expressing bacteria were incubated with 100 ng of recombinant Ficolin-3 (R&D System, USA) in 5 mM Tris–HCl, pH 7.4, containing 0.15 M NaCl, 2 mM CaCl2 , and 2% (w/v) BSA at 37 ◦ C for 4 h and observed in a TE-200U fluorescent microscope (Nikon, Japan). In some experiments (to estimate whether agglutination of bacteria is calcium-dependent), 2 mM EDTA (Sigma–Aldrich, USA) replaced CaCl2 in the assay buffer (Ariki et al. 2011). Agglutination rate was estimated spectrophotometrically by measurement of bacterial sedimentation, as described by Ofek et al. (2001), modified. Briefly, bacteria were suspended to a density of 1.3 OD600 in Tris-buffered saline (TBS), supplemented with 10 mM CaCl2 , 5 mM MgCl2 and 0.1% BSA (pH 7.4). In some experiments, 20 mM EDTA replaced CaCl2 and MgCl2 . Next, 50 ␮l of bacterial suspension was transferred to polystyrene cuvettes (Eppendorf, Germany). After addition of 50 ␮l (2 ␮g) of recombinant Ficolin3 (R&D System) or buffer alone, mixtures were gently rotated at 37 ◦ C for 45 min. The aggregation of bacteria was then monitored (at RT) by recording the optical density at 600 nm for 2 h, using the Eppendorf BioPhotometer. Starting OD was considered as 100%. Bactericidal assay H. alvei PCM 1200 bacteria were grown in LB medium overnight and then diluted in fresh LB (1:25) and further cultured for 20 min. Thereafter, 5 ␮l 100-fold dilutions of bacteria were added to serially diluted sera in a total volume of 105 ␮l in microplates (Nunc, Denmark). After 30 min of incubation at 37 ◦ C, Alamar Blue reagent (Life Technologies, USA) was added. Survival of bacteria was measured according to the manufacturer’s protocol. To inhibit the activity of classical and alternative pathways of complement, serum was pre-treated with sodium polyanethole sulfonate (SPS, Sigma–Aldrich) (0.4 mg/ml) for 20 min on ice (Palarasah et al. 2010). Ficolin-3- (and H. alvei PCM 1200-reactive antibodies)depleted serum was prepared by thrice incubation of pre-diluted 1:10 serum with 5 mg of dry mass of H. alvei PCM 1200 for 30 min at 4 ◦ C, followed by centrifugation (10,000 × g, 2 min). Alternatively, Ficolin-3 was depleted by incubation of serum in an anti-Ficolin-3 mAb (clone 4H5, Hycult, The Netherlands) coated-plate. Antibodydepleted serum was performed similarly, using protein A/G agarose (Sigma–Aldrich). Serum was diluted in a buffer containing 25 mM Hepes, 5 mM CaCl2 , 5 mM MgCl2 and 155 mM NaCl (pH 7.4) unless otherwise indicated. C4 deposition assay Ficolin-3-dependent lectin pathway activation by bacterial cells was estimated in a modified C4 deposition assay (Petersen et al. 2001; Swierzko et al. 2012). Briefly, the microtitre plates (Nunc) were coated with dry mass (10 ␮g/ml) of paraformaldehydeinactivated bacteria (according to Neth et al. 2000) and incubated overnight at 4 ◦ C. The plates were washed with TBS supplemented with 5 mM Ca2+ (TBS-Ca2+ ) and 0.05% Tween-20 and then blocked with 0.1% BSA in TBS-Ca2+ . Normal or Ficolin-3- (and H. alvei PCM 1200-reactive antibodies)-depleted human serum (10-fold diluted in 0.1% BSA/MBL-binding buffer) was added and incubated overnight (4 ◦ C). The MBL-binding buffer, containing 20 mM Tris, 1 M NaCl, 10 mM CaCl2 , pH 7.4 (high ionic strength) was used to prevent the activation of complement via the classical pathway (Petersen et al. 2001). Next, low Ficolin-3 serum (Swierzko et al. 2012), pre-diluted 1:3000 in 0.1% BSA/TBS-Ca2+ , was added as a source of C4. After incubation for 2 h at 37 ◦ C, deposited C4c was detected

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with the use of specific rabbit and HRP-labeled goat anti-rabbit Ig antibodies (both from Dako, Denmark). 2,2 -azino-bis(3ethylbenz-thiazoline-6-sulphonic)acid (ABTS) (Sigma) was used as substrate for HRP. Absorbance (at 405 nm) was read with the use of a Benchmark Plus microplate spectrophotometer (Bio-Rad, USA).

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Statistical analysis Experimental values are given as the mean ± standard deviation (SD) from at least three experiments. The statistical significance of differences was assessed by using Tukey’s multiple comparison test after analysis of variance; p values of <0.05 were considered significant.

Phagocytosis assay – flow cytometry The U-937 cells were distributed in 6-well tissue culture plates (Nunc) (106 cells/well) in DMEM medium (with antibiotics and FBS) containing additionally 20 ng/ml of phorbal myristate acetate (PMA, Sigma–Aldrich), to induce differentiation and adherence. After 48 h of incubation, the medium containing non-adherent cells was aspirated. The adherent cells were washed in PBS, and GFP-tagged bacteria in DMEM (without antibiotics and FBS) were added (bacteria to macrophages ratio: 100:1). Bacteria were preincubated with 10% NHS or 2 ␮g of recombinant Ficolin-3 (R&D System, USA) dissolved in 25 mM Hepes, 5 mM CaCl2 , 5 mM MgCl2 and 155 mM NaCl (pH 7.4) for 20 min, at 37 ◦ C. Next, plates were centrifuged (100 × g, 5 min, RT) and then incubated (1 h, 37 ◦ C, 5% CO2 , 95% humidity) to allow phagocytosis. After washing with PBS, macrophages were detached with trypsin-EDTA, and then fixed with BD fixation buffer (Becton Dickinson, USA). The cells were washed twice with PBS and analyzed in a BD LSRII flow cytometer (Becton Dickinson). Phagocytosis assay – fluorescent microscopy For this approach, phagocytosis was performed in a similar manner to that described above. The U-937 cells were seeded in glass chamber slides (Nunc) at a density of 105 cells/well and cultured as described above. GFP-expressing bacteria were preopsonized with 1% NHS or 200 ng of recombinant Ficolin-3 (R&D System). After phagocytosis, cells were washed three times and fixed with 4% paraformaldehyde (PFA). Then, cells were permeabilized with 0.1% Triton X-100 (Sigma–Aldrich, USA) and stained with DAPI (Life Technologies) and Texas Red-X Phalloidin (Life Technologies). The coverslips were mounted on glass slides using Mowiol (Sigma–Aldrich). Slides were viewed in a TE-200U fluorescent microscope (Nikon), and the percentage of cells containing intracellular GFP-expressing bacteria was determined. The images were taken with the help of a confocal microscope (Nikon D-Eclipse C1 with EZ-C1 version 3.6 software). NF-B translocation For detection of the intracellular location of the NF-␬B p65 subunit, an ArrayScan Reader (Thermo Fisher Scientific, USA) was employed, allowing quantitative analysis of the translocation of NF-␬B p65. HEK293/TLR4-MD2-CD14 or HEK293/null cells were seeded in a 96-well plate (black with clear bottom, Greiner Bio One, Germany; 2 × 104 cells/well) in DMEM medium, without FBS and antibiotics. After overnight incubation, cells were treated with LPS or LPS pre-incubated with various amounts of Ficolin-3 for 40 min. Next, cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 (Sigma–Aldrich) and incubated with NF-␬B p65 DyLight 488 Rabbit Monoclonal Antibody (Abcam, USA). After washing, the nuclei were stained with HCS Nuclear Mask Blue (Life Technologies) and the cells were analyzed using an ArrayScan Reader. The Cytoplasm to Nucleus Translocation BioApplication software (Thermo Fisher Scientific, USA) was used to calculate the difference in NF-␬B staining intensity between nucleus and cytoplasm. An average intensity of 1000 cells/well was quantified.

Fig. 1. Ficolin-3-dependent agglutination of H. alvei PCM 1200 and PCM 1222 bacteria. (A) Data from fluorescent microscopy (GFP-tagged H. alvei PCM1200 strain). (I) Bacteria incubated with recombinant Ficolin-3; (II) bacteria incubated with recombinant Ficolin-3 and EDTA; (III) bacteria incubated with neither Ficolin-3 nor EDTA; (IV) bacteria incubated with EDTA. Data from one of the three independent experiments are presented. (B and C) Data from photometry. Sedimentation of H. alvei PCM 1200 (B) and H. alvei PCM 1222 (C) cells. Graphs show the decrease of optical density of bacterial suspensions incubated with recombinant Ficolin-3 (20 ␮g/ml) in TBS supplemented with Ca2+ and Mg2+ (solid squares), buffer alone (open squares), or recombinant Ficolin-3 (20 ␮g/ml) in TBS containing EDTA instead of divalent cations (circles). Dotted lines indicate incubation at 37 ◦ C when the OD was not measured. Each point is expressed as mean ± SD (n = 2).

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Results Ficolin-3 agglutinates bacteria and contributes to the bactericidal activity of serum Recombinant Ficolin-3 was demonstrated to agglutinate GFPexpressing H. alvei PCM 1200 bacteria, in the presence of CaCl2 (Fig. 1A-I). Addition of EDTA markedly reduced that ability (Fig. 1AII). No agglutination was observed in the absence of Ficolin-3 (Fig. 1A-III and A-IV) or after incubation of Ficolin-3 with H. alvei PCM 1222 bacteria (data not shown). Furthermore, by measuring the optical density of the bacterial suspension, it was shown that Ficolin-3 agglutinated approximately 70% of H. alvei PCM 1200 cells within 2 h (Fig. 1B). A more modest effect (approx. 20%) was also observed with H. alvei PCM 1222 (Fig. 1C). To determine the anti-bacterial activity of Ficolin-3, H. alvei PCM 1200 bacteria were incubated with serially diluted NHS (i), serum depleted of Ficolin-3-MASP complexes and H. alvei PCM 1200-recognizing antibodies (ii), serum depleted of immunoglobulins (iii) or serum pre-treated with SPS (iv). The effectiveness of absorption (Ficolin-3 concentration, normal immunoglobulins concentration and ability of absorbed sera to activate C3) was checked in a specific ELISA (data not shown). The survival of bacteria was estimated spectrophotometrically with the use of Alamar blue reagent. Pre-absorption of serum with H. alvei PCM 1200 dry mass significantly reduced its bactericidal activity in comparison

with NHS (Fig. 2A). A similar effect was observed when Ficolin-3 was removed with specific mAbs (Fig. 2B). In contrast, removal of immunoglobulins by pre-incubation with protein A/G-Sepharose or inhibition of classical and alternative complement pathways by SPS did not influence significantly the anti-bacterial potency of serum (Fig. 2A). Additionally, activation of the lectin pathway of complement on the surface of bacterial cells was confirmed by ELISA (Fig. 2C). C4c deposition was observed for H. alvei PCM 1200 incubated with NHS. Depletion of Ficolin-3 markedly reduced this activity. Practically no effect was observed for H. alvei PCM 1222. Those findings indicated a key role of Ficolin-3 and the lectin pathway of complement activation in elimination of H. alvei PCM 1200 bacteria. Ficolin-3 augments phagocytosis of bacteria To test the possible contribution of Ficolin-3 to opsonophagocytosis, GFP-tagged H. alvei bacteria (PCM 1200 or PCM 1222 strains) were opsonized with recombinant Ficolin-3. The lipopolysaccharide from PCM 1200 strain, in contrast to that from PCM 1222 bacteria, was previously shown to be recognized by Ficolin-3 (Swierzko et al. 2012). Alternatively, NHS was used as a source of Ficolin-3, complexed with MASP. Phagocytosis was analyzed with the use of three different methods. Non-opsonized bacteria were used as control (Fig. 3A-I). Opsonisation of PCM 1200 cells either with NHS (Fig. 3A-II) or recombinant Ficolin-3 (Fig. 3A-III) markedly

Fig. 2. Bactericidal activity of serum Ficolin-3. (A) H. alvei PCM 1200 bacteria were pre-incubated with normal human serum (I), serum depleted of Ficolin-3 (and H. alvei PCM 1200-reactive antibodies) (pre-incubation with dry mass of bacteria) (II), serum depleted of antibodies (III) or serum with inhibited complement classical and alternative pathways (IV). The survival of bacteria was estimated spectrophotometrically with the use of Alamar blue reagent. Results (fold change to NHS) are expressed as mean values ± SD (n = 3). Statistical significance is indicated by * (* p < 0.05). (B) H. alvei PCM 1200 bacteria were pre-incubated with normal human serum (I) and Ficolin-3 depleted serum (by incubation of serum in an anti-Ficolin-3 mAb coated plate) (II). Results (fold change to NHS) are expressed as mean values ± SD (n = 2). (C) Ficolin-3-dependent C4c deposition on the bacterial cells. The plate was coated with H. alvei PCM 1200 (circles) or H. alvei PCM 1222 (squares) and incubated with NHS (open symbols) or Ficolin-3/H. alvei PCM 1200-reactive antibodies-depleted serum (solid symbols). Each point is expressed as mean values ± SD (n = 3). Control, no serum added.

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Fig. 3. Contribution of Ficolin-3 to phagocytosis of GFP-tagged H. alvei PCM 1200 bacteria. (A) Data from confocal microscopy: (I) non-opsonized bacteria; (II) bacteria opsonized with normal human serum; (III) bacteria opsonized with recombinant Ficolin-3. The nuclei and actin filaments of macrophages were stained with DAPI and Red-Phalloidin, respectively. White boxes are shown in lower pictures. (B) Data from flow cytometry. Bacteria (H. alvei PCM 1200 or PCM 1222 strain) were opsonized with normal human serum (NHS) or recombinant Ficolin-3 (F-3). As control, non-opsonized bacteria were used (C). The numbers of cells with engulfed bacteria are presented as mean values ± SD (n = 5). Statistical significance is indicated by * (** p < 0.01 and * p < 0.05). (C) Data from flow cytometry. Bacteria were opsonized with normal human serum (NHS), Ficolin-3 depleted serum or recombinant Ficolin-3 (rF-3). Data from one of two independent experiments are presented.

enhanced their phagocytosis. That was further confirmed in flow cytometry – more than 40% of phagocytes contained engulfed NHS-opsonized bacteria, compared with less than 15% in controls (Fig. 3B). In confirmation, depletion of Ficolin-3 and PCM 1200-reactive antibodies, significantly reduced the effect of serum (Fig. 3C). The rate of phagocytosis of H. alvei PCM 1222 bacteria opsonized with normal serum or recombinant Ficolin-3 was significantly lower when compared with that of the PCM 1200 strain (Fig. 3B). On the other hand, although PCM 1222 LPS was previously demonstrated not to be recognized by Ficolin-3 (Swierzko et al. 2012), a slight (statistically insignificant) effect of the recombinant protein on phagocytosis was observed. Similar results were obtained when the number of phagocytes containing engulfed bacteria was estimated using fluorescent microscopy (data not shown). Ficolin-3 inhibits LPS-dependent cell activation To test the biological consequences of Ficolin-3 interaction with LPS, the influence of Ficolin-3 on H. alvei PCM 1200

LPS-induced NF-␬B activation was analyzed. HEK293 cells, expressing the TLR4/MD-2/CD14 complex, which is crucial for LPS lipid A-dependent NF-␬B activation, were treated with LPS, stained, and the intracellular location of NF-␬B p65 subunit was measured. In preliminary experiments, the optimal period of stimulation was estimated (data not shown). Since maximal cell activation was observed after 50 min, for further experiments 40 min stimulation with 100 ng/ml of LPS was chosen. Pre-incubation of LPS with Ficolin-3 led to dose-dependent inhibition of NF-␬B activation (Fig. 4). No cell activation was observed when HEK293/TLR4-MD2-CD14 cells were incubated with Ficolin-3 only and was minimal upon stimulation of HEK293/null cells with LPS (data not shown). Discussion H. alvei is a Gram-negative, facultatively anaerobic component of normal gut flora that is not normally pathogenic in humans. Nevertheless, it is occasionally an aetiological agent of septicaemia, respiratory infections and peritonitis, and may even complicate graft-versus-host disease (Janda and Abbott 2006; Liu et al. 2007;

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Fig. 4. Effect of Ficolin-3 on LPS-dependent cell activation. HEK293/TLR4/MD2/CD14 cells were stimulated with H. alvei PCM 1200 LPS, H. alvei PCM 1200 LPS pre-incubated with increasing doses of recombinant Ficolin-3 or Ficolin-3 alone. The cell activation was estimated as NF-␬B translocation to nucleus with the use of AssayScan Reader. Results are expressed as mean values ± SD (n = 7). Statistical significance is indicated by * (*** p < 0.001 and ns p > 0.05).

Savini et al. 2008; Yap et al. 2010). Opportunistic infections are by their very nature rare in healthy subjects and are caused by microbes for which most people have adequate protective immunity. Ficolin-3 might therefore be an important part of the molecular basis of normal protection against extraintestinal H. alvei infection. Despite being discovered over 20 years ago (Yae et al. 1991), knowledge about anti-microbial activity of Ficolin-3 is limited and generally negative. This is because it fails to bind to common bacterial pathogens and common opportunistic microorganisms like Pseudomonas aeruginosa (James Chalmers, personal communication). As one of its few known microbial ligands is H. alvei PCM 1200 LPS, we used it (as well as PCM 1222 bacteria) to investigate the participation of Ficolin-3 in Gram-negative bacterial clearance. Here we clearly demonstrate the ability of Ficolin-3 to agglutinate H. alvei PCM 1200 cells in vitro (agglutination is an important anti-microbial activity: it keeps bacteria at the site of infection, inhibits their spreading and helps phagocytes to engulf bacteria). We also demonstrated that in the presence of Ficolin-3, phagocytosis of H. alvei PCM 1200 is increased, and moreover, we demonstrated that Ficolin-3 possesses potent bactericidal activity, presumably via activation of the lectin pathway of complement. Those effects are linked: deposition of complement activation products enhances opsonization, and may intensify the phagocytosis process. We believe this is the first example of protective activity of Ficolin-3 against a human bacterial pathogen to be published (however, Ficolin-3 was reported to participate in killing of the protozoal pathogens, Trypanosoma cruzi (Cestari Idos et al. 2009) and Giardia intestinalis (Evans-Osses et al. 2010), and to inhibit infectivity of influenza A virus (Verma et al. 2012)). Previously we identified H. alvei PCM 1200 LPS O-specific polysaccharide as a target for Ficolin-3 (Swierzko et al. 2012). Generally, LPS polysaccharide appears to be a target structure of other serum factors, including specific antibodies and the collectins, MBL and surfactant protein D (SP-D) (Yokochi et al. 1990; Sahly et al. 2002). Structures on the LPS polysaccharide are also recognized by cell receptors (Parent 1990). Although the lipid A region is commonly considered the toxic principle of LPS, responsible for cell activation via TLR4/MD-2/CD14 complex (Maeshima and Fernandez 2013; Park and Lee 2013), a modulatory effect of the LPS polysaccharide region on inflammatory responses has been

postulated as well (Kotrange et al. 2011). Our experiments indicate that interaction of Ficolin-3 with the polysaccharide part of H. alvei PCM 1200 LPS significantly attenuates the host cell response to TLR4/MD-2/CD14-lipid A-dependent NF-␬B activation. Ficolin-3 concentration is the highest amongst the human plasma proteins that can activate the lectin pathway and its deficiency is extremely rare. Only three genetically confirmed cases have been reported to date (Munthe-Fog et al. 2009; Schlapbach et al. 2011; Michalski et al. 2012). Recently, we found another patient: a man suffering from primary glomerulonephritis with accompanying EBV infection (unpublished). However, the physiological role of Ficolin-3 still remains unclear. Our data provide further evidence that Ficolin-3 has anti-bacterial activity and modulates the host response to LPS and thus should be considered a component of innate immunity. It might be speculated that the crucial role of Ficolin-3 is in controlling normal (commensal) flora rather than protection from obligatory pathogens. The alarming spread of antimicrobial resistance may portend the end of the era of anti-bacterial chemotherapy. Understanding mechanisms of innate immunity may generate innovative ideas that may be useful for treatment. Replacement therapy with MBL has already been used (Valdimarsson 2003) and ficolins are also thought to play an important role in immunity since their deficiencies may increase susceptibility to infection (Eisen et al. 2008; Schlapbach et al. 2009; Ameye et al. 2012; Luo et al. 2013). Substitution therapy with plasma-derived or recombinant proteins may provide an alternative to, or support for, conventional treatment. Conflict of interest None. Acknowledgements This work was supported by Polish Ministry of Science and Higher Education, Grant N N401 267339. Authors are very grateful to Dr. David C. Kilpatrick for critical reading of the manuscript. References Akaiwa, M., Yae, Y., Sugimoto, R., Suzuki, S.O., Iwaki, T., Izuhara, K., Hamasaki, N., 1999. Hakata antigen, a new member of the ficolin/opsonin p35 family, is a novel human lectin secreted into bronchus/alveolus and bile. J. Histochem. Cytochem. 47, 777–786. Ameye, L., Paesmans, M., Thiel, S., Jensenius, J.C., Aoun, M., 2012. M-ficolin levels are associated with the occurrence of severe infections in patients with haematological cancer undergoing chemotherapy. Clin. Exp. Immunol. 167, 303–308. Ariki, S., Kojima, T., Gasa, S., Saito, A., Nishitani, C., Takahashi, M., Shimizu, T., Kurimura, Y., Sawada, N., Fujii, N., Kuroki, Y., 2011. Pulmonary collectins play distinct roles in host defense against Mycobacterium avium. J. Immunol. 187, 2586–2594. Cestari Idos, S., Krarup, A., Sim, R.B., Inal, J.M., Ramirez, M.I., 2009. Role of early lectin pathway activation in the complement-mediated killing of Trypanosoma cruzi. Mol. Immunol. 47, 426–437. Eisen, D.P., Dean, M.M., Boermeester, M.A., Fidler, K.J., Gordon, A.C., Kronborg, G., Kun, J.F., Lau, Y.L., Payeras, A., Valdimarsson, H., Brett, S.J., Ip, W.K., Mila, J., Peters, M.J., Saevarsdottir, S., van Till, J.W., Hinds, C.J., McBryde, E.S., 2008. Low serum mannose-binding lectin level increases the risk of death due to pneumococcal infection. Clin. Infect. Dis. 47, 510–516. Evans-Osses, I., Ansa-Addo, E.A., Inal, J.M., Ramirez, M.I., 2010. Involvement of lectin pathway activation in the complement killing of Giardia intestinalis. Biochem. Biophys. Res. Commun. 395, 382–386. Gunthard, H., Pennekamp, A., 1996. Clinical significance of extraintestinal Hafnia alvei isolates from 61 patients and review of the literature. Clin. Infect. Dis. 22, 1040–1045. Hummelshoj, T., Munthe-Fog, L., Madsen, H.O., Sim, R.B., Garred, P., 2008. Comparative study of the human ficolins reveals unique features of Ficolin-3 (Hakata antigen). Mol. Immunol. 45, 1623–1632. Janda, J.M., Abbott, S.L., 2006. The genus Hafnia: from soup to nuts. Clin. Microbiol. Rev. 19, 12–18. Kotrange, S., Kopp, B., Akhter, A., Abdelaziz, D., Abu Khweek, A., Caution, K., Abdulrahman, B., Wewers, M.D., McCoy, K., Marsh, C., Loutet, S.A., Ortega, X., Valvano,

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