This is the accepted version of the journal article: Perea, Lidia; Rodríguez-Rubio, Lorena; Nieto Sáchica, Juan Camilo; [et al.]. «Bacteriophages immunomodulate the response of monocytes». Experimental Biology and Medicine, Vol. 246, Issue 11 (June 2021), p. 1263-1268. DOI 10.1177/1535370221995154 This version is available at https://ddd.uab.cat/record/269718 under the terms of the license 1 Bacteriophages immunomodulate the response of monocytes: 2 3 Short running title: Immunogenicity of bacteriophages 4 5 Lídia Perea1*, Lorena Rodríguez-Rubio2*, Juan C Nieto1, Carlos Zamora1, Elisabet Cantó1, German 6 Soriano3, Maria Poca3, Pedro Blanco-Picazo2, Ferran Navarro4, Maite Muniesa2, Silvia Vidal1 7 8 Affiliations: 1 9 Inflammatory Diseases, Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau, Biomedical 10 Research Institute Sant Pau (IIB Sant Pau), 08041, Barcelona, Spain. 2 11 Department of Genetics, Microbiology and Statistics, University of Barcelona, Diagonal 643, 12 Prevosti building. Floor 0, 08028 Barcelona, Spain 3 13 Department of Gastroenterology, Hospital de la Santa Creu i Sant Pau, 08041, Barcelona, Spain. 14 CIBERehd, Instituto de Salud Carlos III, Madrid, Spain. Autonomous University of Barcelona, 15 Cerdanyola del Vallès, Spain. 4 16 Microbiology Department, Hospital de la Santa Creu i Sant Pau, 08041, Barcelona, Spain. 17 Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona, Spain. Department of Genetics and 18 Microbiology, Autonomous University of Barcelona, Cerdanyola del Vallès, Spain. 19 * Lídia Perea and Lorena Rodríguez-Rubio contributed equally to this work. 20 21 Corresponding author: 22 Silvia Vidal 23 Inflammatory Diseases, Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau, Biomedical 24 Research Institute Sant Pau (IIB Sant Pau), Barcelona, Spain. 25 svidal@santpau.cat 26 27 1 28 ABSTRACT 29 Bacteriophages are present in fluids from cirrhosis patients. However, their effect on the immune 30 response is unknown. In this work, we explore the role of phages in the phenotype, function and 31 cytokine production of monocytes. We stimulated healthy monocytes with five different butanol- 32 purified phage suspensions infective for Gram-negative and Gram-positive bacteria. We studied the 33 expression of the monocyte markers involved in lipopolysaccharide recognition (LPS; CD14), antigen 34 presentation (HLA-DR) and co-stimulation (CD86), and the concentration of induced cytokines 35 (TNF-α, IFN-α and IL-10) by phages. To confirm the direct role of phages without the interference of 36 contaminating soluble LPS in phage suspensions, polymyxin B was added to the cell cultures. 37 Phagocytosis experiments were assessed by flow cytometry using labelled phage suspensions. We 38 observed that butanol-purified phages reduced the surface levels of CD14 and CD86 in monocytes 39 and increased the secreted levels of TNF-α and IL-10 compared with the control sample containing 40 only butanol buffer. All phage suspensions showed downregulation of HLA-DR expression but only 41 Staphylococcus aureus phage contaminated with Escherichia coli reached statistical significance. The 42 addition of polymyxin B did not restore the monocytic response induced by phages, suggesting that 43 the effect was not caused by the presence of LPS. Monocytes were able to phagocyte phages in a 44 dose- and time-dependent manner. To conclude, the phagocytosis of butanol-purified phages altered 45 the phenotype and cytokine production of monocytes suggesting they become tolerogenic. 46 Keywords: Bacteriophages; Immunity; Cirrhosis 47 48 49 IMPACT STATEMENT 50 It is known that patients with liver cirrhosis have phages in their ascitic fluids. Our work demonstrates 51 that phages induce an immune response in healthy monocytes, in terms of phenotype, production of 52 soluble factors and phagocytosis function. Furthermore, our work validates the usefulness of 53 polymyxin B and 1-butanol protocol as easy strategies to discard the interference of bacterial products 54 in the phage suspensions. 55 56 INTRODUCTION 57 Bacteriophages, also called phages, are viruses present in 45% of the ascitic fluid (AF) samples from 58 patients with spontaneous bacterial peritonitis (SBP).1, 2 SBP, which is the most severe complication 59 in patients with liver cirrhosis, is generated by the translocation of bacteria from the gut to the 60 peritoneal cavity caused by an increased intestinal permeability.3 The diagnosis of SBP is based on a 61 positive bacteriological culture result and a count of polymorphonuclear cells above 250 cells/mm3 in 62 the AF. However, bacteria are not detected in a half of the patients with >250 cells/mm3 in AF. 63 Interestingly, these patients have a similar severity and prognosis than those patients with positive 64 bacteriological result, 4 suggesting that not only the presence of bacteria in AF induces an immune 65 response. 66 Bacterial cells are infected by specific phages through lysogenic cycles and then, bacteria are 67 lysed through lytic cycles.5 Although phages do not infect eukaryotic cells, immune cells can be 68 stimulated directly by phages or indirectly by the massive pathogen-associated molecular patterns 69 (PAMPs), released after the lysis of phage-induced or phage-infected bacteria.6 Phagocytes 70 (monocytes and neutrophils) are the main cell populations found in AF from patients with cirrhosis. 71 We have previously reported that ascitic monocytes from SBP expressed low levels of antigen 72 presentation (HLA-DR) and activation (CD86) markers. Furthermore, they produced high levels of 73 TNF-α and IL-10 and they have a reduced ability to phagocytose Escherichia coli.7 Surprisingly, 74 ascitic monocytes from SBP patients with either positive or negative bacteriological culture show a 75 similar immune response.7 76 In the present study, we hypothesized that the immune response displayed by ascitic monocytes in 77 cirrhosis is not only due to the presence of bacteria but also to the presence of phages in the AF. To 78 confirm our hypothesis, first, healthy monocytes were stimulated with butanol-purified phage 79 suspensions and we studied the markers related with LPS receptor (CD14), antigen presentation 80 (HLA-DR) and co-stimulation (CD86), and the soluble factors (TNF-α, IFN-α and IL-10) observed in 81 the ascitic monocytes. Second, it was analyzed whether the immune response observed was phage- 82 host specific. Third, the interference of soluble LPS in the phage suspensions was discarded through 83 two strategies: by the addition of polymyxin B and using phage suspensions infecting Gram-positive 84 Staphylococcus aureus, which do not contain LPS in their bacterial cell wall. Finally, the 85 phagocytosis was studied by flow cytometry using labelled phages in dose- and time-dependent 86 assays. 87 88 MATERIALS AND METHODS 89 Bacteriophages and bacterial strains 90 Five different phage suspensions were studied. They included three virulent phages infecting E. coli 91 WG5 (ATCC 700078) (phages SOM1, SOM3 and SOM4)8 and one phage infecting S. aureus strain 92 RN4220 (phage ɸ11)9 (Table 1). The fifth phage suspension contained the same S. aureus phage but 93 propagated in a S. aureus RN4220 culture contaminated with a sonicated culture of E. coli strain 94 WG5 (prepared as described in the following section). This phage suspension was included to confirm 95 that the butanol protocol removes the contaminating soluble lipopolysaccharide (LPS) from E. coli. 96 This is in contrast with the non-contaminated ɸ11 phage suspension where LPS could not be present 97 as S. aureus lacks LPS in its cell wall. 98 Butanol-purification of phage suspensions 99 Phage preparations were purified following the butanol protocol described by Szermer-Olearnik B et 100 al.10 For it, bacteria culture in Luria broth was carried at 37°C for 8–16 hours, until the optical density 101 (OD, 600 nm) reached 0.3, which corresponded to about 108 bacterial cells/ml.8 At this point the 102 culture was infected with phage in a proportion of 0.1 PFU/bacterial cells, and incubated at 37°C for 8 103 h. Crude bacterial lysates (5 ml–20 l) were filtered through 0.22 μm low protein binding 104 polyethersulfone (PES) membranes (Millex-GP, Merck). 1-butanol was added (about 40% v/v) to the 105 bacterial lysate and shaken for 1–3 hours at room temperature. Then, the two-phase mixture was 106 cooled to 4°C for 1–3 hours and separated by centrifugation at 4000 ×g, 10 min. The collected 107 aqueous phases were dialyzed in a buffer containing NaCl 0.15M and concentrated with Amicon 108 Ultra-15 Centrifugal Filters 50K (Millipore) to a final volume of 5 mL in NaCl 0.15M. A buffer 109 control using the same bacterial cultures in the absence of phages was processed in parallel and 110 included in the analysis. In parallel, one phage ɸ11 suspension was contaminated with E. coli to 111 include LPS in the suspensions and monitor its removal by the butanol protocol. An overnight culture 112 of E. coli strain WG5 containing 109 bacterial cells/ml was sonicated for 30 sec and placed on dry ice 113 for 30 sec in four consecutive steps to disrupt the cells. One ml of this culture was added to 200 ml 114 culture of S. aureus RN4220 containing phage ɸ11 culture in a proportion of 0.1 plaque-forming unit 115 (PFU)/bacterial cells, and incubated at 37°C for 8 h. Purification of phages was performed after 116 incubation as described above. After purification and concentration of the phage suspensions, phage 117 titer was determined using the double layer agar technique.11 118 SYBR-Gold staining of phage suspensions 119 The phage SOM1 and SOM3 suspensions were stained with SYBR-Gold (Molecular probes, 120 Thermofisher) as previously reported.12 Briefly, 20 µl of SYBR-Gold 100X was added per ml of 121 phage suspension (109 PFU/ml). Suspensions were gently mixed and incubated 1h in the dark. 122 Suspensions were washed four times with NaCl 0.15M using Amicon Ultra-15 Centrifugal Filters 123 50K (Millipore) to remove the excess of SYBR-Gold and the suspension was obtained in a final 124 volume of 1 ml using NaCl 0.15M. After purification and concentration of the phage suspensions, the 125 total number of labelled phages was counted by flow cytometry and adjusted to ca 107 phage 126 particles/ml using buffer NaCl 0.15M. 127 A buffer control using culture media in the absence of phages was processed in parallel for SYBR 128 Gold staining and purification and included in the analysis. 129 PBMCs isolation and stimulation with phage suspensions 130 PBMCs were isolated from 10 ml of peripheral blood of healthy donors using a Lymphoprep gradient 131 (AXIS-SHIELD, PoCAs, Oslo, Norway). The total number of cells was counted by flow cytometry 132 and adjusted to 2x105 monocytes/ml with RPMI medium supplemented with 25mM HEPES buffer 133 (hereafter referred as RPMI-HEPES medium; Sigma-Aldrich, St.Louis, MO). Subsequently, 100.000 134 PBMCs were stimulated with 1/100 of phage suspensions diluted in RPMI-HEPES medium in a total 135 volume of 200 µl in 96-well culture plates. As a negative control, PBMCs were also stimulated with 136 1/100 of the butanol buffer. Furthermore, to discard the possibility that soluble LPS molecules 137 remained in the purifications of phage infecting E. coli, PBMCs were pre-cultured with 10 µg/ml of 138 polymyxin B (Sigma-Aldrich) for 30 minutes at 37ºC before the stimulation with phage suspensions. 139 Then, PBMCs were incubated for 24 hours at 37ºC. After incubation, cells were harvested from wells, 140 stained for 15 minutes at room temperature and darkness with anti-CD14 PECy7 (BioLegend), anti- 141 CD86 PE (BioLegend) and anti-HLA-DR APC (Immunotools), and washed with Phosphate Buffered 142 Saline (PBS 1X) before the acquisition by flow cytometry (MACSQuant Analyzer; Miltenyi, 143 Germany). 144 Soluble factors measured in supernatants of phage stimulated PBMCs 145 Supernatants of PBMCs stimulated with phage suspensions for 24 hours were collected and the levels 146 of TNF-α, IFN-α (BD Biosciences, San Diego, CA) and IL-10 (ImmunoTools, Friesoythe, Germany) 147 were measured by ELISA. The limit of detection was 30 pg/ml for TNF-α, 7 pg/ml for IFN-α and 9.4 148 pg/ml for IL-10. Supernatants were diluted 1/2 for TNF-α, 1/20 for IFN-α, and 1/10 for IL-10. 149 Phagocytosis assay with labelled phage 150 Phage suspensions containing 107 PFU/ml labelled with SYBR-Gold as described above were used 151 for the phagocytosis experiments. Two ml of whole blood were lysed using red blood cell lysis 152 (BioLegend, San Diego, CA). Phagocytes (monocytes and neutrophils) were counted by flow 153 cytometry and adjusted to 200.000 cells with RPMI-HEPES medium. Phagocytes were incubated in 154 96-well culture plates with 1/5, 1/10 and 1/20 labelled phage SOM1 and SOM3 for periods of 20, 40 155 and 120 minutes at 37ºC and at 4ºC to inhibit the phagocytosis process. After the incubation, cells 156 were harvested from wells, stained for 15 minutes at room temperature and darkness with anti-CD14- 157 PECy7 (BioLegend) and washed with PBS 1X before the acquisition by flow cytometry. Phagocytic 158 monocytes were defined as CD14 positive, low granularity and SYBR-Gold positive. 159 Statistical analysis 160 Experimental groups were compared by t-test for paired data with the respective negative controls. 161 Correlations were analysed by Spearman test. Significance was established at p < 0.05. Values were 162 expressed as mean ± SD. 163 164 RESULTS 165 Effect of phages on the expression of monocyte markers 166 It was first confirmed that either the butanol buffer used for the phage purification or the phage 167 suspensions did not affect to the viability, size, and granularity of CD14+ cells (monocytes) (Figure 168 S1). Upon stimulation, we found that all phage suspensions significantly reduced the percentage and 169 the expression of CD14 and CD86 compared with monocytes stimulated with buffer in the absence of 170 phages. Only ɸ11 phage contaminated with E. coli significantly down-regulated HLA-DR 171 expression compared with buffer in absence of phages (Figure 1). 172 To validate that the observed changes in monocytes were not due to contaminating LPS from Gram- 173 negative bacteria in the phage suspensions, we performed the monocyte stimulation in the presence of 174 polymyxin B (polyB). PolyB neutralizes the effect of LPS but at higher concentrations can also down- 175 regulate CD14 13. Therefore, we first tested the concentration of polyB to counteract the effect of LPS 176 without down-regulating CD14. At 10 µg/ml, polyB maintained the expression of CD14 and reverted 177 the downregulation of HLA-DR and CD86 produced by LPS (Figure S2). However, polyB did not 178 revert the reduction of CD14, HLA-DR and CD86 expression on monocytes after phage stimulus, 179 confirming that the specificity of immune response in monocytes was induced by the phages. 180 Furthermore, we did not observe significant differences between the S. aureus ɸ11 phage suspension 181 and S. aureus ɸ11 phage contaminated with E. coli (Figure 1). This observation validates that butanol 182 protocol removed efficiently soluble LPS from the phage suspensions, either those propagated in E. 183 coli or those in S. aureus that were contaminated with E. coli. Only the phage suspension SOM4 184 cultured in presence of polyB increased the levels of CD14 in monocytes compared with the culture 185 without polyB, but without reaching de CD14 levels observed under buffer conditions. However, the 186 effect of polyB in this phage suspension was not observed neither in HLA-DR nor in CD86 levels, 187 suggesting that only a minimal source of residual LPS could exist in this phage suspension. 188 Effect of phages on the production of soluble factors by PBMCs 189 The concentration of IFN-α, TNF-α and IL-10 produced by PBMCs stimulated with the five phage 190 suspensions was measured in the supernatants. IFN-α levels were undetectable in all the conditions in 191 PBMCs cultured neither with buffer alone nor upon stimulation with phage suspensions (Figure 2A). 192 All phage suspensions increased the TNF-α and IL-10 production by PBMCs compared with buffer 193 alone (Figure 2B-C). We have also demonstrated using butanol-purified phages that the presence of 194 polyB during the stimulation with phage suspensions did not revert the TNF-α and IL-10 levels. 195 Taking together the results about phenotype and soluble factors induced by phages, a negative 196 correlation between the expression of CD86 on monocytes stimulated with phage suspensions and the 197 TNF-α levels in their supernatants was observed (rho=-0.66, p=0.007) (Figure S3). 198 Ability of monocytes to phagocytose phages 199 The phage-monocyte interaction using labelled phages infecting Gram-negative (SOM4) and Gram- 200 positive (ɸ11) bacteria was assessed by flow cytometry. We observed an increased phagocytosis at the 201 highest phage concentration used (1/5) compared with the lowest phage concentration (1/20) and at 40 202 minutes of incubation (Figure 3A). Since phagocytosis and unspecific binding of phages to the cell 203 surface cannot be distinguished by flow cytometry, we repeated the experiment at 4ºC, a temperature 204 at which phagocytosis activity does not take place, to confirm that monocytes efficiently phagocyte 205 phages in an early and dose-dependent process (Figure 3B). 206 207 DISCUSSION 208 In the present study, by using butanol-purified phages, we have demonstrated that phages are able to 209 induce in monocytes a tolerant immune response after being phagocytosed. This response is similar to 210 that observed in our previous paper about ascitic monocytes from cirrhotic patients. It is mainly 211 characterized by a reduction in the expression of CD14 and CD86 and an increase in the soluble TNF- 212 α and IL-10 levels. Furthermore, we have validated the butanol purification as a useful protocol to be 213 used in cell stimulation assays without any interference of free endotoxin in phage suspensions. 214 The reduction in CD14 and CD86 observed on monocytes stimulated by phages suggest a strategy of 215 phages to avoid being removed by the immune system through turning cells into tolerogenic state. 216 HLA-DR expression was also down-regulated by ɸ11 phage contaminated with E. coli. During an 217 infection, it is likely that the tolerogenic monocytes induced by phages favors the infection 218 progression. Our previous results in ascitic monocytes from infected SBP patients showed a similarly 219 reduced expression of CD14, HLA-DR and CD86 7 than the response observed in healthy monocytes 220 stimulated by phages. Particularly, low HLA-DR levels in the ascitic monocytes from SBP with the 221 negative bacteriological result were associated with a high bacterial DNA burden.14 However, studies 222 with isolated phages from infected fluids are needed to better understand their role during the 223 infection. It is also well known that soluble LPS from Gram-negative bacteria is a potent inductor of 224 the immune response. Our results demonstrate that polyB did not revert the changes induced by phage 225 suspensions in monocytes. Therefore, our work highlight the use of polyB as an easy strategy to 226 validate that the changes observed in immune cells after phage stimulations are not affected by 227 contaminating soluble LPS in phage suspensions. 228 We did not observe significant differences in the immune response induced in monocytes by the 229 different phages infecting the different bacterial hosts. Van Belleghem JD et al. have also observed 230 comparable induced immune responses by Gram-negative and Gram-positive phages.15 One 231 possibility is that the changes in CD14, HLA-DR and CD86 expression are induced by phage proteins 232 common in all these phages. In any case, differences attributable to remaining LPS fragments from 233 the host bacteria in the phage suspensions can be ruled out. 234 In our assays, we were not able to detect IFN-α levels. This fact could be surprising since monocytes 235 and dendritic cells are able to produce type I IFN during viral infections. However, it could be 236 explained by the fact that IFN-α needs shorter stimulation times to be detected.16 We found that phage 237 suspensions increased the TNF-α and IL-10 levels. IL-10 results are in line with the findings in 238 cirrhosis, since SBP patients had also an increased ascitic IL-10 levels compared with patients without 239 ascitic infection. However, patients with SBP did not show any difference in TNF-α levels compared 240 with patients without ascitic infection.7 This finding can be explained by the elevated production of 241 inflammatory mediators that patients with cirrhosis display regardless of any ascitic infection.17 242 According to our results, both pro- and anti-inflammatory gene expression profiles of PBMCs 243 stimulated with CsCl-purified phages are reported,15 supporting the results about the immunogenicity 244 of phages. Taking together the phenotypic changes and the soluble factors induced, we found a 245 negative correlation between CD86 and TNF-α levels. This finding is consistent with the 246 downregulation of CD86 on monocytes by TNF-α described in patients infected by human 247 immunodeficiency virus (HIV). This phenotypic change induces an altered production of IL-2 and, 248 consequently, results in a deficient proliferative response of lymphocytes.18 It is likely that phages in 249 SBP induce soluble factors that contribute to the tolerant state of ascitic monocytes and, consequently, 250 favor the infection. 251 We have validated a flow cytometry assay to study the phage-monocyte interaction through 252 phagocytosis assays with labelled phage suspensions. We have observed the phagocytosis of phages 253 by monocytes at short times. According to other reports using microscopic analysis, phagocytosis of 254 phages is an early process since, at longer times of incubation, the intracellular destruction of phages 255 begins.19, 20 In line with these results, we did not detect phagocytosis of phages at longer times of 256 incubation (120 minutes). In cirrhosis, we have previously shown that ascitic monocytes from patients 257 with SBP had impaired phagocytosis of E. coli.7 It is possible that the tolerant state induced by phages 258 also contributes to the impaired phagocytosis of monocytes against bacterial infection. The outcome 259 for the phages is to reduce their own elimination by monocytes while promoting the survival of their 260 bacterial host. This hypothesis was demonstrated by Sweere JM et al. when observed that the 261 presence of phages reduced the phagocytosis of bacteria.21 262 Finally, our work suggests that phages infecting Gram-negative and Gram-positive bacteria turn 263 healthy monocytes into a tolerogenic state. We can speculate that in certain infection-associated 264 pathologies, such as cirrhosis, that not only bacteria but also phages in AF, could induce an immune 265 response in monocytes to avoid the clearance of the infection. However, further studies using isolated 266 phages from infected fluids are needed to support our hypothesis. 267 268 Author’s contributions: Conceptualization, L.P., L.R-R., F.N., M.M. and S.V.; methodology, P.B-P, 269 L.R-R, L.P., JC.N., C.Z., E.C., G.S., M.P.; software analysis, L.P., L.R-R; validation, L.P., L.R-R.; 270 formal analysis, L.P., L.R-R., P.B-P, JC.N, C.Z., E.C., G.S., M.P.; investigation, L.P., L.R-R., P.B-P, 271 JC.N, C.Z., E.C., G.S., M.P.; resources, S.V., F.N., M.M.; data curation, L.P., L.R-R., P.B-P, JC.N, 272 C.Z., E.C., G.S., M.P.; writing—original draft preparation, L.P., L.R-R, M.M., S.V.; writing—review 273 and editing, L.P., L. R-R., M.M., F.N., S.V.; visualization, L.P., L.R-R., P.B-P, JC.N, C.Z., E.C., 274 G.S., M.P.; supervision, M.M, S.V., F.N.; project administration, S.V.; funding acquisition, S.V., 275 M.M. All authors have read and agreed to the published version of the manuscript. 276 Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with 277 respect to the research, authorship, and/or publication of this article. 278 Funding: This research was supported by the Spanish Ministerio de Innovación y Ciencia 279 [AGL2016-75536-P]; the Agencia Estatal de Investigación (AEI); the European regional fund (ERF); 280 and the Generalitat de Catalunya [2017SGR170]. P.B.-P. has a grant from the Spanish Ministry of 281 Economy, Industry and Competitiveness [BES-2017-081296]. L.R-R. is a Serra Húnter Fellow. 282 283 FIGURE LEGENDS 284 Figure 1. Changes in monocyte markers expression induced by butanol-purified Gram-negative 285 and Gram-positive phages. (A) Percentage of CD14+, (B) CD14+HLA-DR+ and (C) CD14+CD86+ 286 cells from PBMCs stimulated with 1/100 butanol buffer and 1/100 butanol-purified phages without 287 adding polymixin B (polyB; solid bar) or after adding polyB (stripe pattern). (D) Representative flow 288 cytometry image of CD14+CD86+ cells from PBMCs stimulated with 1/100 butanol buffer or 1/100 289 SOM1 phage with or without polyB. *<0.05; **<0.01; ***<0.001; *Buffer vs Phage; #Buffer with polyB 290 vs Phage with polyB; $Phage vs Phage with polyB. 291 Figure 2. Inflammation induced by butanol-purified Gram-negative and Gram-positive phages. 292 (A) IFN-α (B) TNF-α and (C) IL-10 levels measured by ELISA assays in the supernatants of PBMCs 293 stimulated with 1/100 butanol-purified phages without (solid bar) or after adding polyB (stripe 294 pattern). *<0.05; **<0.01; ***<0.001; *Buffer vs Phage; #Buffer with polyB vs Phage with polyB; $ 295 Phage vs Phage with polyB. 296 Figure 3. Phagocytosis of gram-negative and gram-positive phages by monocytes. (A) Kinetic 297 assay of phagocytosis: Percentage of phagocytic monocytes cultured with 1/5, 1/10 and 1/20 of 298 SYBR-Gold -labeled Gram-negative (SOM4) and Gram-positive (ɸ11) phage for 20 and 40 minutes 299 at 37ºC. Phagocytosis was determined using flow cytometry and was expressed as the percentage of 300 phagocytic monocytes stained with anti-CD14. *<0.05, Phagocytosis 1/5 vs 1/10 or 1/20. (B) 301 Temperature assay to determine the non-specific phagocytosis of phages: monocytes were cultured 302 with 1/5, 1/10 and 1/20 of SYBR-Gold -labeled gram-negative (SOM4) and gram-positive (ɸ11) 303 phage for 40 minutes at 4ºC to inhibit the mechanism of phagocytosis. *<0.05, **<0.01, Phagocytosis 304 at 37ºC vs phagocytosis at 4ºC. 305 306 REFERENCES 307 1. Blanco-Picazo P, Fernández-Orth D, Brown-Jaque M, Miró E, Espinal P, Rodríguez-Rubio L, 308 Muniesa M, Navarro F. 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Bacteriophage Phage Tail type Source Bacterial host Titer after Reference family strain purification (PFU/ml) SOM1 Siphoviridae Curly Sewage E. coli WG5 8.0 x107 (8) Muniesa et al., 2003 ATCC 700078 SOM3 Myoviridae Contractile Sewage E. coli WG5 7.0 x108 (8) Muniesa et al., 2003 ATCC 700078 SOM4 Siphoviridae Flexible Sewage E. coli WG5 4.2 x108 (8) Muniesa et al., 2003 ATCC 700078 ɸ11 Siphoviridae Flexible S. aureus S. aureus 3.2 x108 (22) Ubeda et al., RN451 RN4220 2005 365 1 PFU, Plaque-forming unit 366 367 368 369 370 371 372 373 374 375 376 FIGURES 377 378 Figure 1 379 380 381 382 Figure 2. 383 384 385 386 387 Figure 3. 388 389 390